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Sample records for endogenous precursor cell

  1. Cyclosporin A-Mediated Activation of Endogenous Neural Precursor Cells Promotes Cognitive Recovery in a Mouse Model of Stroke

    Directory of Open Access Journals (Sweden)

    Labeeba Nusrat

    2018-04-01

    Full Text Available Cognitive dysfunction following stroke significantly impacts quality of life and functional independance; yet, despite the prevalence and negative impact of cognitive deficits, post-stroke interventions almost exclusively target motor impairments. As a result, current treatment options are limited in their ability to promote post-stroke cognitive recovery. Cyclosporin A (CsA has been previously shown to improve post-stroke functional recovery of sensorimotor deficits. Interestingly, CsA is a commonly used immunosuppressant and also acts directly on endogenous neural precursor cells (NPCs in the neurogenic regions of the brain (the periventricular region and the dentate gyrus. The immunosuppressive and NPC activation effects are mediated by calcineurin-dependent and calcineurin-independent pathways, respectively. To develop a cognitive stroke model, focal bilateral lesions were induced in the medial prefrontal cortex (mPFC of adult mice using endothelin-1. First, we characterized this stroke model in the acute and chronic phase, using problem-solving and memory-based cognitive tests. mPFC stroke resulted in early and persistent deficits in short-term memory, problem-solving and behavioral flexibility, without affecting anxiety. Second, we investigated the effects of acute and chronic CsA treatment on NPC activation, neuroprotection, and tissue damage. Acute CsA administration post-stroke increased the size of the NPC pool. There was no effect on neurodegeneration or lesion volume. Lastly, we looked at the effects of chronic CsA treatment on cognitive recovery. Long-term CsA administration promoted NPC migration toward the lesion site and rescued cognitive deficits to control levels. This study demonstrates that CsA treatment activates the NPC population, promotes migration of NPCs to the site of injury, and leads to improved cognitive recovery following long-term treatment.

  2. A brain slice culture model for studies of endogenous and exogenous precursor cell migration in the rostral migratory stream

    DEFF Research Database (Denmark)

    Tanvig, Mette; Blaabjerg, Morten; Andersen, Rikke K

    2009-01-01

    The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb (OB) in rodents. This migration has been well studied in vivo, but an organotypic in vitro model would facilitate more experimental investigations. Here we introduce...

  3. Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

    Science.gov (United States)

    Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

    2017-09-02

    Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. The Innate Lymphoid Cell Precursor.

    Science.gov (United States)

    Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

    2016-05-20

    The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

  5. [Endogenous pyrogen formation by bone marrow cells].

    Science.gov (United States)

    Efremov, O M; Sorokin, A V; El'kina, O A

    1978-01-01

    The cells of the rabbit bone marrow produced endogenous pyrogen in response to stimulation with bacterial lipopolysaccharide. Incubation of the cells in medium No 199 containing a 15% homologous serum is optimal for the release of pyrogen. It is supposed that the cells of the bone marrow take part in the formation of endgenous pyrogen and in the mechanism of pyrexia in the organism.

  6. Persistent Inflammation Alters the Function of the Endogenous Brain Stem Cell Compartment

    OpenAIRE

    Pluchino, Stefano; Muzio, Luca; Alfaro-Cervello, Clara; Salani, Giuliana; Porcheri, Cristina; Brambilla, Elena; Cavasinni, Francesca; Bergamaschi, Andrea; Garcia-Verdugo, Jose Manuel; Comi, Giancarlo; Martino, Gianvito; Imitola, Jaime; Deleidi, Michela; Khoury, Samia Joseph

    2008-01-01

    Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain in...

  7. Planar half-cell shaped precursor body

    DEFF Research Database (Denmark)

    2015-01-01

    The invention relates to a half-cell shaped precursor body of either anode type or cathode type, the half-cell shaped precursor body being prepared to be free sintered to form a sintered or pre-sintered half-cell being adapted to be stacked in a solid oxide fuel cell stack. The obtained half......-cell has an improved planar shape, which remains planar also after a sintering process and during temperature fluctuations....

  8. Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I

    International Nuclear Information System (INIS)

    Drago, J.; Murphy, M.; Carroll, S.M.; Harvey, R.P.; Bartlett, P.F.

    1991-01-01

    Fibroblast growth factor stimulates proliferation and subsequent differentiation of precursor cells isolated from the neuroepithelium of embryonic day 10 mice in vitro. Here we show that fibroblast growth factor-induced proliferation is dependent on the presence of insulin-like growth factors (IGFs) and that IGF-I is endogenously produced by the neuroepithelial cells. Blocking of endogenous IGF-I activity with anti-IGF-I antibodies results in complete inhibition of fibroblast growth factor-mediated proliferation and in cell death. IGF-I alone acts as a survival agent. These observations correlate with the detection of transcripts for IGF-I and basic fibroblast growth factor in freshly isolated neuroepithelium and are consistent with an autocrine action of these factors in early brain development in vivo

  9. Evidence for arginine as the endogenous precursor of necines in heliotropium.

    Science.gov (United States)

    Birecka, H; Birecki, M; Frohlich, M W

    1987-05-01

    In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to (14)C-labeled CO(2) for 44 hours, the incorporation of (14)C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with alpha-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on (14)C incorporation into the necines. In contrast, alpha-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of (14)C into the necines of both species; the inhibitor did not affect the absolute incorporation of (14)C from exogenous [1,4-(14)C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-(14)C]arginine, but also exogenous l-[5-(14)C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of (14)C-labeled CO(2) assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed.

  10. Evidence for Arginine as the Endogenous Precursor of Necines in Heliotropium1

    Science.gov (United States)

    Birecka, Helena; Birecki, Mieczyslaw; Frohlich, M. W.

    1987-01-01

    In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to 14C-labeled CO2 for 44 hours, the incorporation of 14C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with α-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on 14C incorporation into the necines. In contrast, α-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of 14C into the necines of both species; the inhibitor did not affect the absolute incorporation of 14C from exogenous [1,4-14C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-14C]arginine, but also exogenous l-[5-14C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of 14C-labeled CO2 assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed. PMID:16665402

  11. Endogenous T-Cell Therapy: Clinical Experience.

    Science.gov (United States)

    Yee, Cassian; Lizee, Greg; Schueneman, Aaron J

    2015-01-01

    Adoptive cellular therapy represents a robust means of augmenting the tumor-reactive effector population in patients with cancer by adoptive transfer of ex vivo expanded T cells. Three approaches have been developed to achieve this goal: the use of tumor-infiltrating lymphocytes or tumor-infiltrating lymphocytess extracted from patient biopsy material; the redirected engineering of lymphocytes using vectors expressing a chimeric antigen receptor and T-cell receptor; and third, the isolation and expansion of often low-frequency endogenous T cells (ETCs) reactive to tumor antigens from the peripheral blood of patients. This last form of adoptive transfer of T cells, known as ETC therapy, requires specialized methods to isolate and expand from peripheral blood the very low-frequency tumor-reactive T cells, methods that have been developed over the last 2 decades, to the point where such an approach may be broadly applicable not only for the treatment of melanoma but also for that of other solid tumor malignancies. One compelling feature of ETC is the ability to rapidly deploy clinical trials following identification of a tumor-associated target epitope, a feature that may be exploited to develop personalized antigen-specific T-cell therapy for patients with almost any solid tumor. With a well-validated antigen discovery pipeline in place, clinical studies combining ETC with agents that modulate the immune microenvironment can be developed that will transform ETC into a feasible treatment modality.

  12. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

    Science.gov (United States)

    Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

    2016-01-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  13. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Therese Wilhelm

    2016-05-01

    Full Text Available Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es. Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3% rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing, and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and

  14. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    Science.gov (United States)

    Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

    2016-05-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  15. Innate lymphoid cells, precursors and plasticity.

    Science.gov (United States)

    Gronke, Konrad; Kofoed-Nielsen, Michael; Diefenbach, Andreas

    2016-11-01

    Innate lymphoid cells (ILC) have only recently been recognized as a separate entity of the lymphoid lineage. Their subpopulations share common characteristics in terms of early development and major transcriptional circuitry with their related cousins of the T cell world. It is currently hypothesized that ILCs constitute an evolutionary older version of the lymphoid immune system. They are found at all primary entry points for pathogens such as mucosal surfaces of the lung and gastrointestinal system, the skin and the liver, which is the central contact point for pathogens that breach the intestinal barrier and enter the circulation. There, ILC contribute to the first line defense as well as to organ homeostasis. However, ILC are not only involved in classical defense tasks, but also contribute to the organogenesis of lymphoid organs as well as tissue remodeling and even stem cell regeneration. ILC may, therefore, implement different functions according to their emergence in ontogeny, their development and their final tissue location. We will review here their early development from precursors of the fetal liver and the adult bone marrow as well as their late plasticity in adaptation to their environment. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  16. Endogenous Cartilage Repair by Recruitment of Stem Cells.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Articular cartilage has a very limited capacity for repair after injury. The adult body has a pool of stem cells that are mobilized during injury or disease. These cells exist inside niches in bone marrow, muscle, adipose tissue, synovium, and other connective tissues. A method that mobilizes this endogenous pool of stem cells will provide a less costly and less invasive alternative if these cells successfully regenerate defective cartilage. Traditional microfracture procedures employ the concept of bone marrow stimulation to regenerate cartilage. However, the regenerated tissue usually is fibrous cartilage, which has very poor mechanical properties compared to those of normal hyaline cartilage. A method that directs the migration of a large number of autologous mesenchymal stem cells toward injury sites, retains these cells around the defects, and induces chondrogenic differentiation that would enhance success of endogenous cartilage repair. This review briefly summarizes chemokines and growth factors that induce recruitment, proliferation, and differentiation of endogenous progenitor cells, endogenous cell sources for regenerating cartilage, scaffolds for delivery of bioactive factors, and bioadhesive materials that are necessary to bring about endogenous cartilage repair.

  17. In situ tissue regeneration: chemoattractants for endogenous stem cell recruitment.

    Science.gov (United States)

    Vanden Berg-Foels, Wendy S

    2014-02-01

    Tissue engineering uses cells, signaling molecules, and/or biomaterials to regenerate injured or diseased tissues. Ex vivo expanded mesenchymal stem cells (MSC) have long been a cornerstone of regeneration therapies; however, drawbacks that include altered signaling responses and reduced homing capacity have prompted investigation of regeneration based on endogenous MSC recruitment. Recent successful proof-of-concept studies have further motivated endogenous MSC recruitment-based approaches. Stem cell migration is required for morphogenesis and organogenesis during development and for tissue maintenance and injury repair in adults. A biomimetic approach to in situ tissue regeneration by endogenous MSC requires the orchestration of three main stages: MSC recruitment, MSC differentiation, and neotissue maturation. The first stage must result in recruitment of a sufficient number of MSC, capable of effecting regeneration, to the injured or diseased tissue. One of the challenges for engineering endogenous MSC recruitment is the selection of effective chemoattractant(s). The objective of this review is to synthesize and evaluate evidence of recruitment efficacy by reported chemoattractants, including growth factors, chemokines, and other more recently appreciated MSC chemoattractants. The influence of MSC tissue sources, cell culture methods, and the in vitro and in vivo environments is discussed. This growing body of knowledge will serve as a basis for the rational design of regenerative therapies based on endogenous MSC recruitment. Successful endogenous MSC recruitment is the first step of successful tissue regeneration.

  18. Therapeutic targeting strategies using endogenous cells and proteins.

    Science.gov (United States)

    Parayath, Neha N; Amiji, Mansoor M

    2017-07-28

    Targeted drug delivery has become extremely important in enhancing efficacy and reducing the toxicity of therapeutics in the treatment of various disease conditions. Current approaches include passive targeting, which relies on naturally occurring differences between healthy and diseased tissues, and active targeting, which utilizes various ligands that can recognize targets expressed preferentially at the diseased site. Clinical translation of these mechanisms faces many challenges including the immunogenic and toxic effects of these non-natural systems. Thus, use of endogenous targeting systems is increasingly gaining momentum. This review is focused on strategies for employing endogenous moieties, which could serve as safe and efficient carriers for targeted drug delivery. The first part of the review involves cells and cellular components as endogenous carriers for therapeutics in multiple disease states, while the second part discusses the use of endogenous plasma components as endogenous carriers. Further understanding of the biological tropism with cells and proteins and the newer generation of delivery strategies that exploits these endogenous approaches promises to provide better solutions for site-specific delivery and could further facilitate clinical translations. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Adult subependymal neural precursors, but not differentiated cells, undergo rapid cathodal migration in the presence of direct current electric fields.

    Directory of Open Access Journals (Sweden)

    Robart Babona-Pilipos

    Full Text Available BACKGROUND: The existence of neural stem and progenitor cells (together termed neural precursor cells in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. METHODS AND FINDINGS: With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. CONCLUSIONS: These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma.

  20. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  1. Dentin and dental pulp regeneration by the patient's endogenous cells.

    Science.gov (United States)

    Kim, Sahng G; Zheng, Ying; Zhou, Jian; Chen, Mo; Embree, Mildred C; Song, Karen; Jiang, Nan; Mao, Jeremy J

    2013-03-01

    The goal of regenerative endodontics is to restore the functions of the dental pulp-dentin complex. Two approaches are being applied toward dental pulp-dentin regeneration: cell transplantation and cell homing. The majority of previous approaches are based on cell transplantation by delivering ex vivo cultivated cells toward dental pulp or dentin regeneration. Many hurdles limit the clinical translation of cell transplantation such as the difficulty of acquiring and isolating viable cells, uncertainty of what cells or what fractions of cells to use, excessive cost of cell manipulation and transportation, and the risk of immune rejection, pathogen transmission, and tumorigenesis in associated with ex vivo cell manipulation. In contrast, cell homing relies on induced chemotaxis of endogenous cells and therefore circumvents many of the difficulties that are associated with cell transplantation. An array of proteins, peptides, and chemical compounds that are yet to be identified may orchestrate endogenous cells to regenerate dental pulp-dentin complex. Both cell transplantation and cell homing are scientifically valid approaches; however, cell homing offers a number of advantages that are compatible with the development of clinical therapies for dental pulp-dentin regeneration.

  2. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

    Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

  3. Control of RFM strain endogenous retrovirus in RFM mouse cells

    International Nuclear Information System (INIS)

    Tennant, R.W.; Otten, J.A.; Wang, T.W.; Liou, R.S.; Brown, A.; Yang, W.K.

    1983-01-01

    RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1/sup n/ allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission

  4. Hemopoietic precursor cell regeneration following irradiation and syngeneic marrow transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Melchner, H. von

    1983-01-01

    The transplantation of hemopoietic cells into adequately pretreated recipients represents one of the most promising approaches in the treatment of immunohematological disorders such as aplastic anemia, immunodeficiency diseases, leukemias and malignant lymphomas. The basic property of the hemopoietic cells permitting such therapeutic procedure, namely, the capacity of hemopoietic precursors to actively proliferate and differentiate in recipients suffering the consequences of various kinds of hemopoietic failure, represents the subject of the present review. The main cell populations addressed in the subsequent sections are the hemopoietic precursor cells. Mature end cells and in particular lymphocytes did not receive as much attention.

  5. Meninges harbor cells expressing neural precursor markers during development and adulthood.

    Science.gov (United States)

    Bifari, Francesco; Berton, Valeria; Pino, Annachiara; Kusalo, Marijana; Malpeli, Giorgio; Di Chio, Marzia; Bersan, Emanuela; Amato, Eliana; Scarpa, Aldo; Krampera, Mauro; Fumagalli, Guido; Decimo, Ilaria

    2015-01-01

    Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

  6. Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy.

    Directory of Open Access Journals (Sweden)

    Daniel Rodríguez-Martínez

    Full Text Available Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE, a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80% and yield (>70%. Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.

  7. Laser microdissection of sensory organ precursor cells of Drosophila microchaetes.

    Directory of Open Access Journals (Sweden)

    Eulalie Buffin

    Full Text Available BACKGROUND: In Drosophila, each external sensory organ originates from the division of a unique precursor cell (the sensory organ precursor cell or SOP. Each SOP is specified from a cluster of equivalent cells, called a proneural cluster, all of them competent to become SOP. Although, it is well known how SOP cells are selected from proneural clusters, little is known about the downstream genes that are regulated during SOP fate specification. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the mechanism involved in the specification of these precursor cells, we combined laser microdissection, toisolate SOP cells, with transcriptome analysis, to study their RNA profile. Using this procedure, we found that genes that exhibit a 2-fold or greater expression in SOPs versus epithelial cells were mainly associated with Gene Ontology (GO terms related with cell fate determination and sensory organ specification. Furthermore, we found that several genes such as pebbled/hindsight, scabrous, miranda, senseless, or cut, known to be expressed in SOP cells by independent procedures, are particularly detected in laser microdissected SOP cells rather than in epithelial cells. CONCLUSIONS/SIGNIFICANCE: These results confirm the feasibility and the specificity of our laser microdissection based procedure. We anticipate that this analysis will give new insight into the selection and specification of neural precursor cells.

  8. Neural precursor cells in the ischemic brain - integration, cellular crosstalk and consequences for stroke recovery

    Directory of Open Access Journals (Sweden)

    Dirk M. Hermann

    2014-09-01

    Full Text Available After an ischemic stroke, neural precursor cells (NPCs proliferate within major germinal niches of the brain. Endogenous NPCs subsequently migrate towards the ischemic lesion where they promote tissue remodelling and neural repair. Unfortunately, this restorative process is generally insufficient and thus unable to support a full recovery of lost neurological functions. Supported by solid experimental and preclinical data, the transplantation of exogenous NPCs has emerged as a potential tool for stroke treatment. Transplanted NPCs are thought to act mainly via trophic and immune modulatory effects, thereby complementing the restorative responses initially executed by the endogenous NPC population. Recent studies have attempted to elucidate how the therapeutic properties of transplanted NPCs vary depending on the route of transplantation. Systemic NPC delivery leads to potent immune modulatory actions, which prevent secondary neuronal degeneration, reduces glial scar formation, diminishes oxidative stress and stabilizes blood-brain barrier integrity. On the contrary, local stem cell delivery, allows for the accumulation of large numbers of transplanted NPCs in the brain, thus achieving high levels of locally available tissue trophic factors, which may better induce a strong endogenous NPC proliferative response.Herein we describe the diverse capabilities of exogenous (systemically vs locally transplanted NPCs in enhancing the endogenous neurogenic response after stroke, and how the route of transplantation may affect migration, survival, bystander effects and integration of the cellular graft. It is the authors’ claim that understanding these aspects will be of pivotal importance in discerning how transplanted NPCs exert their therapeutic effects in stroke.

  9. Endogenous retinal neural stem cell reprogramming for neuronal regeneration

    Directory of Open Access Journals (Sweden)

    Romain Madelaine

    2017-01-01

    Full Text Available In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by permanent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Müller glial cells. Following injury, zebrafish Müller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Müller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from zebrafish and mammalian Müller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Müller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases.

  10. Early postradiation recovery of precursor cells of hemopoietic stroma

    International Nuclear Information System (INIS)

    Todriya, T.V.

    1984-01-01

    Ability of stroma precursor cells to early postradiation recovery was studied in male mices using the method of fraction irradiation of bone marrow. Donor mices of bone marrow were irradiated in vivo once by the total dose (nonfraction irradiation) and fractionally with 6 h interval between two irradiation doses. The cumulative irradiation doses equal to 10, 12, 14, 16 Gr were investigated. Irradiation was carried out using gamma facility. Bone marrow of the femur was implanted immediately after irradiation under kidney capsule of nonirradiated syngeneic recipient. The ability of stroma precursor cells to intracellular repair (repair index) was evaluated according to the ratio of the number of hemopoietic cells formed in heterotropic transplants in groups with fraction irradiation to the same one in groups with nonfraction irradiation. The obtained results testify to the fact that slowly regenerated highly radioresistant population of precursor cells of hemopoietic stroma is capable to early postradiation recovery

  11. IL-9-Producing Mast Cell Precursors and Food Allergy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0517 TITLE: IL-9-Producing Mast Cell Precursors and Food Allergy PRINCIPAL INVESTIGATOR: Dr. Simon P. Hogan PhD...IL-9-Producing Mast Cell Precursors and Food Allergy 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Yui Hsi Wang, Sunil...threatening anaphylaxis. We have identified a novel multi-functional IL-9-producing mucosal mast cells (MMC9s) that produce large amounts of IL-9, IL

  12. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  13. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  14. Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

    Science.gov (United States)

    Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

    2017-09-01

    Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells

    NARCIS (Netherlands)

    Maldonado, Paloma P; Angulo, María Cecilia

    The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron-OPC synaptic activity

  16. HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

    Science.gov (United States)

    Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

    1995-03-01

    We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

  17. Are there endogenous stem cells in the spinal cord?

    Science.gov (United States)

    Ferrucci, Michela; Ryskalin, Larisa; Busceti, Carla L; Gaglione, Anderson; Biagioni, Francesca; Fornai, Francesco

    2017-12-01

    Neural progenitor cells (NPC) represent the stem-like niche of the central nervous system that maintains a regenerative potential also in the adult life. Despite NPC in the brain are well documented, the presence of NPC in the spinal cord has been controversial for a long time. This is due to a scarce activity of NPC within spinal cord, which also makes difficult their identification. The present review recapitulates the main experimental studies, which provided evidence for the occurrence of NPC within spinal cord, with a special emphasis on spinal cord injury and amyotrophic lateral sclerosis. By using experimental models, here we analyse the site-specificity, the phenotype and the main triggers of spinal cord NPC. Moreover, data are reported on the effect of specific neurogenic stimuli on these spinal cord NPC in an effort to comprehend the endogenous neurogenic potential of this stem cell niche.

  18. Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches.

    Science.gov (United States)

    Akram, Khondoker M; Patel, Neil; Spiteri, Monica A; Forsyth, Nicholas R

    2016-01-19

    The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

  19. Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

    Directory of Open Access Journals (Sweden)

    Chan Anthony WS

    2008-02-01

    Full Text Available Abstract Background Alzheimer's disease (AD is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD. Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP. Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. Results Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. Conclusion This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue

  20. Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

    OpenAIRE

    Bodel, P; Ralph, P; Wenc, K; Long, J C

    1980-01-01

    Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous ...

  1. Two endogenous proteins that induce cell wall extension in plants

    Science.gov (United States)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  2. Whole-cell fungal transformation of precursors into dyes

    Directory of Open Access Journals (Sweden)

    Jarosz-Wilkołazka Anna

    2010-07-01

    Full Text Available Abstract Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25. Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other

  3. Hemopoietic cell precursor responses to erythropoietin in plasma clot cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, W.L.

    1979-01-01

    The time dependence of the response of mouse bone marrow cells to erythropoietin (Ep) in vitro was studied. Experiments include studies on the Ep response of marrow cells from normal, plethoric, or bled mice. Results with normal marrow reveal: (1) Not all erythroid precursors (CFU-E) are alike in their response to Ep. A significant number of the precursors develop to a mature erythroid colony after very short Ep exposures, but they account for only approx. 13% of the total colonies generated when Ep is active for 48 hrs. If Ep is active more than 6 hrs, a second population of erythroid colonies emerges at a nearly constant rate until the end of the culture. Full erythroid colony production requires prolonged exposure to erythropoietin. (2) The longer erythropoietin is actively present, the larger the number of erythroid colonies that reach 17 cells or more. Two distinct populations of immediate erythroid precursors are also present in marrow from plethoric mice. In these mice, total colony numbers are equal to or below those obtained from normal mice. However, the population of fast-responding CFU-E is consistently decreased to 10 to 20% of that found in normal marrow. The remaining colonies are formed from plethoric marrow at a rate equal to normal marrow. With increasing Ep exposures, the number of large colonies produced increases. From the marrow of bled mice, total erythroid colony production is equal to or above that of normal marrow. Two populations of colony-forming cells are again evident, with the fast-responding CFU-E being below normal levels. The lack of colonies from this group was compensated in bled mice by rapid colony production in the second population. A real increase in numbers of precursors present in this pool increased the rate of colony production in culture to twice that of normal marrow. The number of large colonies obtained from bled mice was again increased as the Ep exposure was lengthened. (ERB)

  4. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  5. The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development

    Science.gov (United States)

    Benz, Claudia; Martins, Vera C.; Radtke, Freddy; Bleul, Conrad C.

    2008-01-01

    T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development. PMID:18458114

  6. Cell adhesion signaling regulates RANK expression in osteoclast precursors.

    Directory of Open Access Journals (Sweden)

    Ayako Mochizuki

    Full Text Available Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK differentiate into osteoclasts following stimulation with the RANK ligand (RANKL. Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition. BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6 in BMMs induced their differentiation into osteoclasts even under the non

  7. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    International Nuclear Information System (INIS)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

    1991-01-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells

  8. [Research progress of intervertebral disc endogenous stem cells for intervertebral disc regeneration].

    Science.gov (United States)

    Liang, Hang; Deng, Xiangyu; Shao, Zengwu

    2017-10-01

    To summarize the research progress of intervertebral disc endogenous stem cells for intervertebral disc regeneration and deduce the therapeutic potential of endogenous repair for intervertebral disc degeneration. The original articles about intervertebral disc endogenous stem cells for intervertebral disc regeneration were extensively reviewed; the reparative potential in vivo and the extraction and identification in vitro of intervertebral disc endogenous stem cells were analyzed; the prospect of endogenous stem cells for intervertebral disc regeneration was predicted. Stem cell niche present in the intervertebral discs, from which stem cells migrate to injured tissues and contribute to tissues regeneration under certain specific microenvironment. Moreover, the migration of stem cells is regulated by chemokines system. Tissue specific progenitor cells have been identified and successfully extracted and isolated. The findings provide the basis for biological therapy of intervertebral disc endogenous stem cells. Intervertebral disc endogenous stem cells play a crucial role in intervertebral disc regeneration. Therapeutic strategy of intervertebral disc endogenous stem cells is proven to be a promising biological approach for intervertebral disc regeneration.

  9. Thin film solar cells by selenization sulfurization using diethyl selenium as a selenium precursor

    Science.gov (United States)

    Dhere, Neelkanth G.; Kadam, Ankur A.

    2009-12-15

    A method of forming a CIGSS absorber layer includes the steps of providing a metal precursor, and selenizing the metal precursor using diethyl selenium to form a selenized metal precursor layer (CIGSS absorber layer). A high efficiency solar cell includes a CIGSS absorber layer formed by a process including selenizing a metal precursor using diethyl selenium to form the CIGSS absorber layer.

  10. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-05-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

  11. Colorimetric detection of endogenous hydrogen sulfide production in living cells

    Science.gov (United States)

    Ahn, Yong Jin; Lee, Young Ju; Lee, Jaemyeon; Lee, Doyeon; Park, Hun-Kuk; Lee, Gi-Ja

    2017-04-01

    Hydrogen sulfide (H2S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H2S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H2S concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of H2S has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective H2S detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO3 concentration, as well as the pH of the Na2S (H2S donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na2S standard solution both at room temperature and at 37 °C in a 5% CO2 incubator. As a result, the sensitivity of the colorimetric assay for H2S at 37 °C in the incubator (0.0056 Abs./μM Na2S, R2 = 0.9948) was similar to that at room temperature (0.0055 Abs./μM Na2S, R2 = 0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, L-cysteine (Cys), and dithiothreitol than to the H2S from Na2S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H2S concentrations in live C6 glioma cells by s-(5‧-adenosyl)-L-methionine stimulation with and without Cys and L-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver

  12. Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system.

    Directory of Open Access Journals (Sweden)

    Yuen Fei Wong

    2011-02-01

    Full Text Available Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle's loop and distal tubules.Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the

  13. Neurogenic and non neurogenic functions of endogenous neural stem cells.

    Directory of Open Access Journals (Sweden)

    Erica eButti

    2014-04-01

    Full Text Available Adult neurogenesis is a lifelong process that occurs in two main neurogenic niches of the brain, namely in the subventricular zone (SVZ of the lateral ventricles and in the subgranular zone (SGZ of the dentate gyrus (DG in the hippocampus. In the 1960s, studies on adult neurogenesis have been hampered by the lack of established phenotypic markers. The precise tracing of neural stem/progenitor cells (NPCs was therefore, not properly feasible. After the (partial identification of those markers, it was the lack of specific tools that hindered a proper experimental elimination and tracing of those cells to demonstrate their terminal fate and commitment. Nowadays, irradia-tion, cytotoxic drugs as well as genetic tracing/ablation procedures have moved the field forward and increased our understanding of neurogenesis processes in both physiological and pathological conditions. Newly formed NPC progeny from the SVZ can replace granule cells in the olfactory bulbs of rodents, thus contributing to orchestrate sophisticated odour behaviour. SGZ-derived new granule cells, instead, integrate within the DG where they play an essential role in memory functions. Furthermore, converging evidence claim that endogenous NPCs not only exert neurogenic functions, but might also have non-neurogenic homeostatic functions by the release of different types of neuroprotective molecules. Remarkably, these non-neurogenic homeostatic functions seem to be necessary, both in healthy and diseased conditions, for example for preventing or limiting tissue damage. In this review, we will discuss the neurogenic and the non-neurogenic functions of adult NPCs both in physiological and pathological conditions.

  14. Content of endogenous thiols and radioresistance of gemmating cells of Saccharomyces ellipsoideus and Saccharomyces cerevisiale yeasts

    International Nuclear Information System (INIS)

    Simonyan, N.V.; Avakyan, Ts.M.; Dzhanpoladyan, N.L.; Stepanyan, L.G.

    1983-01-01

    It has been shown that gemmating cells of ''wild type'' yeasts are more radioresistant and contain more endogenous thiols, than resting cells. Gemmating cells of Saccharomyces cerevisial yeasts, carrying the mutation rad 51, as to radioresistance and content of SH groups do not differ from resting cells. The results obtained testify to a connec-- tion between increased radioresistance of the yeast gemmating cells and increased content of endogenous thiols in them

  15. Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

    Science.gov (United States)

    Pavlath, G K; Thaloor, D; Rando, T A; Cheong, M; English, A W; Zheng, B

    1998-08-01

    Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

  16. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...... proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...

  17. Circulating osteogentic precursor cells in non-hereditary heterotopic ossification.

    Science.gov (United States)

    Egan, Kevin P; Duque, Gustavo; Keenan, Mary Ann; Pignolo, Robert J

    2018-04-01

    Non-hereditary heterotopic ossification (NHHO) may occur after musculoskeletal trauma, central nervous system (CNS) injury, or surgery. We previously described circulating osteogenic precursor (COP) cells as a bone marrow-derived type 1 collagen + CD45 + subpopulation of mononuclear adherent cells that are able of producing extraskeletal ossification in a murine in vivo implantation assay. In the current study, we performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All bone specimens revealed the presence of COP cells at 2-14 cells per high power field. COP cells were localized to early fibroproliferative and neovascular lesions of NHHO with evidence for their circulatory status supported by their presence near blood vessels in examined lesions. This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Endogenous stem cell proliferation induced by intravenous hedgehog agonist administration after contusion in the adult rat spinal cord.

    Science.gov (United States)

    Bambakidis, Nicholas C; Horn, Eric M; Nakaji, Peter; Theodore, Nicholas; Bless, Elizabeth; Dellovade, Tammy; Ma, Chiyuan; Wang, Xukui; Preul, Mark C; Coons, Stephen W; Spetzler, Robert F; Sonntag, Volker K H

    2009-02-01

    Sonic hedgehog (Shh) is a glycoprotein molecule that upregulates the transcription factor Gli1. The Shh protein plays a critical role in the proliferation of endogenous neural precursor cells when directly injected into the spinal cord after a spinal cord injury in adult rodents. Small-molecule agonists of the hedgehog (Hh) pathway were used in an attempt to reproduce these findings through intravenous administration. The expression of Gli1 was measured in rat spinal cord after the intravenous administration of an Hh agonist. Ten adult rats received a moderate contusion and were treated with either an Hh agonist (10 mg/kg, intravenously) or vehicle (5 rodents per group) 1 hour and 4 days after injury. The rats were killed 5 days postinjury. Tissue samples were immediately placed in fixative. Samples were immunohistochemically stained for neural precursor cells, and these cells were counted. Systemic dosing with an Hh agonist significantly upregulated Gli1 expression in the spinal cord (p < 0.005). After spinal contusion, animals treated with the Hh agonist had significantly more nestin-positive neural precursor cells around the rim of the lesion cavity than in vehicle-treated controls (means +/- SDs, 46.9 +/- 12.9 vs 20.9 +/- 8.3 cells/hpf, respectively, p < 0.005). There was no significant difference in the area of white matter injury between the groups. An intravenous Hh agonist at doses that upregulate spinal cord Gli1 transcription also increases the population of neural precursor cells after spinal cord injury in adult rats. These data support previous findings based on injections of Shh protein directly into the spinal cord.

  19. [The role of endothelial cells and endothelial precursor cells in angiogenesis].

    Science.gov (United States)

    Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

    2006-01-01

    Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

  20. Two separate defects affecting true naive or virtual memory T cell precursors combine to reduce naive T cell responses with aging.

    Science.gov (United States)

    Renkema, Kristin R; Li, Gang; Wu, Angela; Smithey, Megan J; Nikolich-Žugich, Janko

    2014-01-01

    Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.

  1. Brain Region-Dependent Rejection of Neural Precursor Cell Transplants

    Directory of Open Access Journals (Sweden)

    Nina Fainstein

    2018-04-01

    Full Text Available The concept of CNS as an immune-privileged site has been challenged by the occurrence of immune surveillance and allogeneic graft rejection in the brain. Here we examined whether the immune response to allogeneic neural grafts is determined by the site of implantation in the CNS. Dramatic regional differences were observed between immune responses to allogeneic neural precursor/stem cell (NPC grafts in the striatum vs. the hippocampus. Striatal grafts were heavily infiltrated with IBA-1+ microglia/macrophages and CD3+ T cells and completely rejected. In contrast, hippocampal grafts exhibited milder IBA-1+ cell infiltration, were not penetrated efficiently by CD3+ cells, and survived efficiently for at least 2 months. To evaluate whether the hippocampal protective effect is universal, astrocytes were then transplanted. Allogeneic astrocyte grafts elicited a vigorous rejection process from the hippocampus. CD200, a major immune-inhibitory signal, plays an important role in protecting grafts from rejection. Indeed, CD200 knock out NPC grafts were rejected more efficiently than wild type NPCs from the striatum. However, lack of CD200 expression did not elicit NPC graft rejection from the hippocampus. In conclusion, the hippocampus has partial immune-privilege properties that are restricted to NPCs and are CD200-independent. The unique hippocampal milieu may be protective for allogeneic NPC grafts, through host-graft interactions enabling sustained immune-regulatory properties of transplanted NPCs. These findings have implications for providing adequate immunosuppression in clinical translation of cell therapy.

  2. A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination

    Science.gov (United States)

    Tran, Lee; Masters, Haley; Roust, Lori R; Katsanos, Christos S

    2015-01-01

    Enrichment from the easily accessible blood amino acid pool is commonly used as precursor enrichment to calculate rates of muscle protein fractional synthesis in relevant human studies in lieu of the less accessible muscle fluid amino acid pool. However, the accuracy of this approach depends largely on the extent to which there is low discrepancy in free amino acid enrichment between blood and muscle. Steady-state gradient (i.e., ratio) of amino acid enrichment between blood and muscle fluid in the basal state and in response to amino acid infusion were determined in five healthy subjects, and in association with two separate tracers: d9-leucine, introduced endogenously by the metabolism of d10-leucine (i.e., l-[2,3,3,4,5,5,5,6,6,6-2H10]leucine) infused in blood, and 13C6-phenylalanine introduced/infused in blood. The blood-to-muscle fluid amino acid enrichment ratio was lower (P enrichment introduced endogenously by intravenous infusion of d10-leucine provides a closer estimate of the muscle fluid amino acid enrichment, and its associated changes, than blood phenylalanine enrichment to calculate rates of muscle protein synthesis in humans. PMID:26243214

  3. GBM secretome induces transient transformation of human neural precursor cells.

    Science.gov (United States)

    Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

    2012-09-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

  4. Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem cell function.

    Science.gov (United States)

    Garaycoechea, Juan I; Crossan, Gerry P; Langevin, Frederic; Daly, Maria; Arends, Mark J; Patel, Ketan J

    2012-09-27

    Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age, the functional quality of HSCs declines, partly owing to the accumulation of damaged DNA. However, the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects, a predisposition to leukaemia, and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia, with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly, we find that only HSPCs, and not more mature blood precursors, require Aldh2 for protection against acetaldehyde toxicity. Additionally, the aldehyde-oxidizing activity of HSPCs, as measured by Aldefluor stain, is due to Aldh2 and correlates with this protection. Finally, there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore, the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs, and define the protective mechanisms that counteract this threat.

  5. Precursors of executive function in infants with sickle cell anemia.

    Science.gov (United States)

    Hogan, Alexandra M; Telfer, Paul T; Kirkham, Fenella J; de Haan, Michelle

    2013-10-01

    Executive dysfunction occurs in sickle cell anemia, but there are few early data. Infants with sickle cell anemia (n = 14) and controls (n = 14) performed the "A-not-B" and Object Retrieval search tasks, measuring precursors of executive function at 9 and 12 months. Significant group differences were not found. However, for the A-not-B task, 7 of 11 sickle cell anemia infants scored in the lower 2 performance categories at 9 months, but only 1 at 12 months (P = .024); controls obtained scores at 12 months that were statistically comparable to the scores they had already obtained at 9 months. On the Object Retrieval task, 9- and 12-month controls showed comparable scores, whereas infants with sickle cell anemia continued to improve (P = .027); at 9 months, those with lower hemoglobin oxygen saturation passed fewer trials (R s = 0.670, P = .024) and took longer to obtain the toy (R s = -0.664, P = .013). Subtle delays in acquiring developmental skills may underlie abnormal executive function in childhood.

  6. Aqueous-Containing Precursor Solutions for Efficient Perovskite Solar Cells.

    Science.gov (United States)

    Liu, Dianyi; Traverse, Christopher J; Chen, Pei; Elinski, Mark; Yang, Chenchen; Wang, Lili; Young, Margaret; Lunt, Richard R

    2018-01-01

    Perovskite semiconductors have emerged as competitive candidates for photovoltaic applications due to their exceptional optoelectronic properties. However, the impact of moisture instability on perovskite films is still a key challenge for perovskite devices. While substantial effort is focused on preventing moisture interaction during the fabrication process, it is demonstrated that low moisture sensitivity, enhanced crystallization, and high performance can actually be achieved by exposure to high water content (up to 25 vol%) during fabrication with an aqueous-containing perovskite precursor. The perovskite solar cells fabricated by this aqueous method show good reproducibility of high efficiency with average power conversion efficiency (PCE) of 18.7% and champion PCE of 20.1% under solar simulation. This study shows that water-perovskite interactions do not necessarily negatively impact the perovskite film preparation process even at the highest efficiencies and that exposure to high contents of water can actually enable humidity tolerance during fabrication in air.

  7. Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells.

    Science.gov (United States)

    Maldonado, Paloma P; Angulo, María Cecilia

    2015-06-01

    The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron-OPC synaptic activity is a signaling pathway controlling OPC proliferation/differentiation, influencing the myelination process. However, new evidences describing non-synaptic mechanisms of communication between neurons and OPCs have revealed that neuron-OPC interactions are more complex than expected. The activation of extrasynaptic receptors by ambient neurotransmitter or local spillover and the ability of OPCs to sense neuronal activity through a potassium channel suggest that distinct modes of communication mediate different functions of OPCs in the CNS. This review discusses different mechanisms used by OPCs to interact with neurons and their potential roles during postnatal development and in brain disorders. © The Author(s) 2014.

  8. Bone Morphogenetic Protein Signaling and Olig1/2 Interact to Regulate the Differentiation and Maturation of Adult Oligodendrocyte Precursor Cells

    OpenAIRE

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R.; Cao, Qilin

    2007-01-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understandin...

  9. Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

    OpenAIRE

    Oken, M M; Peterson, P K; Wilkinson, B J

    1981-01-01

    To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocy...

  10. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    Science.gov (United States)

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  11. Packaging of human endogenous retrovirus sequences is undetectable in porcine endogenous retrovirus particles produced from human cells

    International Nuclear Information System (INIS)

    Suling, Kristen; Quinn, Gary; Wood, James; Patience, Clive

    2003-01-01

    The chronic shortage of human donor organs and tissues for allotransplantation could be relieved if clinical xenotransplantation were to become a viable clinical therapy. Balanced against the benefits of xenotransplantation are the possible consequences of zoonotic infections, and in particular, infection by porcine endogenous retrovirus (PERV). An often-proclaimed risk of PERV infection is the possible recombination of PERV with human endogenous retroviruses (HERV) . To address this issue, we examined the potential for HERV sequences to be cross-packaged into PERV particles produced from infected human 293 cells. Although HERV-K, W, E, R, and ERV-9 RNA transcripts are expressed in 293 cells, we did not detect cross-packaging of any of these HERV groups. Quantitative analysis indicated that less than approximately 1 in 10 4 -10 7 PERV particles might contain HERV sequences. In comparison, we found that murine leukemia virus (MLV)-based vector transcripts were cross-packaged at a rate of approximately one copy in 10 4 PERV particles. Our results indicate that the potential for recombination of PERV and HERV sequences is low and that novel viruses generated by this mechanism are unlikely to represent a significant risk for xenotransplantation

  12. Are Haemopoietic Stem Cells Precursor Cells in Secondary Disease?

    Energy Technology Data Exchange (ETDEWEB)

    Chertkov, J. L. [Central Institute of Haematology and Blood Transfusion, Moscow, USSR (Russian Federation)

    1969-07-15

    The paper gives data on acute secondary disease developing in supra-lethally irradiated dogs and monkeys after transplantation of allogenic bone marrow. On the basis of the experimental data obtained, the author discusses the question whether haemopoietic stem cells play a role as first links in the histogenesis of the lymphoid elements responsible for acute secondary disease. (author)

  13. Endogenous reverse transcriptase (RT) activity and Chromatin remodeling in normal and transformed cells and early embryos

    International Nuclear Information System (INIS)

    Spadafora, C.; Sciamanna, I.; Misteli, T.

    2009-01-01

    Endogenous Reverse Transcriptase (RT) is an enzyme encoded by two classes of genomic retro-elements: retro-transposons and endogenous retroviruses. Basal levels of RT are expressed in all non pathological, differentiated tissues while high RT expression levels characterize tumorigenic cells, germ cells and embryonic tissues. Preliminary studies carried out in our laboratory have shown that RT inhibition using pharmacological inhibitors (nevirapine and efavirenz, two drugs currently used in AIDS therapy) drastically reduces cell proliferation, promotes differentiation of tumorigenic cells in vitro, induces a reprogrammed gene expression and antagonizes tumor progression in nude mice inoculated with tumorigenic human cell lines, including melanoma, prostate and colon carcinoma and microcitoma

  14. Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

    Science.gov (United States)

    Bodel, P; Ralph, P; Wenc, K; Long, J C

    1980-02-01

    Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

  15. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

    Science.gov (United States)

    Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

    2012-03-01

    Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

  16. ENDOGENOUS PYROGEN RELEASE FROM RABBIT BLOOD CELLS INCUBATED IN VITRO WITH PARAINFLUENZA VIRUS.

    Science.gov (United States)

    ATKINS, E; CRONIN, M; ISACSON, P

    1964-12-11

    Rabbit blood cells incubated in vitro with purified parainfluenza-5 virus (DA strain) released a rapidly acting pyrogen. Spleen and lymph node cells were inactive. The pyrogen resembled in behavior a pyrogen extracted from granulocytic exudates. Similar cells in the blood are believed to be activated by virus in vivo to produce the circulating endogenous pyrogen that mediates virus-induced fever.

  17. Exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions.

    Science.gov (United States)

    Montenegro, Mary Lourdes; Ferriani, Rui Alberto; Basse, Per H

    2015-08-29

    Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35%-50%, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions. As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2). Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis. For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track

  18. CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells*

    Science.gov (United States)

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-01-01

    NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

  19. Dynamic equilibrium of endogenous selenium nanoparticles in selenite-exposed cancer cells: a deep insight into the interaction between endogenous SeNPs and proteins.

    Science.gov (United States)

    Bao, Peng; Chen, Song-Can; Xiao, Ke-Qing

    2015-12-01

    Elemental selenium (Se) was recently found to exist as endogenous nanoparticles (i.e., SeNPs) in selenite-exposed cancer cells. By sequestrating critical intracellular proteins, SeNPs appear capable of giving rise to multiple cytotoxicity mechanisms including inhibition of glycolysis, glycolysis-dependent mitochondrial dysfunction, microtubule depolymerization and inhibition of autophagy. In this work, we reveal a dynamic equilibrium of endogenous SeNP assembly and disassembly in selenite-exposed H157 cells. Endogenous SeNPs are observed both in the cytoplasm and in organelles. There is an increase in endogenous SeNPs between 24 h and 36 h, and a decrease between 36 h and 72 h according to transmission electron microscopy results and UV-Vis measurements. These observations imply that elemental Se in SeNPs could be oxidized back into selenite by scavenging superoxide radicals and ultimately re-reduced into selenide; then the assembly and disassembly of SeNPs proceed simultaneously with the sequestration and release of SeNP high-affinity proteins. There is also a possibility that the reduction of elemental Se to selenide pathway may lie in selenite-exposed cancer cells, which results in the assembly and disassembly of endogenous SeNPs. Genome-wide expression analysis results show that endogenous SeNPs significantly altered the expression of 504 genes, compared to the control. The endogenous SeNPs induced mitochondrial impairment and decreasing of the annexin A2 level can lead to inhibition of cancer cell invasion and migration. This dynamic flux of endogenous SeNPs amplifies their cytotoxic potential in cancer cells, thus provide a starting point to design more efficient intracellular self-assembling systems for overcoming multidrug resistance.

  20. Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Marianne; Rasmussen, Mikkel Aabech

    2012-01-01

    Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression...... pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species....... transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1¿µg/mL doxycycline or 4¿µg/mL doxycycline. Our results revealed that endogenous...

  1. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  2. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    International Nuclear Information System (INIS)

    Liu, Te; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe

    2012-01-01

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: ► microRNA-145 inhibits Sox2 expression in human iPS cells. ► microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. ► HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. ► HuAECs feeder layers maintain human iPS cells pluripotency. ► HuAECs negatively regulates the synthesis of

  3. Developmental origin and lineage plasticity of endogenous cardiac stem cells

    Science.gov (United States)

    Santini, Maria Paola; Forte, Elvira; Harvey, Richard P.; Kovacic, Jason C.

    2016-01-01

    Over the past two decades, several populations of cardiac stem cells have been described in the adult mammalian heart. For the most part, however, their lineage origins and in vivo functions remain largely unexplored. This Review summarizes what is known about different populations of embryonic and adult cardiac stem cells, including KIT+, PDGFRα+, ISL1+ and SCA1+ cells, side population cells, cardiospheres and epicardial cells. We discuss their developmental origins and defining characteristics, and consider their possible contribution to heart organogenesis and regeneration. We also summarize the origin and plasticity of cardiac fibroblasts and circulating endothelial progenitor cells, and consider what role these cells have in contributing to cardiac repair. PMID:27095490

  4. Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: Compared kinetics of endogenous and intrathymically transferred progenitors

    International Nuclear Information System (INIS)

    Penit, C.; Ezine, S.

    1989-01-01

    After sublethal (6 Gy) whole-body irradiation, the C57BL/Ba (Thy-1.1) murine thymus regenerated in two waves, on days 3-10 and 25-32, separated by a severe relapse. The second phase of depletion-reconstitution reproduced the first one, in a less synchronous manner. The depletion affected all cell subsets, but CD4+ CD8- cells decreased later than immature cells. Cell proliferation, measured by BrdUrd incorporation, started on day 3 after irradiation and concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sequentially. CD4+ CD8- cells never represented a significant percentage of cycling cells. When irradiation was immediately followed by an intrathymic injection of 10(5) C57BL/Ka (Thy-1.2) bone marrow cells, the relapse in thymus reconstitution was no longer observed. Detected with anti-Thy-1.2 antibodies, donor cells started cycling on day 14 and showed only one wave of proliferation. In these chimeras, recipient thymocytes behave exactly like thymocytes of solely irradiated mice. Intrathymically transferred CD4- CD8- thymocytes 10(5) showed the same proliferation kinetics as endogenous cells, with a peak in number on day 10 but completely disappeared from the thymus on days 14-21. These data reflect maturational differences between intrathymic and bone marrow precursor cells and suggest different radiosensitivities not linked to proliferative status. The resting state of the thymus immigrants was shown by the absence of Thy-1 acquisition by bone marrow cells continuously labeled for 10 days with BrdUrd in vivo before intrathymic transfer. When such labeled bone marrow cells were injected in the thymus, only the minor BrdUrd- subset gave rise to Thy-1+ cells

  5. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination

    Science.gov (United States)

    Sahel, Aurélia; Ortiz, Fernando C.; Kerninon, Christophe; Maldonado, Paloma P.; Angulo, María Cecilia; Nait-Oumesmar, Brahim

    2015-01-01

    Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders. PMID:25852473

  7. Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice

    International Nuclear Information System (INIS)

    Kitamura, Y.; Yokoyama, M.; Sonoda, T.; Mori, K.J.

    1983-01-01

    Although tissue mast cells are derived from the bone marrow, some descendants of bone marrow-derived precursors retain the ability to proliferate and differentiate into mast cells even after localization in the skin. The purpose of the present study was to determine the D 0 values for mast-cell precursors in the bone marrow and those localized in the skin. Bone marrow cells were removed from (WB X C57BL/6)F 1 +/+ mice after various doses of irradiation and injected into the skin of the congenic W/W/sup v/ mice which were genetically without mast cells. Radiosensitivity of mast-cell precursors in the bone marrow was evaluated by determining the proportion of the injection sites at which mast cells did not appear. For the assay of the radiosensitivity of mast-cell precursors localized in the skin, pieces of skin were removed from beige C57BL/6 (bg/sup J//bg/sup J/, Chediak-Higashi syndrome) mice after various doses of irradiation and grafted onto the backs of the normal C57BL/6 mice. Radiosensitivity of mast-cell precursors in the skin was evaluated by determining the decrease of beige-type mast cells which possessed giant granules. Mast-cell precursors in the bone marrow were much more radiosenitive than those localized in the skin. D 0 value was about 100 rad for the former and about 800 rad for the latter

  8. Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice

    International Nuclear Information System (INIS)

    Kitamura, Y.; Yokoyama, M.; Sonoda, T.; Mori, K.J.

    1983-01-01

    Although tissue mast cells are derived from the bone marrow, some descendants of bone marrow-derived precursors retain the ability to proliferate and differentiate into mast cells even after localization in the skin. The purpose of the present study was to determine the D0 values for mast-cell precursors in the bone marrow and those localized in the skin. Bone marrow cells were removed from (WB X C57BL/6)F1-+/+ mice after various doses of irradiation and injected into the skin of the congenic W/Wv mice which were genetically without mast cells. Radiosensitivity of mast-cell precursors in the bone marrow was evaluated by determining the proportion of the injection sites at which mast cells did not appear. For the assay of the radiosensitivity of mast-cell precursors localized in the skin, pieces of skin were removed from beige C57BL/6 (bgJ/bgJ. Chediak-Higashi syndrome) mice after various doses of irradiation and grafted onto the back of the normal C57BL/6 mice. Radiosensitivity of mast-cell precursors in the skin was evaluated by determining the decrease of beige-type mast cells which possessed giant granules. Mast-cell precursors in the bone marrow were much more radiosensitive than those localized in the skin. D0 value was about 100 rad for the former and about 800 rad for the latter

  9. Endogenous Ion Dynamics in Cell Motility and Tissue Regeneration

    International Nuclear Information System (INIS)

    Özkucur, N; Perike, S; Epperlein, H H; Funk, R H W

    2011-01-01

    Directional cell migration is an essential process, including regeneration of tissues, wound healing, and embryonic development. Cells achieve persistent directional migration by polarizing the spatiotemporal components involved in the morphological polarity. Ion transporter proteins situated at the cell membrane generates small electric fields that can induce directional cell motility. Besides them, externally applied direct current electric fields induce similar kind of responses as cell orientation and directional migration. However, the bioelectric mechanisms that lead to cellular directedness are poorly understood. Therefore, understanding the bioelectric signaling cues can serve as a powerful modality in controlling the cell behaviour, which can contribute additional insights for development and regeneration.

  10. Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

    Science.gov (United States)

    Kurzer, Jason H; Weinberg, Olga K

    2018-05-25

    Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  11. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow.

    Science.gov (United States)

    Jung, Younghun; Decker, Ann M; Wang, Jingcheng; Lee, Eunsohl; Kana, Lulia A; Yumoto, Kenji; Cackowski, Frank C; Rhee, James; Carmeliet, Peter; Buttitta, Laura; Morgan, Todd M; Taichman, Russell S

    2016-05-03

    GAS6 and its receptors (Tryo 3, Axl, Mer or "TAM") are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment.We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

  12. GH mediates exercise-dependent activation of SVZ neural precursor cells in aged mice.

    Directory of Open Access Journals (Sweden)

    Daniel G Blackmore

    Full Text Available Here we demonstrate, both in vivo and in vitro, that growth hormone (GH mediates precursor cell activation in the subventricular zone (SVZ of the aged (12-month-old brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation.

  13. GH Mediates Exercise-Dependent Activation of SVZ Neural Precursor Cells in Aged Mice

    Science.gov (United States)

    Blackmore, Daniel G.; Vukovic, Jana; Waters, Michael J.; Bartlett, Perry F.

    2012-01-01

    Here we demonstrate, both in vivo and in vitro, that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. PMID:23209615

  14. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  15. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  16. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  17. Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency

    Science.gov (United States)

    Squirrell, Jayne M.; Fong, Jimmy J.; Ariza, Carlos A.; Mael, Amber; Meyer, Kassondra; Shevde, Nirupama K.; Roopra, Avtar; Lyons, Gary E.; Kamp, Timothy J.; Eliceiri, Kevin W.; Ogle, Brenda M.

    2012-01-01

    The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications. PMID:22952742

  18. Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

    Directory of Open Access Journals (Sweden)

    Pastor Maria

    2010-06-01

    Full Text Available Abstract Background Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. Results Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. Conclusions In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

  19. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    Directory of Open Access Journals (Sweden)

    Harish Babu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  20. Endogenous electric fields as guiding cue for cell migration

    Science.gov (United States)

    Funk, Richard H. W.

    2015-01-01

    This review covers two topics: (1) “membrane potential of low magnitude and related electric fields (bioelectricity)” and (2) “cell migration under the guiding cue of electric fields (EF).”Membrane potentials for this “bioelectricity” arise from the segregation of charges by special molecular machines (pumps, transporters, ion channels) situated within the plasma membrane of each cell type (including eukaryotic non-neural animal cells). The arising patterns of ion gradients direct many cell- and molecular biological processes such as embryogenesis, wound healing, regeneration. Furthermore, EF are important as guiding cues for cell migration and are often overriding chemical or topographic cues. In osteoblasts, for instance, the directional information of EF is captured by charged transporters on the cell membrane and transferred into signaling mechanisms that modulate the cytoskeleton and motor proteins. This results in a persistent directional migration along an EF guiding cue. As an outlook, we discuss questions concerning the fluctuation of EF and the frequencies and mapping of the “electric” interior of the cell. Another exciting topic for further research is the modeling of field concepts for such distant, non-chemical cellular interactions. PMID:26029113

  1. Recruiting endogenous stem cells: a novel therapeutic approach for erectile dysfunction

    Directory of Open Access Journals (Sweden)

    Zhong-Cheng Xin

    2016-01-01

    Full Text Available Transplanted stem cells (SCs, owing to their regenerative capacity, represent one of the most promising methods to restore erectile dysfunction (ED. However, insufficient source, invasive procedures, ethical and regulatory issues hamper their use in clinical applications. The endogenous SCs/progenitor cells resident in organ and tissues play critical roles for organogenesis during development and for tissue homeostasis in adulthood. Even without any therapeutic intervention, human body has a robust self-healing capability to repair the damaged tissues or organs. Therefore, SCs-for-ED therapy should not be limited to a supply-side approach. The resident endogenous SCs existing in patients could also be a potential target for ED therapy. The aim of this review was to summarize contemporary evidence regarding: (1 SC niche and SC biological features in vitro; (2 localization and mobilization of endogenous SCs; (3 existing evidence of penile endogenous SCs and their possible mode of mobilization. We performed a search on PubMed for articles related to these aspects in a wide range of basic studies. Together, numerous evidences hold the promise that endogenous SCs would be a novel therapeutic approach for the therapy of ED.

  2. Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells.

    Science.gov (United States)

    Kishimoto, Seishi; Muramatsu, Mayumi; Gokoh, Maiko; Oka, Saori; Waku, Keizo; Sugiura, Takayuki

    2005-02-01

    2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

  3. Effect of caffeine on induction of endogenous type C virus in mouse cells in vitro

    International Nuclear Information System (INIS)

    Niwa, O.; Sugahara, T.

    1981-01-01

    The effect of caffeine on the expression of murine endogenous virus in mouse cells induced by radiation and chemicals was studied. Postirradiation treatment of K-BALB cells with caffeine enhanced cell killing as well as the induction of xenotropic virus after ultraviolet light irradiation. The degree of enhancement for the virus induction was comparable to that for cell killing. On the other hand, colony-forming ability and the expression of xenotropic virus of K-BALB cells after X-irradiation were unaffected by caffeine. These data suggest a linear relationship between the degree of endogenous virus expression and the amount of lethal damages after irradiation. For induction by halogenated pyrimidines, a 24-hr incubation of AKR2B cells with caffeine after 5-iodo-2'-deoxyuridine treatment resulted in marked suppression of the expression of ecotropic virus. On the contrary, in K-BALB cells, caffeine exerted only a small effect on 5-iodo-2'-deoxyuridine-induced expression of ecotropic and xenotropic viruses. These results indicate that, although using the same inducing agent, the pathway of endogenous virus induction may be different for AKR2B cells and for K-BALB cells

  4. Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: involvement of endogenous neural stem cells in prion diseases.

    Directory of Open Access Journals (Sweden)

    Aroa Relaño-Ginès

    Full Text Available Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.

  5. In Vivo Clotting Breast Cancer Stem Cells and Platelets: A New Endogenous Precursor of Metastasis Progression

    Science.gov (United States)

    2013-05-01

    In this scenario, PA (or PT) signals can be generated using intrinsic chromophores and pigments such as hemoglobin, mela - nin, cytochromes, or...medical inter- vention may provoke metastasis; however, no direct evidences were previously presented. Using label-free PAFC and the mela - noma-bearing

  6. Depletion of endogenous germ cells in tree shrews in preparation for spermatogonial transplantation.

    Science.gov (United States)

    Liu, Tingting; Guo, Ying; Yan, Lanzhen; Sun, Bin; Zheng, Ping; Zhao, Xudong

    2017-09-01

    To achieve successful spermatogonial transplantation, endogenous germ cells must be depleted in recipient animals to allow donor germ cells to colonize efficiently. Busulfan is commonly used for the depletion of endogenous germ cells in recipient males. However, the optimal dose of busulfan is species-specific, and the optimal dose in tree shrews is yet to be determined. The current study aimed to determine the optimal dose of busulfan for effective suppression of endogenous spermatogenesis in tree shrews. Different doses (15, 20, 25, 30, 35, 40 and 45 mg/kg) of busulfan were injected into tree shrews intraperitoneally. Survival rates of the different treatment groups were calculated at 2 weeks and body weights were measured at 4, 6, 8, 10 and 28 weeks post-busulfan treatment. The testes were also removed and weighed at 4, 6, 8, 10 and 28 weeks post-treatment, and the cross and longitude diameters of the testes and diameters of the seminiferous tubules were measured and histologically evaluated. It was observed that there were no significant differences in the survival rates between the 15-35 mg/kg treatment groups and the control group (P>0.05), while the survival rate of the 40 mg/kg treatment group significantly decreased relative to the control group (Pendogenous germ cells in tree shrews. This dose led to maximum suppression of endogenous spermatogenesis while maintaining an acceptable survival rate of >50% of the lethal dose of busulfan for tree shrews.

  7. Endogenous Jaagsiekte Sheep Retrovirus RNA is expressed by different cell types in ovine foetus and placenta

    Directory of Open Access Journals (Sweden)

    E Sanna

    2010-01-01

    Full Text Available The endogenous retroviruses are inherited elements transmitted trough the germline of most animal species and their biological role is still controversial. Ovine Pulmonary Carcinoma (OPC represents a good model for studying the interactions of endogenous retroviruses with their exogenous counterparts. The type D exogenous retrovirus known as Jaagsiekte Sheep Retro-Virus (JSRV is necessary and sufficient to cause OPC in domestic and wild sheep, but both affected and unaffected animals host in their genome 15 to 20 copies of related endogenous retroviruses named endogenous JSRV (enJSRV. In this study we evaluated the expression of enJSRV gag sequences in ovine foetal and placental tissues. RNA in situ hybridisation was performed on tissue sections of thymi, lymph nodes and lungs from ovine foetuses and related placentas, taken at a late stage of development. Reverse transcriptase- in situ polymerase chain reactions were also carried out on placental samples to better define the involved cells. In foetal tissues, specific signals were observed in the thymus medulla, lymph nodes and, at a lesser extent, in foetal bronchiolar cells. In the placental tissues, positive areas were detected in various cell types in the sincythium-and cyto-trophoblast. These data demonstrate that en JSRV RNA is largely expressed in a broad spectrum of cells including tissues which are critical for the development of the immune system.

  8. The endogenous regenerative capacity of the damaged newborn brain: boosting neurogenesis with mesenchymal stem cell treatment.

    Science.gov (United States)

    Donega, Vanessa; van Velthoven, Cindy T J; Nijboer, Cora H; Kavelaars, Annemieke; Heijnen, Cobi J

    2013-05-01

    Neurogenesis continues throughout adulthood. The neurogenic capacity of the brain increases after injury by, e.g., hypoxia-ischemia. However, it is well known that in many cases brain damage does not resolve spontaneously, indicating that the endogenous regenerative capacity of the brain is insufficient. Neonatal encephalopathy leads to high mortality rates and long-term neurologic deficits in babies worldwide. Therefore, there is an urgent need to develop more efficient therapeutic strategies. The latest findings indicate that stem cells represent a novel therapeutic possibility to improve outcome in models of neonatal encephalopathy. Transplanted stem cells secrete factors that stimulate and maintain neurogenesis, thereby increasing cell proliferation, neuronal differentiation, and functional integration. Understanding the molecular and cellular mechanisms underlying neurogenesis after an insult is crucial for developing tools to enhance the neurogenic capacity of the brain. The aim of this review is to discuss the endogenous capacity of the neonatal brain to regenerate after a cerebral ischemic insult. We present an overview of the molecular and cellular mechanisms underlying endogenous regenerative processes during development as well as after a cerebral ischemic insult. Furthermore, we will consider the potential to use stem cell transplantation as a means to boost endogenous neurogenesis and restore brain function.

  9. Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

    Science.gov (United States)

    Oken, M M; Peterson, P K; Wilkinson, B J

    1981-01-01

    To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocytosis of S. aureus and peptidoglycan as compared with that of purified cell walls. Lysostaphin digestion of peptidoglycan markedly reduced its pyrogenicity. To test whether the chemical composition of the ingested particles is important, latex particles were tested as possible stimuli for monocyte endogenous pyrogen release. Although 40 to 68% of monocytes ingested latex particles during the first hour, there was no evidence of endogenous pyrogen activity in the supernatant even when supernatants equivalent to 5.2 X 10(6) monocytes were tested. This study demonstrates that the pyrogenic moiety of the S. aureus cell wall resides in the peptidoglycan component. Phagocytosis is not in itself a pyrogenic stimulus, but rather serves as an effective mechanism to bring about contact between the chemical stimulus and the monocyte.

  10. MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain

    DEFF Research Database (Denmark)

    Vreys, Ruth; Vande Velde, Greetje; Krylychkina, Olga

    2010-01-01

    The adult rodent brain contains neural progenitor cells (NPCs), generated in the subventricular zone (SVZ), which migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into neurons. The aim of this study was to visualize endogenous NPC migration...... by a longitudinal MRI study and validated with histology. Here, we visualized endogenous NPC migration in the mouse brain by in vivo MRI and demonstrated accumulation of MPIO-labeled NPCs in the OB over time with ex vivo MRI. Furthermore, we investigated the influence of in situ injection of MPIOs on adult...

  11. Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

    Directory of Open Access Journals (Sweden)

    Matthew P. Krause

    2014-07-01

    Full Text Available Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs, a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

  12. Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells.

    Science.gov (United States)

    Angelici, Bartolomeo; Mailand, Erik; Haefliger, Benjamin; Benenson, Yaakov

    2016-08-30

    One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

  14. Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

    Science.gov (United States)

    Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

    2008-09-01

    PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

  15. Potential of bursa-immigrated hematopoietic precursor cells to differentiate to functional B and T cells

    International Nuclear Information System (INIS)

    Weber, W.T.; Alexander, J.E.

    1978-01-01

    The potential of hematopoietic precursor cells, recently immigrated into the 13- and 14-day-old embryonic bursa, to migrate to the thymus and to differentiate to functional T cells was investigated. Chromosomally marked cell populations obtained from 13- and 14-day-old embryonic bursas were transferred i.v. to 780 R γ-irradiated chick embryos of equivalent age. When appropriate chimeras were examined at 4 to 12 weeks after cell transfer, donor cells were found to proliferate primarily in the bursa. Significant donor cell influx into the thymus was not detected. In correlation with these findings, Con A- and PHA-responsive T cells in thymus and spleen cell cultures of recipients remained of host origin whereas the number of anti-CIg responsive B cells of donor type increased gradually in the spleens of recipients. An initial lag period preceded the accumulation of functional donor B cells in the spleens of recipients, despite the predominant presence of dividing donor cells in the bursa. This suggests that the transferred bursal cell population required substantially longer to mature and emigrate from the bursa as functional B cells than the host cell population remaining in the irradiated bursas at time of cell transfer. The failure to detect significant influx of donor cells into the thymus and their failure to differentiate to functional T cells suggest that the recently bursa-immigrated hematopoietic stem cells of 13- and 14-day-old embryos may not be pluripotential cells, but rather cells already committed to the B cell line of differentiation

  16. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    International Nuclear Information System (INIS)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille; Rochel, Natacha; Mély, Yves; Didier, Pascal; Weiss, Etienne

    2013-01-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins

  17. Neural stem cells in the ischemic and injured brain: endogenous and transplanted.

    Science.gov (United States)

    Dong, Jing; Liu, Baohua; Song, Lei; Lu, Lei; Xu, Haitao; Gu, Yue

    2012-12-01

    Neural stem cells functions as the pool of new neurons in adult brain, and plays important roles in normal brain function. Additionally, this pool reacts to brain ischemia, hemorrhage, trauma and many kinds of diseases, serving as endogenous repair mechanisms. The present manuscript discussed the responses of adult neurogenesis to brain ischemia and other insults, then the potential of neural stem cell transplantation therapy to treat such brain injury conditions.

  18. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  19. Contribution of constitutively proliferating precursor cell subtypes to dentate neurogenesis after cortical infarcts

    Directory of Open Access Journals (Sweden)

    Oberland Julia

    2010-11-01

    Full Text Available Abstract Background It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion. Results Quantification of BrdU-expressing precursor cell populations revealed no alteration in number of radial glia-like type 1 cells but a sequential increase of later precursor cell subtypes in lesioned animals (type 2a cells at day 7, type 3 cells/immature neurons at day 14. These alterations result in an enhanced survival of mature neurons 4 weeks postinfarct. Conclusions Focal cortical infarcts recruit dentate precursor cells generated already before the infarct and significantly contribute to an enhanced neurogenesis. Our findings thereby increase our understanding of the complex cellular mechanisms of postlesional neurogenesis.

  20. Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

    Science.gov (United States)

    Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-01-01

    Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

  1. Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

    Science.gov (United States)

    Dumbali, Sandeep P; Mei, Lanju; Qian, Shizhi; Maruthamuthu, Venkat

    2017-10-01

    Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

  2. On the origins of the universal dynamics of endogenous granules in mammalian cells.

    Science.gov (United States)

    Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

    2009-12-01

    Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

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

    Science.gov (United States)

    2014-01-01

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

  4. C-reactive protein bearing cells are a subpopulation of natural killer cell precursors

    International Nuclear Information System (INIS)

    Baum, L.L.; Krueger, N.X.

    1986-01-01

    Cell surface C-reactive protein (S-CRP) is expressed on the surface membrane of a small percentage of lymphocytes. Anti-CRP inhibits natural killer (NK) function. Since NK effectors are heterogeneous, they suspected that the cells expressing S-CRP (CRP + ) might respond differently to stimulation than the NK effectors lacking S-CRP (CRP - ). Methods were developed to separate CRP + and CRP - lymphocytes and their functional responses were examined and compared. These techniques are dependent upon the binding of CRP to its ligands, C-polysaccharide (CPS) or Phosphocholine (PC). The first method involves rosette formation with CPS coupled autologous red blood cells; the second method utilizes the binding of CRP + lymphocytes to PC-sepharose. Lymphocytes separated using either of these techniques yield similar results. CRP - lymphocytes respond to 3 day incubation with PHA or Il-2 by producing effectors which kill 51 Cr labeled K562 tumor cells, CRP + precursors do not. CRP + lymphocytes respond to a 5 day incubation with inactivated K562 by producing effectors which kill K562; CRP - precursors do not. NK functional activity of both is increased by incubation with interferon. This ability to respond differently to stimulation suggests that CRP + and CRP - cells are functionally distinct

  5. Naturally death-resistant precursor cells revealed as the origin of retinoblastoma

    DEFF Research Database (Denmark)

    Trinh, Emmanuelle; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retinal...... precursors. The authors show that a subset of these precursors contain an inherent resistance to apoptosis, and that while most terminally differentiate, some are likely to acquire additional mutations, leading to tumor formation. Thus, this work defines the cell-of-origin of retinoblastoma and suggests...... that mutations giving increased proliferative capacity are required for retinoblastoma development....

  6. Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

    Science.gov (United States)

    Wertheimer, Tobias; Velardi, Enrico; Tsai, Jennifer; Cooper, Kirsten; Xiao, Shiyun; Kloss, Christopher C.; Ottmüller, Katja J.; Mokhtari, Zeinab; Brede, Christian; deRoos, Paul; Kinsella, Sinéad; Palikuqi, Brisa; Ginsberg, Michael; Young, Lauren F.; Kreines, Fabiana; Lieberman, Sophia R.; Lazrak, Amina; Guo, Peipei; Malard, Florent; Smith, Odette M.; Shono, Yusuke; Jenq, Robert R.; Hanash, Alan M.; Nolan, Daniel J.; Butler, Jason M.; Beilhack, Andreas; Manley, Nancy R.; Rafii, Shahin; Dudakov, Jarrod A; van den Brink, Marcel RM

    2018-01-01

    The thymus is extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood and this capacity diminishes considerably with age. Here we show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration, via their production of BMP4. ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signalling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration; and its downstream targets such as Dll4, itself a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity. PMID:29330161

  7. A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

    Science.gov (United States)

    Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

    2017-06-01

    As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

  8. CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

    Science.gov (United States)

    Hardman, Clare S; Chen, Yi-Ling; Salimi, Maryam; Jarrett, Rachael; Johnson, David; Järvinen, Valtteri J; Owens, Raymond J; Repapi, Emmanouela; Cousins, David J; Barlow, Jillian L; McKenzie, Andrew N J; Ogg, Graham

    2017-12-22

    Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis

    Science.gov (United States)

    Quintana, Francisco J.; Murugaiyan, Gopal; Farez, Mauricio F.; Mitsdoerffer, Meike; Tukpah, Ann-Marcia; Burns, Evan J.; Weiner, Howard L.

    2010-01-01

    The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3+ Treg, Tr1 cells, and IL-17–producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3+ Treg compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3+ Treg in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3+ Treg in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3+ Treg that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3+ Treg differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders. PMID:21068375

  10. A New Approach to Establish a Cell Line with Reduced Risk of Endogenous Retroviruses

    Science.gov (United States)

    Fukuma, Aiko; Yoshikawa, Rokusuke; Miyazawa, Takayuki; Yasuda, Jiro

    2013-01-01

    Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV. PMID:23585909

  11. A new approach to establish a cell line with reduced risk of endogenous retroviruses.

    Directory of Open Access Journals (Sweden)

    Aiko Fukuma

    Full Text Available Endogenous retroviruses (ERVs are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells. The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.

  12. A new approach to establish a cell line with reduced risk of endogenous retroviruses.

    Science.gov (United States)

    Fukuma, Aiko; Yoshikawa, Rokusuke; Miyazawa, Takayuki; Yasuda, Jiro

    2013-01-01

    Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.

  13. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

    Directory of Open Access Journals (Sweden)

    Bin-Bin Xie

    Full Text Available The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs. The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  14. Endogenous laminin is required for human airway smooth muscle cell maturation

    Directory of Open Access Journals (Sweden)

    Tran Thai

    2006-09-01

    Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

  15. Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

    Science.gov (United States)

    Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

    2017-06-06

    Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. IL-9-Producing Mast Cell Precursors and Food Allergy

    Science.gov (United States)

    2016-10-01

    that Stat6-/- BM progenitors in sensitized wild type recipients that were competent in GFP- CD4+ST2+TH2 and ILC2s ( innate lymphoid cells ) generation, and...report demonstrated that type 2 innate lymphoid cells (ILC2s) lack cell lineage markers and have the potential to pro- duce IL-9 (Wilhelm et al., 2011...Fujii, H., and Koyasu, S. (2010). Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells . Nature 463, 540–544

  17. T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

    DEFF Research Database (Denmark)

    Dunon, D; Kaufman, J; Salomonsen, J

    1990-01-01

    beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

  18. Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect.

    Science.gov (United States)

    Wang, Xingmin; Yang, Yonghong; Huycke, Mark M

    2015-03-01

    Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect. Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis. Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice. These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  19. Influence of the extracellular matrix on endogenous and transplanted stem cells after brain damage

    Directory of Open Access Journals (Sweden)

    Lars eRoll

    2014-08-01

    Full Text Available The limited regeneration capacity of the adult central nervous system requires strategies to improve recovery of patients. In this context, the interaction of endogenous as well as transplanted stem cells with their environment is crucial. An understanding of the molecular mechanisms could help to improve regeneration by targeted manipulation.In the course of reactive gliosis, astrocytes upregulate Glial fibrillary acidic protein (GFAP and start, in many cases, to proliferate. Beside GFAP, subpopulations of these astroglial cells coexpress neural progenitor markers like Nestin. Although cells express these markers, the proportion of cells that eventually give rise to neurons is limited in many cases in vivo compared to the situation in vitro. In the first section, we present the characteristics of endogenous progenitor-like cells and discuss the differences in their neurogenic potential in vitro and in vivo.As the environment plays an important role for survival, proliferation, migration, and other processes, the second section of the review describes changes in the extracellular matrix (ECM, a complex network that contains numerous signaling molecules. It appears that signals in the damaged central nervous system lead to an activation and de-differentiation of astrocytes, but do not effectively promote neuronal differentiation of these cells. Factors that influence stem cells during development are upregulated in the damaged brain as part of an environment resembling a stem cell niche. We give a general description of the ECM composition, with focus on stem cell-associated factors like the glycoprotein Tenascin-C.Stem cell transplantation is considered as potential treatment strategy. Interaction of transplanted stem cells with the host environment is critical for the outcome of stem cell-based therapies. Possible mechanisms involving the ECM by which transplanted stem cells might improve recovery are discussed in the last section.

  20. Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow

    International Nuclear Information System (INIS)

    Park, Y.-H.; Osmond, D.G.

    1989-01-01

    To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, (iii) μ heavy chains in the cytoplasm (cμ) and at the cell surface (sμ). Three populations of μ - cells (TdT + 14.8 - ; TdT + 14.8 + ; TdT - 14.8 + ) have been proposed to be early B-cell precursors which would give rise to cμ + sμ - pre-B cells and to sμ + B lymphocytes. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT + 14.8 - , TdT + 14.8 + and TdT - 14.8 + cells form three successive stages in B-cell differentiation before the expression of μ chains, presumptively including the stage of μ chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow. (author)

  1. Enhanced endogenous type I interferon cell-driven survival and inhibition of spontaneous apoptosis by Riluzole

    International Nuclear Information System (INIS)

    Achour, Ammar; M'Bika, Jean-Pierre; Biquard, Jean-Michel

    2009-01-01

    Highly active antiretroviral therapy (HAART), although effective in improving the survival of HIV-1-infected individuals, has not been able to reconstitute the adaptive immune response. We have described the use of novel chemical agents to restore T-cell survival/proliferation by inducing cytokine production. Due to its cationic amphiphilic structure, these molecules appear to enhance immune restoration. In this study, we investigated the action of Riluzole (2-amino-6-trifuromethoxybenzothiazole) in HIV-1 infection. Riluzole is able to increase (effective dose from 1 to 1000 nM) the cell-survival of T cells from HIV-1-infected patients and inhibit spontaneous apoptosis. The immunomodulatory effect of riluzole-sensitized cells was ascribed to endogenous type I interferon (IFN) derived from monocytes. Riluzole might be used for restoring the cell survival of immunocompromised patients and eliminating latent infected cells upon HIV-1 reactivation

  2. Lymphoid Precursor Cells and their Differentiation; Kletki-predshestvenniki v limfoidnoj tkani i ikh differentsirovka

    Energy Technology Data Exchange (ETDEWEB)

    Chertkov, I. L. [Central' Nyj Institut Gematologii I Perelivanija Krovi, Moskva, SSSR (Russian Federation); Fridenshtejn, A. Ja. [Institut Epidemiologii, Mikrobiologii i Immunologii Im. N.F.Gamaleja, Moskva, SSSR (Russian Federation)

    1968-08-15

    The authors discuss present-day ideas concerning precursor cells in lymphoid tissues and suggest that the stem cells' decision in regard to proliferation and the path of further differentiation may be a multi-stage process, occurring at various successive phases in the histogenesis of lymphoid cells. They propose that only those elements which are capable of actively deciding (under the influence of suitable stimuli or factors inherent in the cell) in which direction the successor cells are to differentiate should be regarded as precursor cells. It is shown that there are in lymphoid tissue at least three stages with the properties of precursor cells: the stem haemopoietic cell, the antigen-sensitive cell and the immunological memory cell. The authors suggest that the working cells of immune response are transformed into immunological memory cells, and discuss the morphological characteristics of the precursor cells. (author) [Russian] V doklade rassmatrivajutsja sovremennye predstavlenija o kletkah-pred- shestvennjakah v limfoidnoj tkani. Predpolagaetsja, chto prinjatie stvolovymi kletkami reshenija o proliferacii i napravlenii dal'nejshej differencirovki mozhet predstavljat' soboj mnogostadijnyj process, proishodjashhij posledovatel'no na raznyh jetapah gistogenez a limfoidnyh kletok. Predlagaetsja rassmatrivat' v kach estv e kletok-predshestvennikov tol'ko te jelementy, kotorye sposobny aktivno (pod vlijaniem sootvetstvujushhih induktorov ili vnutrennih dlja kletki faktorov) prinimat' reshenie o napravlenii differencirovki kletok-potomkov. Dok azyvaet sja nalichie v limfoidnoj tkani, po men'shej mere, treh stadij so svojstvami kletok-predshestvennikov: stvolovaja krovetvornaja kletka, antigen-chuvstvitel'naja kletka i kletka immunologicheskoj pamjati. Vyskazyvaet sja gi poteza o transformacii rabochih kletok immunnogo otvet a v kletki immunologicheskoj pamjati. Obsuzhdaetsja morfologicheskaja harakteristika kletok-predshestvennikov. (author)

  3. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    Science.gov (United States)

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

  4. Isolation of Precursor Cells from Waste Solid Fat Tissue

    Science.gov (United States)

    Byerly, Diane; Sognier, Marguerite A.

    2009-01-01

    A process for isolating tissue-specific progenitor cells exploits solid fat tissue obtained as waste from such elective surgical procedures as abdominoplasties (tummy tucks) and breast reductions. Until now, a painful and risky process of aspiration of bone marrow has been used to obtain a limited number of tissue- specific progenitor cells. The present process yields more tissue-specific progenitor cells and involves much less pain and risk for the patient. This process includes separation of fat from skin, mincing of the fat into small pieces, and forcing a fat saline mixture through a sieve. The mixture is then digested with collagenase type I in an incubator. After centrifugation tissue-specific progenitor cells are recovered and placed in a tissue-culture medium in flasks or Petri dishes. The tissue-specific progenitor cells can be used for such purposes as (1) generating three-dimensional tissue equivalent models for studying bone loss and muscle atrophy (among other deficiencies) and, ultimately, (2) generating replacements for tissues lost by the fat donor because of injury or disease.

  5. Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

    Directory of Open Access Journals (Sweden)

    Avery S. Walker

    2010-01-01

    Full Text Available Embryonic neuroepithelia and adult subventricular zone (SVZ stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes.

  6. Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

    Science.gov (United States)

    Martínez, Constanza; Cornejo, Víctor Hugo; Lois, Pablo; Ellis, Tammy; Solis, Natalia P; Wainwright, Brandon J; Palma, Verónica

    2013-01-01

    The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

  7. Early postradition recovery of hematopoietic stromal precursor cells

    Energy Technology Data Exchange (ETDEWEB)

    Todriya, T.V.

    1985-04-01

    The aim of this investigation was an immunohistochemical study of alpha-endorphin-producing cells and also a study of rat mast cells (MC in the antral mucosa of the human stomach. Men aged 18 to 30 years undergoing in-patient treatment wre studied. According to the results of radioimmunoassay, antibodies against alpha-endorphin did not react with enkephalins, beta-endorphin, or the C-terminal fragment of beta-endorphin, but had cross reactivity of about 10% with gammaendorphin. Results were subjected to statistical analysis by Student's test at a 85% level of significance and they are shown. The facts presented here suggest that MC of human gastric mucosa include argyrophilic cells which contain alpha-endorphin.

  8. Identifying endogenous neural stem cells in the adult brain in vitro and in vivo: novel approaches.

    Science.gov (United States)

    Rueger, Maria Adele; Androutsellis-Theotokis, Andreas

    2013-01-01

    In the 1960s, Joseph Altman reported that the adult mammalian brain is capable of generating new neurons. Today it is understood that some of these neurons are derived from uncommitted cells in the subventricular zone lining the lateral ventricles, and the dentate gyrus of the hippocampus. The first area generates new neuroblasts which migrate to the olfactory bulb, whereas hippocampal neurogenesis seems to play roles in particular types of learning and memory. A part of these uncommitted (immature) cells is able to divide and their progeny can generate all three major cell types of the nervous system: neurons, astrocytes, and oligodendrocytes; these properties define such cells as neural stem cells. Although the roles of these cells are not yet clear, it is accepted that they affect functions including olfaction and learning/memory. Experiments with insults to the central nervous system also show that neural stem cells are quickly mobilized due to injury and in various disorders by proliferating, and migrating to injury sites. This suggests a role of endogenous neural stem cells in disease. New pools of stem cells are being discovered, suggesting an even more important role for these cells. To understand these cells and to coax them to contribute to tissue repair it would be very useful to be able to image them in the living organism. Here we discuss advances in imaging approaches as well as new concepts that emerge from stem cell biology with emphasis on the interface between imaging and stem cells.

  9. Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts

    Directory of Open Access Journals (Sweden)

    Maucksch C

    2012-01-01

    Full Text Available Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP-expressing astrocytes. This study represents a novel virus-free approach for direct reprogramming of human fibroblasts to a neural precursor fate.

  10. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Gabriel, S. E.; Stutts, M. J.

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath...

  11. Effect of ionizing radiation on human skeletal muscle precursor cells

    International Nuclear Information System (INIS)

    Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

    2013-01-01

    Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions

  12. Expression and regulation of the endogenous retrovirus 3 (ERV3 in Hodgkin’s lymphoma cells

    Directory of Open Access Journals (Sweden)

    Stefanie eKewitz

    2013-07-01

    Full Text Available Human endogenous retroviruses (ERV are an integral part of our genome. Expression of ERV is usually switched off but reactivation of ERV has been observed in varying human diseases including cancer. Recently, reactivation of ERV associated promoters in Hodgkin’s lymphoma (HL cells has been described. Despite relatively good prognosis, not all patients with HL can be cured with the established therapy and this therapy is associated with severe late side effects. Therefore, new targets are required for the development of future treatment strategies. Reactivated ERV might represent such target structures. Therefore, we asked which ERV loci are expressed in HL cells. Using DNA microarray analysis, we found no evidence for a general activation of ERV transcription in HL cells. In contrast, we observed down-regulation of ERV3, an ERV with potential tumor suppressor function, in HL cells in comparison to normal blood cells. Interestingly, ERV3 was also differentially expressed in published DNA microarray data from resting versus cycling B cells. Treatment of HL cells with the histone deacetylase inhibitor vorinostat strongly up-regulated ERV3 expression. In addition, we observed up-regulation in HL cells after treatment with hypoxia-mimetic cobalt(II chloride. Like vorinostat, cobalt(II chloride inhibited cell growth of HL cells. Our results suggest that cell cycle inhibition of HL cells is accompanied by up-regulation of ERV3.

  13. Comparison of neurosphere-like cell clusters derived from dental follicle precursor cells and retinal Müller cells

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Felthaus, Oliver

    2011-01-01

    Unrelated cells such as dental follicle precursor cells (DFPCs) and retinal Müller cells (MCs) make spheres after cultivation in serum-replacement medium (SRM). Until today, the relation and molecular processes of sphere formation from different cell types remain undescribed. Thus in this study we...... compared proteomes of spheres derived from MCs and DFPCs. 73% of 676 identified proteins were similar expressed in both cell types and many of them are expressed in the brain (55%). Moreover proteins are overrepresented that are associated with pathways for neural diseases such as Huntington disease...... or Alzheimer disease. Interestingly up-regulated proteins in DFPCs are involved in the biosynthesis of glycosphingolipids. These lipids are components of gangliosides such as GD3, which is a novel neural stem cell marker. In conclusion spheres from different types of cells have highly similar proteomes...

  14. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  15. Hybrid ZnO:polymer bulk heterojunction solar cells from a ZnO precursor

    NARCIS (Netherlands)

    Beek, W.J.E.; Slooff, L.H.; Wienk, M.M.; Kroon, J.M.; Janssen, R.A.J.; Kafafi, Z.H.

    2005-01-01

    We describe a simple and new method to create hybrid bulk heterojunction solar cells consisting of ZnO and conjugated polymers. A gel-forming ZnO precursor, blended with conjugated polymers, is converted into crystalline ZnO at temperatures as low as 110 °C. In-situ formation of ZnO in MDMO-PPV

  16. Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies

    NARCIS (Netherlands)

    van Dam, V.; Olrichs, N.K.; Breukink, E.J.

    2009-01-01

    Wall chart: The predominant component of the bacterial cell wall, peptidoglycan, consists of long alternating stretches of aminosugar subunits interlinked in a large three-dimensional network and is formed from precursors through several cytosolic and membrane-bound steps. The high tolerance of the

  17. Endogenous electromagnetic forces emissions during cell respiration as additional factor in cancer origin.

    Science.gov (United States)

    Embi, Abraham A

    2016-01-01

    Seven decades ago, a seminal paper by Dr. Denham Harman in (J Gerontol 11(3):298-300, 1956), introduced a theory stating that there are good reasons for assuming that endogenous irradiation in the living cells could lead to cancer via an obscure mechanism. The main purpose of this manuscript is to shed some light in said mechanism by proposing a five-step eukaryotic cell cancer triggering cycle. In other words, a new factor is introduced, namely the recently found emissions of electromagnetic forces (EMFs) as a possible causing agent in diseases, including cancer. Introduced is an eukaryotic cell cancer inducing cycle. It includes five sequential steps of endogenous biological process that are backed by published scientific reports. It is a known fact that in order to achieve homeostasis, toxic reactive oxygen species (ROS) i.e. H2O2 molecules are broken down by the protein enzyme catalase. During this reaction EMFs are generated (Embi in AIS Physics 2(3):226-230, 2016). The EMFs recording breakthrough was possible due to the introduction of a novel table top microscopy technique to detect EMFs by using Prussian Blue Stain and nano-sized iron particles. There are different roots in molecular and clinical biology through which DNA damage could be programmed, EMFs emitted (during cell respiration) are herein proposed as an additional cause.

  18. Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification

    Directory of Open Access Journals (Sweden)

    Thomas Zerjatke

    2017-05-01

    Full Text Available Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations.

  19. THE POTENTIAL ROLE OF ENDOGENOUS STEM CELLS IN REGENERATION OF THE INNER EAR

    Science.gov (United States)

    Martinez-Monedero, Rodrigo; Oshima, Kazuo; Heller, Stefan; Edge, Albert S.B.

    2007-01-01

    Stem cells in various mammalian tissues retain the capacity to renew themselves and may be able to restore damaged tissue. Their existence has been proven by genetic tracer studies that demonstrate their differentiation into multiple tissue types and by their ability to self-renew through proliferation. Stem cells from the adult nervous system proliferate to form clonal floating colonies called spheres in vitro, and recent studies have demonstrated sphere formation by cells in the cochlea in addition to the vestibular system and the auditory ganglia, indicating that these tissues contain cells with stem cell properties. The presence of stem cells in the inner ear raises the hope of regeneration of mammalian inner ear cells but is difficult to correlate with the lack spontaneous regeneration seen in the inner ear after tissue damage. Loss of stem cells postnatally in the cochlea may correlate with the loss of regenerative capacity and may limit our ability to stimulate regeneration. Retention of sphere forming capacity in adult vestibular tissues suggests that the limited capacity for repair may be attributed to the continued presence of progenitor cells. Future strategies for regeneration must consider the distribution of endogenous stem cells in the inner ear and whether cells with the capacity for regeneration are retained. PMID:17321086

  20. Endogenous superoxide dismutase and catalase activities and radiation resistance in mouse cell lines

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Ostrand-Rosenberg, S.

    1988-01-01

    The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by γ-radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (Dsub(q)), or Dsub(o). None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent Ksub(i) of potassium cyanide for mouse CuZn-superoxide dismutase was determined (Ksub(i) = 6.5 μmol dm -3 ). (author)

  1. Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators

    Directory of Open Access Journals (Sweden)

    Mandë Holford

    2009-10-01

    Full Text Available Neuronal circuits depend on the precise regulation of cell-surface receptors and ion channels. An ongoing challenge in neuroscience research is deciphering the functional contribution of specific receptors and ion channels using engineered modulators. A novel strategy, termed “tethered toxins”, was recently developed to characterize neuronal circuits using the evolutionary derived selectivity of venom peptide toxins and endogenous peptide ligands, such as lynx1 prototoxins. Herein, the discovery and engineering of cell-surface tethered peptides is reviewed, with particular attention given to their cell-autonomy, modular composition, and genetic targeting in different model organisms. The relative ease with which tethered peptides can be engineered, coupled with the increasing number of neuroactive venom toxins and ligand peptides being discovered, imply a multitude of potentially innovative applications for manipulating neuronal circuits and tissue-specific cell networks, including treatment of disorders caused by malfunction of receptors and ion channels.

  2. Exploiting endogenous fibrocartilage stem cells to regenerate cartilage and repair joint injury

    Science.gov (United States)

    Embree, Mildred C.; Chen, Mo; Pylawka, Serhiy; Kong, Danielle; Iwaoka, George M.; Kalajzic, Ivo; Yao, Hai; Shi, Chancheng; Sun, Dongming; Sheu, Tzong-Jen; Koslovsky, David A.; Koch, Alia; Mao, Jeremy J.

    2016-01-01

    Tissue regeneration using stem cell-based transplantation faces many hurdles. Alternatively, therapeutically exploiting endogenous stem cells to regenerate injured or diseased tissue may circumvent these challenges. Here we show resident fibrocartilage stem cells (FCSCs) can be used to regenerate and repair cartilage. We identify FCSCs residing within the superficial zone niche in the temporomandibular joint (TMJ) condyle. A single FCSC spontaneously generates a cartilage anlage, remodels into bone and organizes a haematopoietic microenvironment. Wnt signals deplete the reservoir of FCSCs and cause cartilage degeneration. We also show that intra-articular treatment with the Wnt inhibitor sclerostin sustains the FCSC pool and regenerates cartilage in a TMJ injury model. We demonstrate the promise of exploiting resident FCSCs as a regenerative therapeutic strategy to substitute cell transplantation that could be beneficial for patients suffering from fibrocartilage injury and disease. These data prompt the examination of utilizing this strategy for other musculoskeletal tissues. PMID:27721375

  3. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

    Science.gov (United States)

    Wang, Kai; Li, Yan; Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S; Song, Jia-Sheng; Zheng, Jing

    2013-10-28

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. CD1d-Restricted Type II NKT Cells Reactive With Endogenous Hydrophobic Peptides.

    Science.gov (United States)

    Nishioka, Yusuke; Masuda, Sakiko; Tomaru, Utano; Ishizu, Akihiro

    2018-01-01

    NKT cells belong to a distinct subset of T cells that recognize hydrophobic antigens presented by major histocompatibility complex class I-like molecules, such as CD1d. Because NKT cells stimulated by antigens can activate or suppress other immunocompetent cells through an immediate production of a large amount of cytokines, they are regarded as immunological modulators. CD1d-restricted NKT cells are classified into two subsets, namely, type I and type II. CD1d-restricted type I NKT cells express invariant T cell receptors (TCRs) and react with lipid antigens, including the marine sponge-derived glycolipid α-galactosylceramide. On the contrary, CD1d-restricted type II NKT cells recognize a wide variety of antigens, including glycolipids, phospholipids, and hydrophobic peptides, by their diverse TCRs. In this review, we focus particularly on CD1d-restricted type II NKT cells that recognize endogenous hydrophobic peptides presented by CD1d. Previous studies have demonstrated that CD1d-restricted type I NKT cells usually act as pro-inflammatory cells but sometimes behave as anti-inflammatory cells. It has been also demonstrated that CD1d-restricted type II NKT cells play opposite roles to CD1d-restricted type I NKT cells; thus, they function as anti-inflammatory or pro-inflammatory cells depending on the situation. In line with this, CD1d-restricted type II NKT cells that recognize type II collagen peptide have been demonstrated to act as anti-inflammatory cells in diverse inflammation-induction models in mice, whereas pro-inflammatory CD1d-restricted type II NKT cells reactive with sterol carrier protein 2 peptide have been demonstrated to be involved in the development of small vessel vasculitis in rats.

  5. Primary orbital precursor T-cell lymphoblastic lymphoma

    DEFF Research Database (Denmark)

    Stenman, Lisa; Persson, Marta; Enlund, Fredrik

    2016-01-01

    Primary T-cell lymphoblastic lymphoma (T-LBL) in the eye region is very rare. The present study described a unique case of T-LBL involving the extraocular muscles. A 22-year-old male patient presented with a 3-week history of headache, reduced visual acuity and edema of the left eye. Clinical...... knowledge, this is the first report of a case of T-LBL involving the extraocular muscles. Although primary T-LBL in the eye region is very rare, our findings demonstrate that lymphoma should be considered in the differential diagnosis of patients with similar symptoms....

  6. Endogenous WNT Signals Mediate BMP-Induced and Spontaneous Differentiation of Epiblast Stem Cells and Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Dorota Kurek

    2015-01-01

    Full Text Available Therapeutic application of human embryonic stem cells (hESCs requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs. WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs.

  7. Primary orbital precursor T-cell lymphoblastic lymphoma

    DEFF Research Database (Denmark)

    Stenman, Lisa; Persson, Marta; Enlund, Fredrik

    2016-01-01

    Primary T-cell lymphoblastic lymphoma (T-LBL) in the eye region is very rare. The present study described a unique case of T-LBL involving the extraocular muscles. A 22-year-old male patient presented with a 3-week history of headache, reduced visual acuity and edema of the left eye. Clinical...... examination revealed left-sided exophthalmus, periorbital edema, chemosis, and reduced motility of the left eye. A magnetic resonance imaging scan revealed thickening of the left orbital muscles and a positron emission tomography-computed tomography scan also demonstrated activity in a subclavicular lymph....... There was no involvement of the bone marrow. Based on the clinical and histopathological findings, a diagnosis of T-LBL was made. There was no evidence of NOTCH1 mutation or rearrangements of the ETV6 and MLL genes and high-resolution array-based comparative genomic hybridization (arrayCGH) analysis revealed a normal...

  8. [Dinitrosyl iron complexes are endogenous signaling agents in animal and human cells and tissues (a hypothesis)].

    Science.gov (United States)

    Vanin, A F

    2004-01-01

    The hypothesis was advanced that dinitrosyl iron complexes generated in animal and human cells and tissues producing nitric oxide can function as endogenous universal regulators of biochemical and physiological processes. This function is realized by the ability of dinitrosyl iron complexes to act as donors of free nitric oxide molecules interacting with the heme groups of proteins, nitrosonium ions, or Fe+(NO+)2 interacting with the thiol groups of proteins. The effect of dinitrosyl iron complexes on the activity of some enzymes and the expression of the genome at the translation and transcription levels was considered.

  9. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  10. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    Science.gov (United States)

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-06

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    OpenAIRE

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2013-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-spe...

  12. Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

    Czech Academy of Sciences Publication Activity Database

    Zjablovskaja, Polina; Daněk, Petr; Kardošová, Miroslava; Alberich-Jorda, Meritxell

    č. 132 (2018), č. článku e57033. ISSN 1940-087X R&D Projects: GA ČR GA15-03796S Institutional support: RVO:68378050 Keywords : 32D/G-CSF-R cells * murine myeloid precursor cells * liquid culture * differentiation * neutrophils * proliferation * cytokines * IL-3 * G-CSF Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.232, year: 2016

  13. Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

    Science.gov (United States)

    Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

    1978-01-01

    A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

  14. CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Sharma, Arun; Toepfer, Christopher N; Ward, Tarsha; Wasson, Lauren; Agarwal, Radhika; Conner, David A; Hu, Johnny H; Seidman, Christine E

    2018-01-24

    Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  15. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

    Science.gov (United States)

    Quinn, Kyle P.; Sridharan, Gautham V.; Hayden, Rebecca S.; Kaplan, David L.; Lee, Kyongbum; Georgakoudi, Irene

    2013-12-01

    The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

  16. Cure of murine thalassemia by bone marrow transplantation without eradication of endogenous stem cells

    International Nuclear Information System (INIS)

    Wagemaker, G.; Visser, T.P.; van Bekkum, D.W.

    1986-01-01

    alpha-Thalassemic heterozygous (Hbath/+) mice were used to investigate the possible selective advantage of transplanted normal (+/+) hemopoietic cells. Without conditioning by total-body irradiation (TBI), infusion of large numbers of normal bone marrow cells failed to correct the thalassemic peripheral blood phenotype. Since the recipients' stem cells are normal with respect to number and differentiation capacity, it was thought that the transplanted stem cells were not able to lodge, or that they were not stimulated to proliferate. Therefore, a nonlethal dose of TBI was given to temporarily reduce endogenous stem cell numbers and hemopoiesis. TBI doses of 2 or 3 Gy followed by infusion of normal bone marrow cells proved to be effective in replacing the thalassemic red cells by normal red cells, whereas a dose of 1 Gy was ineffective. It is concluded that cure of thalassemia by bone marrow transplantation does not necessarily require eradication of thalassemic stem cells. Consequently, the objectives of conditioning regimens for bone marrow transplantation of thalassemic patients (and possibly other nonmalignant hemopoietic disorders) should be reconsidered

  17. Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

    Directory of Open Access Journals (Sweden)

    Kazuya Takahashi

    Full Text Available Prognosis of childhood acute lymphoblastic leukemia (ALL has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ, a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+ ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ, a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19 ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19 ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19 ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good

  18. Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead.

    Science.gov (United States)

    Bhartiya, Deepa; Shaikh, Ambreen; Anand, Sandhya; Patel, Hiren; Kapoor, Sona; Sriraman, Kalpana; Parte, Seema; Unni, Sreepoorna

    2016-12-01

    more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Broholm, Christa; Brandt, Claus; Schultz, Ninna S

    2012-01-01

    The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response...... nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function......RNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts...

  20. Leukemia inhibitory factor favours neurogenic differentiation of long-term propagated human midbrain precursor cells

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Widmer, Hans R; Zimmer, Jens

    2009-01-01

    There is a lot of excitement about the potential use of multipotent neural stem cells for the treatment of neurodegenerative diseases. However, the strategy is compromised by the general loss of multipotency and ability to generate neurons after long-term in vitro propagation. In the present study......, human embryonic (5 weeks post-conception) ventral mesencephalic (VM) precursor cells were propagated as neural tissue-spheres (NTS) in epidermal growth factor (EGF; 20 ng/ml) and fibroblast growth factor 2 (FGF2; 20 ng/ml). After more than 325 days, the NTS were transferred to media containing either...... EGF+FGF2, EGF+FGF2+heparin or leukemia inhibitory factor (LIF; 10 ng/ml)+FGF2+heparin. Cultures were subsequently propagated for more than 180 days with NTS analyzed at various time-points. Our data show for the first time that human VM neural precursor cells can be long-term propagated as NTS...

  1. Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration

    DEFF Research Database (Denmark)

    Saclier, Marielle; Yacoub-Youssef, Houda; Mackey, Abigail

    2013-01-01

    , we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory...... anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates......Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here...

  2. IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

    Science.gov (United States)

    Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank

    2018-01-01

    Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626

  3. Endogenous Tim-1 (Kim-1) promotes T-cell responses and cell-mediated injury in experimental crescentic glomerulonephritis.

    Science.gov (United States)

    Nozaki, Yuji; Nikolic-Paterson, David J; Snelgrove, Sarah L; Akiba, Hisaya; Yagita, Hideo; Holdsworth, Stephen R; Kitching, A Richard

    2012-05-01

    The T-cell immunoglobulin mucin 1 (Tim-1) modulates CD4(+) T-cell responses and is also expressed by damaged proximal tubules in the kidney where it is known as kidney injury molecule-1 (Kim-1). We sought to define the role of endogenous Tim-1 in experimental T-cell-mediated glomerulonephritis induced by sheep anti-mouse glomerular basement membrane globulin acting as a planted foreign antigen. Tim-1 is expressed by infiltrating activated CD4(+) cells in this model, and we studied the effects of an inhibitory anti-Tim-1 antibody (RMT1-10) on immune responses and glomerular disease. Crescentic glomerulonephritis, proliferative injury, and leukocyte accumulation were attenuated following treatment with anti-Tim-1 antibodies, but interstitial foxp3(+) cell accumulation and interleukin-10 mRNA were increased. T-cell proliferation and apoptosis decreased in the immune system along with a selective reduction in Th1 and Th17 cellular responses both in the immune system and within the kidney. The urinary excretion and renal expression of Kim-1 was reduced by anti-Tim-1 antibodies reflecting diminished interstitial injury. The effects of anti-Tim-1 antibodies were not apparent in the early phase of renal injury, when the immune response to sheep globulin was developing. Thus, endogenous Tim-1 promotes Th1 and Th17 nephritogenic immune responses and its neutralization reduces renal injury while limiting inflammation in cell-mediated glomerulonephritis.

  4. Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.

    Science.gov (United States)

    Wang, Jichang; Xie, Gangcai; Singh, Manvendra; Ghanbarian, Avazeh T; Raskó, Tamás; Szvetnik, Attila; Cai, Huiqiang; Besser, Daniel; Prigione, Alessandro; Fuchs, Nina V; Schumann, Gerald G; Chen, Wei; Lorincz, Matthew C; Ivics, Zoltán; Hurst, Laurence D; Izsvák, Zsuzsanna

    2014-12-18

    Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

  5. Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

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    Tetsuro Yasui

    2017-06-01

    Full Text Available Human neural precursor cells (hNPCs derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

  6. Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway

    Directory of Open Access Journals (Sweden)

    Lü He-Zuo

    2009-10-01

    Full Text Available Abstract Background Neural precursor cells (NPCs are defined by their ability to proliferate, self-renew, and retain the potential to differentiate into neurons and glia. Deciphering the factors that regulate their behaviors will greatly aid in their use as potential therapeutic agents or targets. Chondroitin sulfate proteoglycans (CSPGs are prominent components of the extracellular matrix (ECM in the central nervous system (CNS and are assumed to play important roles in controlling neuronal differentiation and development. Results In the present study, we demonstrated that CSPGs were constitutively expressed on the NPCs isolated from the E16 rat embryonic brain. When chondroitinase ABC was used to abolish the function of endogenous CSPGs on NPCs, it induced a series of biological responses including the proliferation, differentiation and migration of NPCs, indicating that CSPGs may play a critical role in NPC development and differentiation. Finally, we provided evidence suggesting that integrin signaling pathway may be involved in the effects of CSPGs on NPCs. Conclusion The present study investigating the influence and mechanisms of CSPGs on the differentiation and migration of NPCs should help us to understand the basic biology of NPCs during CNS development and provide new insights into developing new strategies for the treatment of the neurological disorders in the CNS.

  7. Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells

    Science.gov (United States)

    Li, Peilin; Fujimoto, Katsuya; Bourguingnon, Lilly; Yukl, Steven; Deeks, Steven; Wong, Joseph K

    2014-01-01

    Preventing mucosal transmission of HIV is critical to halting the HIV epidemic. Novel approaches to preventing mucosal transmission are needed. Hyaluronic acid (HA) is a major extracellular component of mucosa and the primary ligand for the cell surface receptor CD44. CD44 enhances HIV infection of CD4+ T cells, but the role of HA in this process is not clear. To study this, virions were generated with CD44 (HIVCD44) or without CD44 (HIVmock). Exogenous HA reduced HIV infection of unstimulated CD4+ T cells in a CD44-dependent manner. Conversely, hyaluronidase-mediated reduction of endogenous HA on the cell surface enhanced HIV binding to and infection of unstimulated CD4+ T cells. Exogenous HA treatment reduced activation of protein kinase C alpha via CD44 on CD4+ T cells during infection with HIVCD44. These results reveal new roles for HA during the interaction of HIV with CD4+ T cells that may be relevant to mucosal HIV transmission and could be exploitable as a future strategy to prevent HIV infection. PMID:24957217

  8. Long Noncoding RNA HOTAIR Controls Cell Cycle by Functioning as a Competing Endogenous RNA in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Kewei Ren

    2016-12-01

    Full Text Available Recent studies have shown that long noncoding RNAs (lncRNAs play pivotal roles in the initiation and progression of cancer, including esophageal squamous cell carcinoma (ESCC. The lncRNA HOX transcript antisense RNA (HOTAIR was reported to be dysregulated and correlated with the progression of ESCC. However, the biological role and the underlying mechanism of HOTAIR in the development of ESCC remain unclear. Herein, we found that HOTAIR was aberrantly upregulated in ESCC cells and that HOTAIR depletion inhibited proliferation and led to G1 cell cycle arrest in ESCC cells. Besides, we found that HOTAIR acted as an endogenous sponge to downregulate miR-1 expression by directly binding to miR-1. Furthermore, HOTAIR overturned the effect of miR-1 on the proliferation and cell cycle profile in ESCC cells, which involved the derepression of cyclin D1 (CCND1 expression, a target of miR-1. Taken together, our study elucidated a novel HOTAIR /miR-1/CCND1 regulatory axis in which HOTAIR acted as a competing endogenous RNA by sponging miR-1 and upregulated CCND1 expression, thereby facilitating the tumorigenesis of ESCC. Investigation of this lncRNA/miRNA/mRNA pathway may contribute to a better understanding of ESCC pathogenesis and facilitate the development of lncRNA-directed therapy against this disease.

  9. Mesenchymal stem cells promote augmented response of endogenous neural stem cells in spinal cord injury of rats

    Directory of Open Access Journals (Sweden)

    Marta Rocha Araujo

    2016-06-01

    Full Text Available Traumatic spinal cord injury results in severe neurological deficits, mostly irreversible. The cell therapy represents a strategy for treatment particularly with the use of stem cells with satisfactory results in several experimental models. The aim of the study was to compare the treatment of spinal cord injury (SCI with and without mesenchymal stem cells (MSC, to investigate whether MSCs migrate and/or remain at the site of injury, and to analyze the effects of MSCs on inflammation, astrocytic reactivity and activation of endogenous stem cells. Three hours after SCI, animals received bone marrow-derived MSCs (1×107 in 1mL PBS, IV. Animals were euthanized 24 hours, 7 and 21 days post-injury. The MSC were not present in the site of the lesion and the immunofluorescent evaluation showed significant attenuation of inflammatory response with reduction in macrophages labeled with anti-CD68 antibody (ED1, decreased immunoreactivity of astrocytes (GFAP+ and greater activation of endogenous stem cells (nestin+ in the treated groups. Therefore, cell transplantation have a positive effect on recovery from traumatic spinal cord injury possibly due to the potential of MSCs to attenuate the immune response.

  10. Effect of adrenalectomy on recipients of allogeneic lymphocytes on inactivation of endogenous colony-forming cells in mice

    International Nuclear Information System (INIS)

    Semenkov, V.F.

    1985-01-01

    This paper presents a study of the killer functions of lymph node cells directed against endogenous colony-forming cells in adrenalectomized recipients in a genetic system with one-way incompatibility: parental line - F 1 hybrid. Mice were irradiated with Co 60 gamma rays on the EGO-2 apparatus with dose rate from 200 to 250 R/min. The results were subjected to statistical analysis by Student's test. It can be tentatively suggested that the killer action of T lymphocytes on endogenous colonies was intensified in adrenal-ectomized recipients with endogenous hypocorticism, as a result of cooperation with the cortisol-sensitive subpopulation of T helper cells, of a change in the properties of the antigen-recognizing receptors, or an increase in the sensitivity of target cells to the killer action of T lymphocytes

  11. Endogenous hepatitis C virus homolog fragments in European rabbit and hare genomes replicate in cell culture.

    Directory of Open Access Journals (Sweden)

    Eliane Silva

    Full Text Available Endogenous retroviruses, non-retroviral RNA viruses and DNA viruses have been found in the mammalian genomes. The origin of Hepatitis C virus (HCV, the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans, remains unclear since its discovery. Here we show that fragments homologous to HCV structural and non-structural (NS proteins present in the European rabbit (Oryctolagus cuniculus and hare (Lepus europaeus genomes replicate in bovine cell cultures. The HCV genomic homolog fragments were demonstrated by RT-PCR, PCR, mass spectrometry, and replication in bovine cell cultures by immunofluorescence assay (IFA and immunogold electron microscopy (IEM using specific MAbs for HCV NS3, NS4A, and NS5 proteins. These findings may lead to novel research approaches on the HCV origin, genesis, evolution and diversity.

  12. Nanodiamonds with silicon vacancy defects for nontoxic photostable fluorescent labeling of neural precursor cells.

    Science.gov (United States)

    Merson, Tobias D; Castelletto, Stefania; Aharonovich, Igor; Turbic, Alisa; Kilpatrick, Trevor J; Turnley, Ann M

    2013-10-15

    Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiV-containing NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

  13. Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

    Science.gov (United States)

    Darabi, Radbod; Perlingeiro, Rita C R

    2016-01-01

    Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

  14. "The Lower Threshold" phenomenon in tumor cells toward endogenous digitalis-like compounds: Responsible for tumorigenesis?

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    Heidrun Weidemann

    2012-01-01

    Full Text Available Since their first discovery as potential anti-cancer drugs decades ago, there is increasing evidence that digitalis-like compounds (DLC have anti-tumor effects. Less is known about endogenous DLC (EDLC metabolism and regulation. As stress hormones synthesized in and secreted from the adrenal gland, they likely take part in the hypothalamo-pituitary-adrenal (HPA axis. In a previous study, we revealed reduced EDLC concentrations in plasma and organs from immune-compromised animals and proposed that a similar situation of a deregulated HPA axis with "adrenal EDLF exhaustion" may contribute to tumorigenesis in chronic stress situations. Here, we put forward the hypothesis that a lowered EDLC response threshold of tumor cells as compared with normal cells increases the risk of tumorigenesis, especially in those individuals with reduced EDLC plasma concentrations after chronic stress exposure. We will evaluate this hypothesis by (a summarizing the effects of different DLC concentrations on tumor as compared with normal cells and (b reviewing some essential differences in the Na/K-ATPase of tumor as compared with normal cells (isoform pattern, pump activity, mutations of other signalosome receptors. We will conclude that (1 tumor cells, indeed, seem to have their individual "physiologic" EDLC response range that already starts at pmolar levels and (2 that individuals with markedly reduced (pmolar EDLC plasma levels are predisposed to cancer because these EDLC concentrations will predominantly stimulate the proliferation of tumor cells. Finally, we will summarize preliminary results from our department supporting this hypothesis.

  15. Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription*

    Science.gov (United States)

    E, Guangqi; Cao, Ying; Bhattacharya, Santanu; Dutta, Shamit; Wang, Enfeng; Mukhopadhyay, Debabrata

    2012-01-01

    Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis. PMID:22167188

  16. Precursor B-cell lymphoblastic leukemia of the arm mimicking neurogenic tumor: case report

    Directory of Open Access Journals (Sweden)

    Sui Xiu-fang

    2012-07-01

    Full Text Available Abstract Precursor B-cell lymphoblastic lymphoma (PBLL is an infrequent subtype of lymphoblastic lymphoma (LBL that commonly affected site for the diagnosis is the skin, followed by the head and neck. In this report, we presented a special case of PBLL located at the left arm and detected with magnetic resonance imaging (MRI and ultrasonography (US. This kind of PBLL is similar to a peripheral nerve tumor in clinical and radiographic manifestation.

  17. Human haemato-endothelial precursors: cord blood CD34+ cells produce haemogenic endothelium.

    Directory of Open Access Journals (Sweden)

    Elvira Pelosi

    Full Text Available Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-, triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45- capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

  18. Endogenous adaptation to low oxygen modulates T-cell regulatory pathways in EAE.

    Science.gov (United States)

    Esen, Nilufer; Katyshev, Vladimir; Serkin, Zakhar; Katysheva, Svetlana; Dore-Duffy, Paula

    2016-01-19

    In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease. C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis. Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells. Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.

  19. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    Science.gov (United States)

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  20. Moscatilin Inhibits Lung Cancer Cell Motility and Invasion via Suppression of Endogenous Reactive Oxygen Species

    Directory of Open Access Journals (Sweden)

    Akkarawut Kowitdamrong

    2013-01-01

    Full Text Available Lung cancer is the leading cause of death among cancer patients worldwide, and most of them have died from metastasis. Migration and invasion are prerequisite processes associated with high metastasis potential in cancers. Moscatilin, a bibenzyl derivative isolated from the Thai orchid Dendrobium pulchellum, has been shown to have anticancer effect against numerous cancer cell lines. However, little is known regarding the effect of moscatilin on cancer cell migration and invasion. The present study demonstrates that nontoxic concentrations of moscatilin were able to inhibit human nonsmall cell lung cancer H23 cell migration and invasion. The inhibitory effect of moscatilin was associated with an attenuation of endogenous reactive oxygen species (ROS, in which hydroxyl radical (OH∙ was identified as a dominant species in the suppression of filopodia formation. Western blot analysis also revealed that moscatilin downregulated activated focal adhesion kinase (phosphorylated FAK, Tyr 397 and activated ATP-dependent tyrosine kinase (phosphorylated Akt, Ser 473, whereas their parental counterparts were not detectable changed. In conclusion, our results indicate the novel molecular basis of moscalitin-inhibiting lung cancer cell motility and invasion and demonstrate a promising antimetastatic potential of such an agent for lung cancer therapy.

  1. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie

    2014-01-01

    nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells......Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...... was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. Results: Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells...

  2. Electroacupuncture-Induced Dynamic Processes of Gene Expression Levels of Endogenous Opioid Peptide Precursors and Opioid Receptors in the CNS of Goats

    Directory of Open Access Journals (Sweden)

    Li-Li Cheng

    2013-01-01

    Full Text Available In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, μ-, and κ-receptor induced by electroacupuncture (EA in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P<0.01 with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

  3. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

    Science.gov (United States)

    Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C

    2018-04-16

    The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

  4. Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells

    International Nuclear Information System (INIS)

    Limoli, C. L.; Giedzinski, E.; Baure, J.; Doctrow, S. R.; Rola, R.; Fike, J. R.

    2006-01-01

    Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neuro-genesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse per-oxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H 2 O 2 , as is known for CuZnSOD, may be

  5. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  6. Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

    Science.gov (United States)

    Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

    2015-01-01

    The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

  7. Endogenous and exogenous porphyrins as photosensitizers in the Hep-2 human carcinoma cell line.

    Science.gov (United States)

    Alvarez, M G; Milanesio, M; Rivarola, V; Durantini, E; Batlle, A; Fukuda, H

    2009-07-01

    The photodynamic activity of three photosensitizers (PS): AL-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6- trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C(60) (P-C(60)), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. ALA-induced accumulation of the endogenous PS PPIX, reached saturation values between 5 and 24 h incubation time; the maximal PPIX content was 5.7 nmol/106 cells. The same intracellular level was accumulated when the cationic porphyrin CP was used, while the amount of P-C(60) attained was 1.5 nmol/106 cells. Under violet-blue exciting light, the fluorescence of PPIX and P-C(60) was found in the cytoplasm showing a granular appearance indicating lysosomal localization. CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. No dark cytotoxicity was observed using 1mM ALA, 5 microM CP and 1 microM P-C(60) after 24 h incubation. Cell morphology was analyzed using Hoechst-33258, toluidine blue staining, TUNEL assay and DNA fragmentation, 24 h after irradiation with 54 J/cm2. When photosensitized with ALA and P-C(60), chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PS analyzed, the molecular dyad P-C(60) was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose.

  8. Endogenous repair mechanisms enhanced in Parkinson's disease following stem cell therapy

    Directory of Open Access Journals (Sweden)

    Eleonora Napoli

    2017-01-01

    Full Text Available This mini-review highlights the innovative observation that transplanted human neural stem cells can bring about endogenous brain repair through the stimulation of multiple regenerative processes in the neurogenic area (i.e., subventricular zone [SVZ] in an animal model of Parkinson's disease (PD. In addition, we convey that identifying anti-inflammatory cytokines, therapeutic proteomes, and neurotrophic factors within the SVZ may be essential to induce brain repair and behavioral recovery. This work opens up a new area of research for further understanding the pathology and treatment of PD. This paper is a review article. Referred literature in this paper has been listed in the references section. The datasets supporting the conclusions of this article are available online by searching various databases, including PubMed. Some original points in this article come from the laboratory practice in our research center and the authors' experiences.

  9. Genomic Amplification of an Endogenous Retrovirus in Zebrafish T-Cell Malignancies

    Directory of Open Access Journals (Sweden)

    J. Kimble Frazer

    2012-01-01

    Full Text Available Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH, a technique to detect numeric variations between different DNA samples, we examined genomes from zebrafish (Danio rerio T-cell leukemias of three cancer-prone lines. In all malignancies tested, we identified recurring amplifications of a zebrafish endogenous retrovirus. This retrovirus, ZFERV, was first identified due to high expression of proviral transcripts in thymic tissue from larval and adult fish. We confirmed ZFERV amplifications by quantitative PCR analyses of DNA from wild-type fish tissue and normal and malignant D. rerio T cells. We also quantified ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplification and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERV’s phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.

  10. Release of an endogenous pyrogen in vitro from rabbit mononuclear cells.

    Science.gov (United States)

    Atkins, E; Bodel, P; Francis, L

    1967-08-01

    The capacity of rabbit mononuclear cells to release an endogenous pyrogen (EP) in vitro has been studied. After incubation with tuberculin, preparations of predominantly monocytic cells, derived from the respiratory passages of the lungs of rabbits sensitized with BCG, were activated to release EP. Pyrogen production occurred more slowly with lung monocytes than with blood leukocytes of similarly sensitized rabbits and 9 to 10 hr incubation in a fully supportive medium was required to produce clear-cut results. As previously reported with blood leukocytes, mononuclear cells from the lungs of normal animals were also activated by tuberculin but to a lesser degree than were those from specifically sensitized rabbits. Under a variety of conditions, mononuclear cells from either spleen or lymph nodes of the same sensitized rabbits failed to release detectable amounts of pyrogen when incubated with tuberculin in vitro but were activated in a majority of instances when phagocytosis of heat-killed staphylococci was used as the stimulus. Release of pyrogen from lung monocytes appears to be an active process that is both temperature-dependent and requires protein synthesis. Neither serum antibody nor complement appears to play a role in this process. Evidence is presented that the granulocyte is the main source of pyrogen evolved by blood leukocytes incubated in vitro with OT or heat-killed staphylococci, whereas the lung macrophage and/or monocyte is responsible for most of the pyrogen released from the lung cell preparations. From these studies, it is concluded that mononuclear cells can be activated in vitro by several microbial stimuli and must be considered an additional cellular source of EP. The clinical implications of these findings for the pathogenesis of fever in granulomatous diseases where the monocyte is the predominant cell are discussed.

  11. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    Science.gov (United States)

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2014-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP), and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

  12. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    Science.gov (United States)

    Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

    2013-12-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. © 2013 Elsevier Inc. All rights reserved.

  13. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

    Science.gov (United States)

    Svingen, T; Jørgensen, A; Rajpert-De Meyts, E

    2014-08-01

    The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress

    Science.gov (United States)

    Gould, Elizabeth; Tanapat, Patima; McEwen, Bruce S.; Flügge, Gabriele; Fuchs, Eberhard

    1998-01-01

    Although granule cells continue to be added to the dentate gyrus of adult rats and tree shrews, this phenomenon has not been demonstrated in the dentate gyrus of adult primates. To determine whether neurons are produced in the dentate gyrus of adult primates, adult marmoset monkeys (Callithrix jacchus) were injected with BrdU and perfused 2 hr or 3 weeks later. BrdU is a thymidine analog that is incorporated into proliferating cells during S phase. A substantial number of cells in the dentate gyrus of adult monkeys incorporated BrdU and ≈80% of these cells had morphological characteristics of granule neurons and expressed a neuronal marker by the 3-week time point. Previous studies suggest that the proliferation of granule cell precursors in the adult dentate gyrus can be inhibited by stress in rats and tree shrews. To test whether an aversive experience has a similar effect on cell proliferation in the primate brain, adult marmoset monkeys were exposed to a resident-intruder model of stress. After 1 hr in this condition, the intruder monkeys were injected with BrdU and perfused 2 hr later. The number of proliferating cells in the dentate gyrus of the intruder monkeys was compared with that of unstressed control monkeys. We found that a single exposure to this stressful experience resulted in a significant reduction in the number of these proliferating cells. Our results suggest that neurons are produced in the dentate gyrus of adult monkeys and that the rate of precursor cell proliferation can be affected by a stressful experience. PMID:9501234

  15. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

    Full Text Available Proteins containing polyglutamine (polyQ regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD. To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

  16. Extravascular red blood cells and hemoglobin promote tumor growth and therapeutic resistance as endogenous danger signals.

    Science.gov (United States)

    Yin, Tao; He, Sisi; Liu, Xiaoling; Jiang, Wei; Ye, Tinghong; Lin, Ziqiang; Sang, Yaxiong; Su, Chao; Wan, Yang; Shen, Guobo; Ma, Xuelei; Yu, Min; Guo, Fuchun; Liu, Yanyang; Li, Ling; Hu, Qiancheng; Wang, Yongsheng; Wei, Yuquan

    2015-01-01

    Hemorrhage is a common clinical manifestation in patients with cancer. Intratumor hemorrhage has been demonstrated to be a poor prognostic factor for cancer patients. In this study, we investigated the role of RBCs and hemoglobin (Hb) in the process of tumor progression and therapeutical response. RBCs and Hb potently promoted tumor cell proliferation and syngenic tumor growth. RBCs and Hb activated the reactive oxygen species-NF-κB pathway in both tumor cells and macrophages. RBCs and Hb also induced chemoresistance mediated, in part, by upregulating ABCB1 gene expression. Tumor growth induced by RBCs was accompanied by an inflammatory signature, increased tumor vasculature, and influx of M2 macrophages. In both the peritoneal cavity and tumor microenvironment, extravascular RBCs rapidly recruited monocyte-macrophages into the lesion sites. In addition, RBCs and Hb increased several nucleotide-binding oligomerization domain-like receptors' expression and induced IL-1β release. Our results provide novel insights into the protumor function of RBCs and Hb as endogenous danger signals, which can promote tumor cell proliferation, macrophage recruitment, and polarization. Hemorrhage may represent a useful prognostic factor for cancer patients because of its role in tumor promotion and chemoresistance. Copyright © 2014 by The American Association of Immunologists, Inc.

  17. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  18. Optimizing a multifunctional microsphere scaffold to improve neural precursor cell transplantation for traumatic brain injury repair.

    Science.gov (United States)

    Skop, Nolan B; Calderon, Frances; Cho, Cheul H; Gandhi, Chirag D; Levison, Steven W

    2016-10-01

    Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film. Fetal rat neural precursors plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2. To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range 30-100 µm, mean 64 µm) that were subsequently modified. Radial glial cells seeded onto these multifunctional microspheres proliferated for at least 7 days in culture and the microspheres containing cells were small enough to be injected, using 23 Gauge Hamilton syringes, into the brains of adult rats that had previously sustained cortical contusion injuries. When analysed 3 days later, the transplanted RGCs were positive for the stem cell/progenitor marker Nestin. These results demonstrate that this multifunctional scaffold can be used as a cellular and growth factor delivery vehicle for the use in developing cell transplantation therapies for traumatic brain injuries. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation

    Directory of Open Access Journals (Sweden)

    Yanli Li

    2016-09-01

    Full Text Available Omega-3 polyunsaturated fatty acids (n-3 PUFAs exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A was administered intravenously to wild-type (WT and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase (ALT activity, and inhibited production of pro-inflammatory cytokines (e.g. TNF-α, IL-6, IL-17A and IFN-γ. In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism, and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  20. Endogenous Protection Derived from Activin A/Smads Transduction Loop Stimulated via Ischemic Injury in PC12 Cells

    OpenAIRE

    Mang, Jing; Mei, Chun-Li; Wang, Jiao-Qi; Li, Zong-Shu; Chu, Ting-Ting; He, Jin-Ting; Xu, Zhong-Xin

    2013-01-01

    Activin A (ActA), a member of transforming growth factor-beta (TGF-b) super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD) injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate isch...

  1. A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

    Directory of Open Access Journals (Sweden)

    Ichiro Yajima

    Full Text Available BACKGROUND: Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA, a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2 and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.

  2. A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

    Science.gov (United States)

    Puig, Isabel; Champeval, Delphine; Kumasaka, Mayuko; Belloir, Elodie; Bonaventure, Jacky; Mark, Manuel; Yamamoto, Hiroaki; Taketo, Mark M.; Choquet, Philippe; Etchevers, Heather C.; Beermann, Friedrich; Delmas, Véronique; Monassier, Laurent; Larue, Lionel

    2013-01-01

    Background Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes. PMID:23382837

  3. Lipofection indirectly increases expression of endogenous major histocompatibility complex class I molecules on tumor cells.

    Science.gov (United States)

    Fox, B A; Drury, M; Hu, H M; Cao, Z; Huntzicker, E G; Qie, W; Urba, W J

    1998-01-01

    Direct intratumoral injection of a lipid/DNA complex encoding an allogeneic major histocompatibility complex (MHC) class I molecule leads to regression of both an immunogenic murine tumor and also melanoma lesions in some patients. We have sought to understand the mechanism(s) for this augmentation of antitumor activity. While optimizing parameters for in vitro gene transfer into the D5 subclone of B16BL6, it was noted that lipofected tumors not only expressed the new alloantigen but also exhibited increased expression of endogenous MHC class I, both H-2 Kb and H-2 Db. This increase in expression was not restricted to the small percentage of cells that expressed the transfected gene, but appeared to affect the majority of cells in culture. Class I expression was not increased by lipopolysaccharide, DNA alone, lipid, or lipid/lipopolysaccharide mixtures. Enhanced class I expression required a DNA/lipid complex and was greatest when parameters optimized for gene transfer of the alloantigen were used. All DNA plasmids tested had this effect, including one plasmid whose DNA was not transcribed because it lacked an expression cassette. Because of the critical role that MHC class I antigens play in immune recognition, we propose that lipid complex-mediated gene transfer may provide immunological advantages beyond those that are attributable to expression of the specific gene transferred.

  4. Enriched Endogenous Omega-3 Fatty Acids in Mice Ameliorate Parenchymal Cell Death After Traumatic Brain Injury.

    Science.gov (United States)

    Ren, Huixia; Yang, Zhen; Luo, Chuanming; Zeng, Haitao; Li, Peng; Kang, Jing X; Wan, Jian-Bo; He, Chengwei; Su, Huanxing

    2017-07-01

    Currently no effective therapies are available for the treatment of traumatic brain injury (TBI). Early intervention that specifically provides neuroprotection is of most importance which profoundly influences the outcome of TBI. In the present study, we adopted a closed-skull mild TBI model to investigate potential roles of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in protecting against TBI. Using two-photon laser scanning microscopy (2PLSM), parenchymal cell death and reactive oxidative species (ROS) expression were directly observed and recorded after TBI through a thinned skull bone window. Fat-1 mice with high endogenous ω-3 PUFAs significantly inhibited ROS expression and attenuated parenchymal cell death after compression injury during the early injury phase. Elevated generation of glutathione (GSH) and neuroprotectin D1 (NPD1) in the parenchyma of fat-1 mice could be the contributor to the beneficial role of ω-3 PUFAs in TBI. The results of the study suggest that ω-3 PUFAs is an effective neuroprotectant as an early pharmacological intervention for TBI and the information derived from this study may help guide dietary advice for those who are susceptible to repetitive mild TBI.

  5. Tumefactive intracranial presentation of precursor B-cell acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Forester, Craig M.; Braunreiter, Chi L.; Yaish, Hasan; Afify, Zeinab; Hedlund, Gary L.

    2009-01-01

    In children, leukemia is the most common malignancy, and approximately 75% of leukemias are acute lymphoblastic leukemia (ALL). Central nervous system leukemia is found at diagnosis in fewer than 5% of children with ALL. Leukemic intracranial masses have been described with acute myeloid leukemia, but ALL presenting as a mass lesion is rare. We describe a unique case of an intracranial confirmed precursor B cell (pre-B) ALL mass in a 13-year-old girl that was diagnosed by brain CT, MRI and cerebral angiography, and confirmed by biopsy. This report details pertinent history and distinguishing imaging features of an intracranial ALL tumefaction. (orig.)

  6. Tumefactive intracranial presentation of precursor B-cell acute lymphoblastic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Forester, Craig M. [University of Utah, Salt Lake City, UT (United States); Braunreiter, Chi L. [University of Utah, Division of Pediatric Hematology Oncology, Primary Children' s Medical Center, Salt Lake City, UT (United States); Helen DeVos Children' s Hospital, Department of Pediatric Hematology Oncology, Grand Rapids, MI (United States); Yaish, Hasan; Afify, Zeinab [University of Utah, Division of Pediatric Hematology Oncology, Primary Children' s Medical Center, Salt Lake City, UT (United States); Hedlund, Gary L. [Primary Children' s Medical Center, Department of Pediatric Radiology, Salt Lake City, UT (United States)

    2009-11-15

    In children, leukemia is the most common malignancy, and approximately 75% of leukemias are acute lymphoblastic leukemia (ALL). Central nervous system leukemia is found at diagnosis in fewer than 5% of children with ALL. Leukemic intracranial masses have been described with acute myeloid leukemia, but ALL presenting as a mass lesion is rare. We describe a unique case of an intracranial confirmed precursor B cell (pre-B) ALL mass in a 13-year-old girl that was diagnosed by brain CT, MRI and cerebral angiography, and confirmed by biopsy. This report details pertinent history and distinguishing imaging features of an intracranial ALL tumefaction. (orig.)

  7. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Science.gov (United States)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  8. Endogenous Stem Cells Were Recruited by Defocused Low-Energy Shock Wave in Treating Diabetic Bladder Dysfunction.

    Science.gov (United States)

    Jin, Yang; Xu, Lina; Zhao, Yong; Wang, Muwen; Jin, Xunbo; Zhang, Haiyang

    2017-04-01

    Defocused low-energy shock wave (DLSW) has been shown effects on activating mesenchymal stromal cells (MSCs) in vitro. In this study, recruitment of endogenous stem cells was firstly examined as an important pathway during the healing process of diabetic bladder dysfunction (DBD) treated by DLSW in vivo. Neonatal rats received intraperitoneal injection of 5-ethynyl-2-deoxyuridine (EdU) and then DBD rat model was created by injecting streptozotocin. Four weeks later, DLSW treatment was performed. Afterward, their tissues were examined by histology. Meanwhile, adipose tissue-derived stem cells (ADSCs) were treated by DLSW in vitro. Results showed DLSW ameliorated voiding function of diabetic rats by recruiting EdU + Stro-1 + CD34 - endogenous stem cells to release abundant nerve growth factor (NGF) and vascular endothelial growth factor (VEGF). Some EdU + cells overlapped with staining of smooth muscle actin. After DLSW treatment, ADSCs showed higher migration ability, higher expression level of stromal cell-derived factor-1 and secreted more NGF and VEGF. In conclusion, DLSW could ameliorate DBD by recruiting endogenous stem cells. Beneficial effects were mediated by secreting NGF and VEGF, resulting into improved innervation and vascularization in bladder.

  9. ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

    International Nuclear Information System (INIS)

    Fujita, Yuko; Hiroyama, Masami; Sanbe, Atsushi; Yamauchi, Junji; Murase, Shoko; Tanoue, Akito

    2008-01-01

    While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway

  10. Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury.

    Directory of Open Access Journals (Sweden)

    Jianqin Ye

    Full Text Available BACKGROUND: Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI. However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear. METHODOLOGY/PRINCIPAL FINDING: Using "middle aged" mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1(+CD45(- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1 in Sca-1(+CD45(- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1(+CD45(- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts. CONCLUSIONS/SIGNIFICANCE: These studies demonstrate that cloned Sca-1(+CD45(- cells derived from CSs from infarcted "middle aged" hearts are enriched for second heart field (i.e., Isl-1(+ precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.

  11. HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

    Science.gov (United States)

    Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

    1999-06-18

    To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

  12. Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

    Science.gov (United States)

    Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

    2014-05-01

    In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

  13. Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan)

    2014-09-26

    Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.

  14. γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation

    Science.gov (United States)

    D’Avanzo, Carla; Sliwinski, Christopher; Wagner, Steven L.; Tanzi, Rudolph E.; Kim, Doo Yeon; Kovacs, Dora M.

    2015-01-01

    Soluble γ-secretase modulators (SGSMs) selectively decrease toxic amyloid β (Aβ) peptides (Aβ42). However, their effect on the physiologic functions of γ-secretase has not been tested in human model systems. γ-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures with γ-secretase inhibitors (GSIs), but not SGSMs, induced a 2- to 4-fold increase in the expression of the neuronal markers Tuj1 and doublecortin. GSI treatment also induced neuronal marker protein expression, as shown by Western blot analysis. In the same conditions, SGSM treatment selectively reduced endogenous Aβ42 levels by ∼80%. Mechanistically, we found that Notch target gene expressions were selectively inhibited by a GSI, not by SGSM treatment. We can assert, for the first time, that SGSMs do not affect the neuronal differentiation of hNPCs while selectively decreasing endogenous Aβ42 levels in the same conditions. Our results suggest that our hNPC differentiation system can serve as a useful model to test the impact of GSIs and SGSMs on both endogenous Aβ levels and γ-secretase physiologic functions including endogenous Notch signaling.—D’Avanzo, C., Sliwinski, C., Wagner, S. L., Tanzi, R. E., Kim, D. Y., Kovacs, D. M. γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation. PMID:25903103

  15. In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors.

    Directory of Open Access Journals (Sweden)

    Matteo Vecellio

    Full Text Available Adult human cardiac mesenchymal-like stromal cells (CStC represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS in the presence of 5 µM all-trans Retinoic Acid (ATRA, 5 µM Phenyl Butyrate (PB, and 200 µM diethylenetriamine/nitric oxide (DETA/NO, to create a novel epigenetically active cocktail (EpiC. Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

  16. An immortal cell line to study the role of endogenous CFTR in electrolyte absorption.

    Science.gov (United States)

    Bell, C L; Quinton, P M

    1995-01-01

    The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Diabetes Accelerates Retinal neuronal cell Death in A Mouse Model of endogenous Hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Preethi S. Ganapathy

    2009-01-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase ( cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs +/– mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs +/– and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs +/– non-DB cbs +/– DB cbs +/+ ; non-DB cbs +/+ . One group of diabetic cbs +/– mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs +/– had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs +/– and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 μm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  18. Diabetes Accelerates Retinal Neuronal Cell Death In A Mouse Model of Endogenous Hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Preethi S. Ganapathy

    2009-07-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase (cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs+/- mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs+/- and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs+/-; non-DB cbs+/-; DB cbs+/+; non-DB cbs+/+. One group of diabetic cbs+/- mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs+/- had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs+/- and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  19. DNA precursor compartmentation in mammalian cells: distribution and rates of equilibration between nucleus and cytoplasm

    International Nuclear Information System (INIS)

    Leeds, J.M.

    1986-01-01

    A rapid nuclear isolation technique was adapted in order to examine the question of DNA precursor compartmentation in mammalian cells. By using this method a reproducible proportion of the cellular nucleotides remained associated with the isolated nuclei. Examination, at several different cell densities, of exponentially growing HeLa cells showed that the nuclei contained a constant but distinct proportion of each dNTP. The nuclear dATP and dTTP concentrations were equal at all densities examined even though the dTTP pool was 150% of the dATP whole-cell pool. The nuclear portion of the whole-cell pools was roughly equal to the volume occupied by the nucleus. The nuclear-cytoplasmic dNTP pool distribution did not change throughout the cell cycle of synchronized Chinese hamster ovary (CHO) cells. The rates at which either radiolabeled cytidine or deoxycytidine equilibrated with the nuclear and whole-cell dCTP pools of G1 and S phase CHO cells were compared. Experiments comparing the labeling kinetics of 3 H-thymidine in G1, S phase, and exponentially growing cells revealed that the S phase dTTP pool equilibrated with exogenously added thymidine faster than the G1 phase pool. The rate of equilibration in exponentially growing cells appeared to be a combination of that seen in G1 and S phases. A linear rate of 3 H-thymidine incorporation into DNA occurred at the same rate in S phase and exponentially growing cells

  20. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang [East Hospital, Tongji University School of Medicine, Shanghai (China); Dong, Chuanming [East Hospital, Tongji University School of Medicine, Shanghai (China); Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong (China); Sun, Chenxi; Ma, Rongjie; Yang, Danjing [East Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Hongwen, E-mail: hongwen_zhu@hotmail.com [Tianjin Hospital, Tianjin Academy of Integrative Medicine, Tianjin (China); Xu, Jun, E-mail: xunymc2000@yahoo.com [East Hospital, Tongji University School of Medicine, Shanghai (China)

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  1. Measuring telomere length for the early detection of precursor lesions of esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Lin, Shih-Wen; Wang, Guo-Qing; Wei, Wen-Qiang; Lu, Ning; Taylor, Philip R; Qiao, You-Lin; Dawsey, Sanford M; Abnet, Christian C; Freedman, Neal D; Murphy, Gwen; Risques, Rosana; Prunkard, Donna; Rabinovitch, Peter; Pan, Qin-Jing; Roth, Mark J

    2013-01-01

    Esophageal cancer is the sixth leading cause of cancer death worldwide; current early detection screening tests are inadequate. Esophageal balloon cytology successfully retrieves exfoliated and scraped superficial esophageal epithelial cells, but cytologic reading of these cells has poor sensitivity and specificity for detecting esophageal squamous dysplasia (ESD), the precursor lesion of esophageal squamous cell carcinoma (ESCC). Measuring telomere length, a marker for chromosomal instability, may improve the utility of balloon cytology for detecting ESD and early ESCC. We examined balloon cytology specimens from 89 asymptomatic cases of ESD (37 low-grade and 52 high-grade) and 92 age- and sex-matched normal controls from an esophageal cancer early detection screening study. All subjects also underwent endoscopy and biopsy, and ESD was diagnosed histopathologically. DNA was extracted from the balloon cytology cells, and telomere length was measured by quantitative PCR. A receiver operating characteristic (ROC) curve was plotted for telomere length as a diagnostic marker for high-grade dysplasia. Telomere lengths were comparable among the low- and high-grade dysplasia cases and controls, with means of 0.96, 0.96, and 0.92, respectively. The area under the ROC curve was 0.55 for telomere length as a diagnostic marker for high-grade dysplasia. Further adjustment for subject characteristics, including sex, age, smoking, drinking, hypertension, and body mass index did not improve the use of telomere length as a marker for ESD. Telomere length of esophageal balloon cytology cells was not associated with ESCC precursor lesions. Therefore, telomere length shows little promise as an early detection marker for ESCC in esophageal balloon samples

  2. Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

    Science.gov (United States)

    Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

    2017-02-01

    Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    Science.gov (United States)

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-06-01

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 118: 1531-1546, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep

    Science.gov (United States)

    Lee, Chang H.; Rodeo, Scott A.; Fortier, Lisa Ann; Lu, Chuanyong; Erisken, Cevat

    2015-01-01

    Regeneration of complex tissues, such as kidney, liver, and cartilage, continues to be a scientific and translational challenge. Survival of ex vivo cultured, transplanted cells in tissue grafts is among one of the key barriers. Meniscus is a complex tissue consisting of collagen fibers and proteoglycans with gradient phenotypes of fibrocartilage and functions to provide congruence of the knee joint, without which the patient is likely to develop arthritis. Endogenous stem/progenitor cells regenerated the knee meniscus upon spatially released human connective tissue growth factor (CTGF) and transforming growth factor–β3 (TGFβ3) from a three-dimensional (3D)–printed biomaterial, enabling functional knee recovery. Sequentially applied CTGF and TGFβ3 were necessary and sufficient to propel mesenchymal stem/progenitor cells, as a heterogeneous population or as single-cell progenies, into fibrochondrocytes that concurrently synthesized procollagens I and IIα. When released from microchannels of 3D–printed, human meniscus scaffolds, CTGF and TGFβ3 induced endogenous stem/progenitor cells to differentiate and synthesize zone-specific type I and II collagens. We then replaced sheep meniscus with anatomically correct, 3D–printed scaffolds that incorporated spatially delivered CTGF and TGFβ3. Endogenous cells regenerated the meniscus with zone-specific matrix phenotypes: primarily type I collagen in the outer zone, and type II collagen in the inner zone, reminiscent of the native meniscus. Spatiotemporally delivered CTGF and TGFβ3 also restored inhomogeneous mechanical properties in the regenerated sheep meniscus. Survival and directed differentiation of endogenous cells in a tissue defect may have implications in the regeneration of complex (heterogeneous) tissues and organs. PMID:25504882

  5. In Situ Passivation for Efficient PbS Quantum Dot Solar Cells by Precursor Engineering.

    Science.gov (United States)

    Wang, Yongjie; Lu, Kunyuan; Han, Lu; Liu, Zeke; Shi, Guozheng; Fang, Honghua; Chen, Si; Wu, Tian; Yang, Fan; Gu, Mengfan; Zhou, Sijie; Ling, Xufeng; Tang, Xun; Zheng, Jiawei; Loi, Maria Antonietta; Ma, Wanli

    2018-04-01

    Current efforts on lead sulfide quantum dot (PbS QD) solar cells are mostly paid to the device architecture engineering and postsynthetic surface modification, while very rare work regarding the optimization of PbS synthesis is reported. Here, PbS QDs are successfully synthesized using PbO and PbAc 2  · 3H 2 O as the lead sources. QD solar cells based on PbAc-PbS have demonstrated a high power conversion efficiency (PCE) of 10.82% (and independently certificated values of 10.62%), which is significantly higher than the PCE of 9.39% for PbO-PbS QD based ones. For the first time, systematic investigations are carried out on the effect of lead precursor engineering on the device performance. It is revealed that acetate can act as an efficient capping ligands together with oleic acid, providing better surface coverage and replace some of the harmful hydroxyl (OH) ligands during the synthesis. Then the acetate on the surface can be exchanged by iodide and lead to desired passivation. This work demonstrates that the precursor engineering has great potential in performance improvement. It is also pointed out that the initial synthesis is an often neglected but critical stage and has abundant room for optimization to further improve the quality of the resultant QDs, leading to breakthrough efficiency. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Directory of Open Access Journals (Sweden)

    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

  7. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Science.gov (United States)

    Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

    2015-01-01

    MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions.

  8. Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

    Directory of Open Access Journals (Sweden)

    Jessberger Sebastian

    2006-11-01

    Full Text Available Abstract Background In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. Results We found that (1 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2 the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3 positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. Conclusion These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.

  9. Native and enzymatically modified wheat (Triticum aestivum L.) endogenous lipids in bread making: a focus on gas cell stabilization mechanisms.

    Science.gov (United States)

    Gerits, Lien R; Pareyt, Bram; Masure, Hanne G; Delcour, Jan A

    2015-04-01

    Lipopan F and Lecitase Ultra lipases were used in straight dough bread making to study how wheat lipids affect bread loaf volume (LV) and crumb structure setting. Lipase effects on LV were dose and dough piece weight dependent. The bread quality improving mechanisms exerted by endogenous lipids were studied in terms of gluten network strengthening, which indirectly stabilizes gas cells, and in terms of direct interfacial gas cell stabilization. Unlike diacetyl tartaric esters of mono- and diacylglycerols (DATEM, used as control), lipase use did not impact dough extensibility. The effect on dough extensibility was therefore related to its lipid composition at the start of mixing. Both lipases and DATEM strongly increase the levels of polar lipids in dough liquor and their availability for and potential accumulation at gas cell interfaces. Lipases form lysolipids that emulsify other lipids. We speculate that DATEM competes with (endogenous) polar lipids for interacting with gluten proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Pulsed DC Electric Field-Induced Differentiation of Cortical Neural Precursor Cells.

    Directory of Open Access Journals (Sweden)

    Hui-Fang Chang

    Full Text Available We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz. The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders.

  11. Pulsed DC Electric Field-Induced Differentiation of Cortical Neural Precursor Cells.

    Science.gov (United States)

    Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K; Cheng, Ji-Yen

    2016-01-01

    We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders.

  12. Pathologic bladder microenvironment attenuates smooth muscle differentiation of skin derived precursor cells: implications for tissue regeneration.

    Directory of Open Access Journals (Sweden)

    Cornelia Tolg

    Full Text Available Smooth muscle cell containing organs (bladder, heart, blood vessels are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.

  13. Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt

    Directory of Open Access Journals (Sweden)

    Yanfang Zong

    2015-01-01

    Full Text Available Background. The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC apoptosis. Methods. Cultured human umbilical vein endothelial cells (HUVECs were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE, cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc. Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL methods. Results. High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt. Conclusion. H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

  14. Radiosensitizing Effects of Ectopic miR-101 on Non-Small-Cell Lung Cancer Cells Depend on the Endogenous miR-101 Level

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Susie; Wang Hongyan; Ng, Wooi Loon; Curran, Walter J. [Department of Radiation Oncology, School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, GA (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, GA (United States)

    2011-12-01

    Purpose: Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101. Methods and Materials: Fourteeen non-small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription-polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein-lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model. Results: The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice. Conclusions: A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.

  15. Radiosensitizing Effects of Ectopic miR-101 on Non–Small-Cell Lung Cancer Cells Depend on the Endogenous miR-101 Level

    International Nuclear Information System (INIS)

    Chen, Susie; Wang Hongyan; Ng, Wooi Loon; Curran, Walter J.; Wang Ya

    2011-01-01

    Purpose: Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101. Methods and Materials: Fourteeen non–small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription–polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein–lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model. Results: The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice. Conclusions: A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.

  16. Mobilization of endothelial precursor cells: systemic vascular response to musculoskeletal trauma.

    LENUS (Irish Health Repository)

    Laing, A J

    2012-02-03

    Postnatal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells (EPC) migrate, differentiate, and incorporate into the nacent endothelium contributing to physiological and pathological neovascularization, has stimulated much interest. Its contribution to tumor nonvascularization, wound healing, and revascularization associated with skeletal and cardiac muscles ischaemia is established. We evaluated the mobilization of EPCs in response to musculoskeletal trauma. Blood from patients (n = 15) following AO type 42a1 closed diaphyseal tibial fractures was analyzed for CD34 and AC133 cell surface marker expression. Immunomagnetically enriched CD34+ mononuclear cell (MNC(CD34+)) populations were cultured and examined for phenotypic and functional vascular endothelial differentiation. Circulating MNC(CD34+) levels increased sevenfold by day 3 postinjury. Circulating MNC(AC133+) increased 2.5-fold. Enriched MNC(CD34+) populations from day 3 samples in culture exhibited cell cluster formation with sprouting spindles. These cells bound UEA-1 and incorporated fluorescent DiI-Ac-LDL intracellularily. Our findings suggest a systemic provascular response is initiated in response to musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of fracture healing.

  17. Alignment of muscle precursor cells on the vertical edges of thick carbon nanotube films

    Energy Technology Data Exchange (ETDEWEB)

    Holt, Ian, E-mail: ian.holt@rjah.nhs.uk [Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, Shropshire SY10 7AG (United Kingdom); Institute for Science and Technology in Medicine, Keele University, Keele, Staffordshire ST5 5BG (United Kingdom); Gestmann, Ingo, E-mail: Ingo.Gestmann@fei.com [FEI Europe B.V., Achtseweg Noord 5, 5651 Eindhoven (Netherlands); Wright, Andrew C., E-mail: a.wright@glyndwr.ac.uk [Advanced Materials Research Laboratory, Glyndwr University, Plas Coch, Mold Rd, Wrexham LL11 2AW (United Kingdom)

    2013-10-15

    The development of scaffolds and templates is an essential aspect of tissue engineering. We show that thick (> 0.5 mm) vertically aligned carbon nanotube films, made by chemical vapour deposition, can be used as biocompatible substrates for the directional alignment of mouse muscle cells where the cells grow on the exposed sides of the films. Ultra high resolution scanning electron microscopy reveals that the films themselves consist mostly of small diameter (10 nm) multi-wall carbon nanotubes of wavy morphology with some single wall carbon nanotubes. Our findings show that for this alignment to occur the nanotubes must be in pristine condition. Mechanical wiping of the films to create directional alignment is detrimental to directional bioactivity. Larger areas for study have been formed from a composite of multiply stacked narrow strips of nanotubes wipe-transferred onto elastomer supports. These composite substrates appear to show a useful degree of alignment of the cells. Highlights: • Highly oriented muscle precursor cells grown on edges of carbon nanotube pads • Mechanical treatment of nanotube pads highly deleterious to cell growth on edges • Larger areas created from wipe-transfer of narrow strips of nanotubes onto elastomer supports • Very high resolution SEM reveals clues to aligned cell growth.

  18. The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

    Science.gov (United States)

    Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

    2011-12-01

    We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Alignment of muscle precursor cells on the vertical edges of thick carbon nanotube films

    International Nuclear Information System (INIS)

    Holt, Ian; Gestmann, Ingo; Wright, Andrew C.

    2013-01-01

    The development of scaffolds and templates is an essential aspect of tissue engineering. We show that thick (> 0.5 mm) vertically aligned carbon nanotube films, made by chemical vapour deposition, can be used as biocompatible substrates for the directional alignment of mouse muscle cells where the cells grow on the exposed sides of the films. Ultra high resolution scanning electron microscopy reveals that the films themselves consist mostly of small diameter (10 nm) multi-wall carbon nanotubes of wavy morphology with some single wall carbon nanotubes. Our findings show that for this alignment to occur the nanotubes must be in pristine condition. Mechanical wiping of the films to create directional alignment is detrimental to directional bioactivity. Larger areas for study have been formed from a composite of multiply stacked narrow strips of nanotubes wipe-transferred onto elastomer supports. These composite substrates appear to show a useful degree of alignment of the cells. Highlights: • Highly oriented muscle precursor cells grown on edges of carbon nanotube pads • Mechanical treatment of nanotube pads highly deleterious to cell growth on edges • Larger areas created from wipe-transfer of narrow strips of nanotubes onto elastomer supports • Very high resolution SEM reveals clues to aligned cell growth

  20. Transcriptional Modulation of Human Endogenous Retroviruses in Primary CD4+ T Cells Following Vorinostat Treatment

    Directory of Open Access Journals (Sweden)

    Cory H. White

    2018-04-01

    Full Text Available The greatest obstacle to a cure for HIV is the provirus that integrates into the genome of the infected cell and persists despite antiretroviral therapy. A “shock and kill” approach has been proposed as a strategy for an HIV cure whereby drugs and compounds referred to as latency-reversing agents (LRAs are used to “shock” the silent provirus into active replication to permit “killing” by virus-induced pathology or immune recognition. The LRA most utilized to date in clinical trials has been the histone deacetylase (HDAC inhibitor—vorinostat. Potentially, pathological off-target effects of vorinostat may result from the activation of human endogenous retroviruses (HERVs, which share common ancestry with exogenous retroviruses including HIV. To explore the effects of HDAC inhibition on HERV transcription, an unbiased pharmacogenomics approach (total RNA-Seq was used to evaluate HERV expression following the exposure of primary CD4+ T cells to a high dose of vorinostat. Over 2,000 individual HERV elements were found to be significantly modulated by vorinostat, whereby elements belonging to the ERVL family (e.g., LTR16C and LTR33 were predominantly downregulated, in contrast to LTR12 elements of the HERV-9 family, which exhibited the greatest signal, with the upregulation of 140 distinct elements. The modulation of three different LTR12 elements by vorinostat was confirmed by droplet digital PCR along a dose–response curve. The monitoring of LTR12 expression during clinical trials with vorinostat may be indicated to assess the impact of this HERV on the human genome and host immunity.

  1. Bone marrow origin of decidual cell precursors in the pseudopregnant mouse uterus

    International Nuclear Information System (INIS)

    Kearns, M.; Lala, P.K.

    1982-01-01

    Decidual cells are considered to be the endproduct of a hormonally induced transformation of endometrial stromal cells of the uterus. However, the source of these precursors remains unknown. This study of evaluated the possibility of their bone marrow origin by an examination of the H-2 phenotype of decidual cells in pseudopregnant bone marrow chimeras. These chimeras were produced by repopulating lethally irradiated CBA/J female (H-2k) mice with bone marrow from (CBA/J x C57BL/6J) F1 female (H-2kb) mice. Pseudopregnancy was produced with a hormonal regimen followed by an oil-induced decidual stimulus. Chimerism was evaluated radioautographically by an identification of the donor-specific Kb phenotype on cells with an immunolabeling technique with monospecific anti-H-2 serum followed by radioiodinated protein A. The extent of chimerism as indicated by the degree of Kb labeling on decidual cells as well as macrophages contained within the decidual nodules was quantitatively compared with that seen on splenic lymphocytes. Fair to good chimerism, as reflected by labeling for the donor-specific marker (Kb), was seen on splenic lymphocytes and macrophages within the decidual nodules in 6 out of 11 animals. A similar level of chimerism was detected on decidual cells in all but one of these six, in which case this was low. One animal showed low chimerism in the spleen but good chimerism on the decidual cells. The remaining four mice were nonchimeric for all three cell types. These results indicate that decidual cells and macrophages appearing within the decidual nodules of pseudopregnant mice are ultimate descendants of bone marrow cells

  2. Pericytes and endothelial precursor cells: cellular interactions and contributions to malignancy.

    Science.gov (United States)

    Bagley, Rebecca G; Weber, William; Rouleau, Cecile; Teicher, Beverly A

    2005-11-01

    Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.

  3. Effect of heat absorbing powder addition on cell morphology of porous titanium composite manufactured by reactive precursor method

    International Nuclear Information System (INIS)

    Kobashi, Makoto; Kamiya, Yoshinori; Kanetake, Naoyuki

    2012-01-01

    Open-cell structured porous titanium/ceramics composite was synthesized by a reactive precursor method using titanium and boron carbide (B 4 C) as reactant powders. Pore morphology was controlled by adding heat absorbing powder (titanium diboride: TiB 2 ) in the Ti+B 4 C blended powder. The effects of molar blending ratio of titanium and B 4 C and the amount of heat absorbing powder addition on the cell morphology (either open or closed) were investigated. Fine and homogeneous open-cell structure was achieved by adding appropriate amount of heat absorbing agent powder (>15 vol%), and the relative density of the specimen after the reaction became closer to that of the precursor by increasing TiB 2 volume fraction. When the volume fraction of TiB 2 addition was 20%, the open-cell fraction was maintained as 1.0 regardless of the relative density of the precursor.

  4. Knock-Down of Endogenous Bornavirus-Like Nucleoprotein 1 Inhibits Cell Growth and Induces Apoptosis in Human Oligodendroglia Cells

    Directory of Open Access Journals (Sweden)

    Peng He

    2016-03-01

    Full Text Available Endogenous bornavirus-like nucleoprotein elements (EBLNs have been discovered in the genomes of various animals including humans, whose functions have been seldom studied. To explore the biological functions of human EBLNs, we constructed a lentiviral vector expressing a short-hairpin RNA against human EBLN1, which successfully inhibited EBLN1 expression by above 80% in infected human oligodendroglia cells (OL cells. We found that EBLN1 silencing suppressed cell proliferation, induced G2/M phase arrest, and promoted apoptosis in OL cells. Gene expression profiling demonstrated that 1067 genes were up-regulated, and 2004 were down-regulated after EBLN1 silencing. The top 10 most upregulated genes were PI3, RND3, BLZF1, SOD2, EPGN, SBSN, INSIG1, OSMR, CREB3L2, and MSMO1, and the top 10 most-downregulated genes were KRTAP2-4, FLRT2, DIDO1, FAT4, ESCO2, ZNF804A, SUV420H1, ZC3H4, YAE1D1, and NCOA5. Pathway analysis revealed that these differentially expressed genes were mainly involved in pathways related to the cell cycle, the mitogen-activated protein kinase pathway, p53 signaling, and apoptosis. The gene expression profiles were validated by using quantitative reverse transcription polymerase chain reaction (RT-PCR for detecting these 20 most-changed genes. Three genes closely related to glioma, RND3, OSMR, and CREB3L2, were significantly upregulated and might be the key factors in EBLN1 regulating the proliferation and apoptosis of OL cells. This study provides evidence that EBLN1 plays a key role in regulating cell life and death, thereby opening several avenues of investigation regarding EBLN1 in the future.

  5. Radiation-induced apoptosis of neural precursors cell cultures: early modulation of the response mediated by reactive oxygen and nitrogen species (ROS/RNS)

    Energy Technology Data Exchange (ETDEWEB)

    Gisone, P.; Dubner, D.; Robello, E.; Michelin, S.; Perez, M. R.

    2004-07-01

    Apoptosis, the typical mode of radiation-induced cell death in developing Central Nervous System (CNS), is closely related with the oxidative status. Enhanced radiation-induced generation of ROS/RNS has been observed after exposures to low radiation doses leading to cellular amplification of signal transduction and further molecular and cellular radiation-responses. Moreover Nitric oxide (NO) and hydroxyl radical are implicated in dopaminergic neurotoxicity in different parading. This study is an attempt to address the participation of radiation-induced free radicals production, the contribution of endogenous NO generation, and the excitonic pathway, in the radiation-induced apoptosis of neural cortical precursors. Cortical cells obtained from at 17 gestational day (gd) were irradiated with doses from 0,2 Gy to 2 Gy at a dose-rate of 0.3 Gy/m. A significant decrease of Luminol-dependent Chemiluminescence was evident 30 m after irradiation reaching basal levels at 120 m follow for a tendency to increasing values Incubations with Superoxide Dismatuse (SOD) decreased significantly the chemiluminescence in irradiated samples NO content estimated by measuring the stable products NO{sub 2} and NO{sub 3} released to the culture medium in the same period, has shown a time-dependent accumulation from 1 h post-irradiation. the apoptosis, determined 24 h post-irradiation by flow cytometry, morphology and DNA fragmentation revealed a dose-effect relationship with significant differences from 0.4 Gy. The samples pre-treated with 10 mM of N-acetyl cysteine (NAC) a precursor of intracellular GSH synthesis, shown a significant decrease of the apoptosis. Apoptosis was significantly increased in irradiated cells after inhibition of nitric oxide synthase (NOS) byL-NAME. We conclude that ROS/RNS play a pivotal role in the early signaling pathways leading to a radiation-induced cell death. (Author) 40 refs.

  6. Radiation-induced apoptosis of neural precursors cell cultures: early modulation of the response mediated by reactive oxygen and nitrogen species (ROS/RNS)

    International Nuclear Information System (INIS)

    Gisone, P.; Dubner, D.; Robello, E.; Michelin, S.; Perez, M. R.

    2004-01-01

    Apoptosis, the typical mode of radiation-induced cell death in developing Central Nervous System (CNS), is closely related with the oxidative status. Enhanced radiation-induced generation of ROS/RNS has been observed after exposures to low radiation doses leading to cellular amplification of signal transduction and further molecular and cellular radiation-responses. Moreover Nitric oxide (NO) and hydroxyl radical are implicated in dopaminergic neurotoxicity in different parading. This study is an attempt to address the participation of radiation-induced free radicals production, the contribution of endogenous NO generation, and the excitonic pathway, in the radiation-induced apoptosis of neural cortical precursors. Cortical cells obtained from at 17 gestational day (gd) were irradiated with doses from 0,2 Gy to 2 Gy at a dose-rate of 0.3 Gy/m. A significant decrease of Luminol-dependent Chemiluminescence was evident 30 m after irradiation reaching basal levels at 120 m follow for a tendency to increasing values Incubations with Superoxide Dismatuse (SOD) decreased significantly the chemiluminescence in irradiated samples NO content estimated by measuring the stable products NO 2 and NO 3 released to the culture medium in the same period, has shown a time-dependent accumulation from 1 h post-irradiation. the apoptosis, determined 24 h post-irradiation by flow cytometry, morphology and DNA fragmentation revealed a dose-effect relationship with significant differences from 0.4 Gy. The samples pre-treated with 10 mM of N-acetyl cysteine (NAC) a precursor of intracellular GSH synthesis, shown a significant decrease of the apoptosis. Apoptosis was significantly increased in irradiated cells after inhibition of nitric oxide synthase (NOS) byL-NAME. We conclude that ROS/RNS play a pivotal role in the early signaling pathways leading to a radiation-induced cell death. (Author) 40 refs

  7. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Directory of Open Access Journals (Sweden)

    Ming-Wai Poon

    Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

  8. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Science.gov (United States)

    Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

    2015-01-01

    A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

  9. Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines.

    Directory of Open Access Journals (Sweden)

    Troels R Petersen

    2011-03-01

    Full Text Available Transfer of antigen between antigen-presenting cells (APCs is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs, were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs, suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.

  10. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

    Directory of Open Access Journals (Sweden)

    Andrea Buchfellner

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP1 is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa, termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

  11. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

    Science.gov (United States)

    Buchfellner, Andrea; Yurlova, Larisa; Nüske, Stefan; Scholz, Armin M; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E; Drexler, Guido A; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A; Rothbauer, Ulrich

    2016-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

  12. A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

    Science.gov (United States)

    Nüske, Stefan; Scholz, Armin M.; Bogner, Jacqueline; Ruf, Benjamin; Zolghadr, Kourosh; Drexler, Sophie E.; Drexler, Guido A.; Girst, Stefanie; Greubel, Christoph; Reindl, Judith; Siebenwirth, Christian; Romer, Tina; Friedl, Anna A.; Rothbauer, Ulrich

    2016-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair. PMID:26950694

  13. Endogenous protection derived from activin A/Smads transduction loop stimulated via ischemic injury in PC12 cells.

    Science.gov (United States)

    Mang, Jing; Mei, Chun-Li; Wang, Jiao-Qi; Li, Zong-Shu; Chu, Ting-Ting; He, Jin-Ting; Xu, Zhong-Xin

    2013-10-17

    Activin A (ActA), a member of transforming growth factor-beta (TGF-b) super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD) injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate ischemic injury on neurons in vitro. Cells were pre-treated by monoclonal antibody against activin receptor type IIA (ActRII-Ab). We found that ActRII-Ab augments ischemic injury in PC12 cells. Further, the extracellular secretion of ActA as well as phosphorylation of smad3 in PC12 cells was also up-regulated by OGD, but suppressed by ActRII-Ab. Taken together, our results show that ActRII-Ab may augment ischemic injury via blocking of transmembrane signal transduction of ActA, which confirmed the existence of endogenous neuroprotective effects derived from the ActA/Smads pathway. ActRIIA plays an important role in transferring neuronal protective signals inside. It is highly possible that ActA transmembrance signaling is a part of the positive feed-back loop for extracellular ActA secretion.

  14. Endogenous Protection Derived from Activin A/Smads Transduction Loop Stimulated via Ischemic Injury in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Zhong-Xin Xu

    2013-10-01

    Full Text Available Activin A (ActA, a member of transforming growth factor-beta (TGF-b super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate ischemic injury on neurons in vitro. Cells were pre-treated by monoclonal antibody against activin receptor type IIA (ActRII-Ab. We found that ActRII-Ab augments ischemic injury in PC12 cells. Further, the extracellular secretion of ActA as well as phosphorylation of smad3 in PC12 cells was also up-regulated by OGD, but suppressed by ActRII-Ab. Taken together, our results show that ActRII-Ab may augment ischemic injury via blocking of transmembrane signal transduction of ActA, which confirmed the existence of endogenous neuroprotective effects derived from the ActA/Smads pathway. ActRIIA plays an important role in transferring neuronal protective signals inside. It is highly possible that ActA transmembrance signaling is a part of the positive feed-back loop for extracellular ActA secretion.

  15. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

    Energy Technology Data Exchange (ETDEWEB)

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K., E-mail: mcloo001@tc.umn.edu

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

  16. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

    International Nuclear Information System (INIS)

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K.

    2011-01-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin -/- (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of

  17. Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Michael A. [Oxiage Cosmeceutical Research Institute, Virginia (United States)

    2013-05-15

    Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD{sub 50}) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative

  18. Effects of Ionizing Radiation and Glutathione Precursor on Antioxidant Enzyme and Cell Survival in Yeast

    International Nuclear Information System (INIS)

    Kim, Jinkyu; Roh, Changhyun; Ryu, Taeho; Park, Jiyoung; Nili, Michael A.

    2013-01-01

    Cells react to such an induced oxidative stress through scavenging the generated reactive oxygen species to reduce oxidative damage. Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase are immediately triggered for reactive oxygen species. N-acetyl-L-cysteine (NAC), a precursor of glutathione, is one of the antioxidants. The effect of NAC as an antioxidant and/or a cell rescue agent was investigated in the present study. Glutathione (GSH) is the most abundant intracellular thiol, which involves in antioxidant defense via direct interaction with ROS or via activities of detoxication enzymes like glutathione peroxidases (GPx). NAC flowed in the cell is converted to cysteine by deacetylation, that is supplied to the depleted GSH by oxidative stress. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity such as GPx and alkaline phosphatase. Cell growth and survivorship and transcriptional level of glutathione gene are analyzed in two yeast strains exposed to combined treatment of NAC with gamma-rays. The effect of NAC on cell growth was measured during 72 hours. The cell growth was hampered by higher concentrations of NAC at stationary phase. NAC, however, didn't affect the cell division at the exponential phase. The survival of the cells decreased with radiation dose. The cell viability of the strain W303-1A was reduced significantly at the low dose (10 and 30 Gy). By comparison, the strain W303-1A was more sensitive to radiation with having a half lethal dose (LD 50 ) of about 20 Gy. The quantitative RT-PCR analysis showed that the transcriptional expression of antioxidant enzyme gene GPX1 increased after irradiation while the expression of the gene decreased by the combined treatment of NAC with 100 Gy radiation. The present study shows that NAC can directly scavenge ROS against oxidative stress in vivo. In conclusion, NAC can prevent radiation-induced oxidative stress by

  19. Reduced Osteogenesis of Human Osteogenic Precursors' Cells Cultured in the Random Positioning Machine

    Science.gov (United States)

    Gershovich, J. G.; Buravkova, L. B.

    2008-06-01

    Recent studies have shown that simulated microgravity (SMG) results in altered proliferation and differentiation not only osteoblasts but also affects on osteogenic capacity of mesenchymal stem cells (MSCs) from various sources. For present study we used system that simulates effects of microgravity produced by the Random Positioning Machine (RPM). Cultured MCSs from human bone marrow and human osteoblasts (OBs) were exposed to SMG at RPM for 10-40 days. Induced osteogenesis of these progenitor cells was compared with the appropriate static (1g) and dynamic (horizontal shaker) controls. Clinorotated OBs and MSCs showed proliferation rate lower than static and dynamic control groups of cells in the early terms of SMG. Significant reduction of ALP activity was detected after 10 days of clinorotation of MSCs. There was no such dramatic difference in ALP activity of MSCs derived cells between SMG and control groups after 20 days of clinorotation but the expression of ALP was still reduced. However, virtually no matrix mineralization was found in OBs cultured under SMG conditions in the presence of differentiation stimuli. The similar effect was observed when we assayed matrix calcification of MSCs derived cultures. Thus, our results confirm low gravity mediated reduction of osteogenesis of different osteogenic precursors' cells and can clarify the mechanisms of bone loss during spaceflight.

  20. Human Neural Precursor Cells Promote Neurologic Recovery in a Viral Model of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Lu Chen

    2014-06-01

    Full Text Available Using a viral model of the demyelinating disease multiple sclerosis (MS, we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4+CD25+FOXP3+ regulatory T cells (Tregs within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments.

  1. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    International Nuclear Information System (INIS)

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos; Cerpa, Waldo; Cambiazo, Veronica; Inestrosa, Nibaldo C.; Gonzalez, Mauricio

    2009-01-01

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu 2+ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu 2+ reduction and 64 Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu 2+ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu 2+ ions. Moreover, wild-type cells exposed to both Cu 2+ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu 2+ reductase activity and increased 64 Cu uptake. We conclude that Cu 2+ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

  2. Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Pierre-François Lesault

    Full Text Available Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i myoblast survival by limiting their massive death, ii myoblast expansion within the tissue and iii myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

  3. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence

  4. Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells.

    Directory of Open Access Journals (Sweden)

    Min-Jeong Kang

    2016-10-01

    Full Text Available Hippocalcin (Hpca is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs. When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3, neurotrophin-4/5 (NT-4/5 and brain-derived neurotrophic factor (BDNF, together with the proneural basic helix loop helix (bHLH transcription factors neuroD and neurogenin 1 (ngn1, increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP, an astrocyte marker, and in dendrite outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, neuroD and ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727, and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201, suggesting that STAT3 (Ser727 activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and dendrite outgrowth in HNPCs.

  5. Cetuximab modified collagen scaffold directs neurogenesis of injury-activated endogenous neural stem cells for acute spinal cord injury repair.

    Science.gov (United States)

    Li, Xing; Zhao, Yannan; Cheng, Shixiang; Han, Sufang; Shu, Muya; Chen, Bing; Chen, Xuyi; Tang, Fengwu; Wang, Nuo; Tu, Yue; Wang, Bin; Xiao, Zhifeng; Zhang, Sai; Dai, Jianwu

    2017-08-01

    Studies have shown that endogenous neural stem cells (NSCs) activated by spinal cord injury (SCI) primarily generate astrocytes to form glial scar. The NSCs do not differentiate into neurons because of the adverse microenvironment. In this study, we defined the activation timeline of endogenous NSCs in rats with severe SCI. These injury-activated NSCs then migrated into the lesion site. Cetuximab, an EGFR signaling antagonist, significantly increased neurogenesis in the lesion site. Meanwhile, implanting cetuximab modified linear ordered collagen scaffolds (LOCS) into SCI lesion sites in dogs resulted in neuronal regeneration, including neuronal differentiation, maturation, myelination, and synapse formation. The neuronal regeneration eventually led to a significant locomotion recovery. Furthermore, LOCS implantation could also greatly decrease chondroitin sulfate proteoglycan (CSPG) deposition at the lesion site. These findings suggest that endogenous neurogenesis following acute complete SCI is achievable in species ranging from rodents to large animals via functional scaffold implantation. LOCS-based Cetuximab delivery system has a promising therapeutic effect on activating endogenous neurogenesis, reducing CSPGs deposition and improving motor function recovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Changing paradigms in cranio-facial regeneration: current and new strategies for the activation of endogenous stem cells

    Directory of Open Access Journals (Sweden)

    Luigi eMele

    2016-02-01

    Full Text Available Craniofacial area represent a unique district of human body characterized by a very high complexity of tissues, innervation and vascularization, and being deputed to many fundamental function such as eating, speech, expression of emotions, delivery of sensations such as taste, sight and earing. For this reasons, tissue loss in this area following trauma or for example oncologic resection, have a tremendous impact on patients’ quality of life. In the last 20 years regenerative medicine has emerged as one of the most promising approach to solve problem related to trauma, tissue loss, organ failure etc. One of the most powerful tools to be used for tissue regeneration is represented by stem cells, which have been successfully implanted in different tissue/organs with exciting results. Nevertheless both autologous and allogeneic stem cell transplantation raise many practical and ethical concerns that make this approach very difficult to apply in clinical practice. For this reason different cell free approaches have been developed aiming to the mobilization, recruitment and activation of endogenous stem cells into the injury site avoiding exogenous cells implant but instead stimulating patients’ own stem cells to repair the lesion. To this aim many strategies have been used including functionalized bioscaffold, controlled release of stem cell chemoattractants, growth factors, BMPs, Platelet–Rich-Plasma and other new strategies such as ultrasound wave and laser are just being proposed. Here we review all the current and new strategies used for activation and mobilization of endogenous stem cells in the regeneration of craniofacial tissue.

  7. Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells.

    Science.gov (United States)

    Ashouri, Judith F; Weiss, Arthur

    2017-01-15

    Distinguishing true Ag-stimulated lymphocytes from bystanders activated by the inflammatory milieu has been difficult. Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling in murine T cells and human thymocytes. Nur77-GFP transgenes serve as specific TCR and BCR signaling reporters in murine transgenic models. In this study, we demonstrate that endogenous Nur77 protein expression can serve as a reporter of TCR and BCR specific signaling in human PBMCs. Nur77 protein amounts were assessed by immunofluorescence and flow cytometry in T and B cells isolated from human PBMCs obtained from healthy donors that had been stimulated by their respective Ag receptors. We demonstrate that endogenous Nur77 is a more specific reporter of Ag-specific signaling events than the commonly used CD69 activation marker in both human T and B cells. This is reflective of the disparity in signaling pathways that regulate the expression of Nur77 and CD69. Assessing endogenous Nur77 protein expression has great potential to identify Ag-activated lymphocytes in human disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  8. Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei.

    Science.gov (United States)

    Pérez-Moreno, Guiomar; Sealey-Cardona, Marco; Rodrigues-Poveda, Carlos; Gelb, Michael H; Ruiz-Pérez, Luis Miguel; Castillo-Acosta, Víctor; Urbina, Julio A; González-Pacanowska, Dolores

    2012-10-01

    Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  9. Allogeneic mesenchymal precursor cells (MPCs): an innovative approach to treating advanced heart failure.

    Science.gov (United States)

    Westerdahl, Daniel E; Chang, David H; Hamilton, Michele A; Nakamura, Mamoo; Henry, Timothy D

    2016-09-01

    Over 37 million people worldwide are living with Heart Failure (HF). Advancements in medical therapy have improved mortality primarily by slowing the progression of left ventricular dysfunction and debilitating symptoms. Ultimately, heart transplantation, durable mechanical circulatory support (MCS), or palliative care are the only options for patients with end-stage HF. Regenerative therapies offer an innovative approach, focused on reversing myocardial dysfunction and restoring healthy myocardial tissue. Initial clinical trials using autologous (self-donated) bone marrow mononuclear cells (BMMCs) demonstrated excellent safety, but only modest efficacy. Challenges with autologous stem cells include reduced quality and efficacy with increased patient age. The use of allogeneic mesenchymal precursor cells (MPCs) offers an "off the shelf" therapy, with consistent potency and less variability than autologous cells. Preclinical and initial clinical trials with allogeneic MPCs have been encouraging, providing the support for a large ongoing Phase III trial-DREAM-HF. We provide a comprehensive review of preclinical and clinical data supporting MPCs as a therapeutic option for HF patients. The current data suggest allogeneic MPCs are a promising therapy for HF patients. The results of DREAM-HF will determine whether allogeneic MPCs can decrease major adverse clinical events (MACE) in advanced HF patients.

  10. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    Science.gov (United States)

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  11. Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications.

    Science.gov (United States)

    Sahoo, Sambit; Ang, Lay-Teng; Cho-Hong Goh, James; Toh, Siew-Lok

    2010-02-01

    Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications. 2009 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  12. JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia

    Science.gov (United States)

    de Goffau-Nobel, Willemieke; Hoogkamer, Alex Q.; Boer, Judith M.; Boeree, Aurélie; van de Ven, Cesca; Koudijs, Marco J.; Besselink, Nicolle J.M.; de Groot-Kruseman, Hester A.; Zwaan, Christian Michel; Horstmann, Martin A.; Pieters, Rob; den Boer, Monique L.

    2017-01-01

    JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy. JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL. PMID:29163799

  13. NFIL3 Orchestrates the Emergence of Common Helper Innate Lymphoid Cell Precursors

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2015-03-01

    Full Text Available Innate lymphoid cells (ILCs are a family of effectors that originate from a common innate lymphoid cell progenitor. However, the transcriptional program that sets the identity of the ILC lineage remains elusive. Here, we show that NFIL3 is a critical regulator of the common helper-like innate lymphoid cell progenitor (CHILP. Cell-intrinsic Nfil3 ablation led to variably impaired development of fetal and adult ILC subsets. Conditional gene targeting demonstrated that NFIL3 exerted its function prior to ILC subset commitment. Accordingly, NFIL3 ablation resulted in loss of ID2+ CHILP and PLZF+ ILC progenitors. Nfil3 expression in lymphoid progenitors was under the control of the mesenchyme-derived hematopoietin IL-7, and NFIL3 exerted its function via direct Id2 regulation in the CHILP. Moreover, ectopic Id2 expression in Nfil3-null precursors rescued defective ILC lineage development in vivo. Our data establish NFIL3 as a key regulator of common helper-like ILC progenitors as they emerge during early lymphopoiesis.

  14. Langerhans cell precursors acquire RANK/CD265 in prenatal human skin.

    Science.gov (United States)

    Schöppl, Alice; Botta, Albert; Prior, Marion; Akgün, Johnnie; Schuster, Christopher; Elbe-Bürger, Adelheid

    2015-01-01

    The skin is the first barrier against foreign pathogens and the prenatal formation of a strong network of various innate and adaptive cells is required to protect the newborn from perinatal infections. While many studies about the immune system in healthy and diseased adult human skin exist, our knowledge about the cutaneous prenatal/developing immune system and especially about the phenotype and function of antigen-presenting cells such as epidermal Langerhans cells (LCs) in human skin is still scarce. It has been shown previously that LCs in healthy adult human skin express receptor activator of NF-κB (RANK), an important molecule prolonging their survival. In this study, we investigated at which developmental stage LCs acquire this important molecule. Immunofluorescence double-labeling of cryostat sections revealed that LC precursors in prenatal human skin either do not yet [10-11 weeks of estimated gestational age (EGA)] or only faintly (13-15 weeks EGA) express RANK. LCs express RANK at levels comparable to adult LCs by the end of the second trimester. Comparable with adult skin, dermal antigen-presenting cells at no gestational age express this marker. These findings indicate that epidermal leukocytes gradually acquire RANK during gestation - a phenomenon previously observed also for other markers on LCs in prenatal human skin. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  15. Differential proliferation rhythm of neural progenitor and oligodendrocyte precursor cells in the young adult hippocampus.

    Directory of Open Access Journals (Sweden)

    Yoko Matsumoto

    Full Text Available Oligodendrocyte precursor cells (OPCs are a unique type of glial cells that function as oligodendrocyte progenitors while constantly proliferating in the normal condition from rodents to humans. However, the functional roles they play in the adult brain are largely unknown. In this study, we focus on the manner of OPC proliferation in the hippocampus of the young adult mice. Here we report that there are oscillatory dynamics in OPC proliferation that differ from neurogenesis in the subgranular zone (SGZ; the former showed S-phase and M-phase peaks in the resting and active periods, respectively, while the latter only exhibited M-phase peak in the active period. There is coincidence between different modes of proliferation and expression of cyclin proteins that are crucial for cell cycle; cyclin D1 is expressed in OPCs, while cyclin D2 is observed in neural stem cells. Similar to neurogenesis, the proliferation of hippocampal OPCs was enhanced by voluntary exercise that leads to an increase in neuronal activity in the hippocampus. These data suggest an intriguing control of OPC proliferation in the hippocampus.

  16. Efficiency Enhancement of Perovskite Solar Cells by Pumping Away the Solvent of Precursor Film Before Annealing.

    Science.gov (United States)

    Xu, Qing-Yang; Yuan, Da-Xing; Mu, Hao-Ran; Igbari, Femi; Bao, Qiaoliang; Liao, Liang-Sheng

    2016-12-01

    A new approach to improve the quality of MAPbI3 - x Cl x perovskite film was demonstrated. It involves annealing the precursor film after pumping away the solvent, which can decrease the influence of solvent evaporation rate for the growth of the MAPbI3 - x Cl x perovskite film. The resulting film showed improved morphology, stronger absorption, fewer crystal defects, and smaller charge transfer resistance. The corresponding device demonstrated enhanced performance when compared with a reference device. The averaged value of power conversion efficiency increased from 10.61 to 12.56 %, and a champion efficiency of 14.0 % was achieved. This work paves a new way to improve the efficiency of perovskite solar cells.

  17. Vitreous humor and albumin augment the proliferation of cultured retinal precursor cells

    DEFF Research Database (Denmark)

    Yang, Jing; Klassen, Henry; Pries, Mette

    2008-01-01

    concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined...... Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment...... proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors. (c) 2008 Wiley-Liss, Inc....

  18. Intrinsic differences in adipocyte precursor cells from different white fat depots

    DEFF Research Database (Denmark)

    Macotela, Yazmín; Emanuelli, Brice; Mori, Marcelo A

    2012-01-01

    Obesity and body fat distribution are important risk factors for the development of type 2 diabetes and metabolic syndrome. Evidence has accumulated that this risk is related to intrinsic differences in behavior of adipocytes in different fat depots. In the current study, we demonstrate...... that adipocyte precursor cells (APCs) isolated from visceral and subcutaneous white adipose depots of mice have distinct patterns of gene expression, differentiation potential, and response to environmental and genetic influences. APCs derived from subcutaneous fat differentiate well in the presence of classical...... induction cocktail, whereas those from visceral fat differentiate poorly but can be induced to differentiate by addition of bone morphogenetic protein (BMP)-2 or BMP-4. This difference correlates with major differences in gene expression signature between subcutaneous and visceral APCs. The number of APCs...

  19. Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.

    Science.gov (United States)

    Prodon, François; Chenevert, Janet; Hébras, Céline; Dumollard, Rémi; Faure, Emmanuel; Gonzalez-Garcia, Jose; Nishida, Hiroki; Sardet, Christian; McDougall, Alex

    2010-06-01

    Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

  20. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

    Science.gov (United States)

    Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

    2008-08-01

    Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

  1. Endogenously produced Indian Hedgehog regulates TGFβ-driven chondrogenesis of human bone marrow stromal/stem cells.

    Science.gov (United States)

    Handorf, Andrew M; Chamberlain, Connie S; Li, Wan-Ju

    2015-04-15

    Human bone marrow stromal/stem cells (hBMSCs) have an inherent tendency to undergo hypertrophy when induced into the chondrogenic lineage using transforming growth factor-beta 1 (TGFβ) in vitro, reminiscent of what occurs during endochondral ossification. Surprisingly, Indian Hedgehog (IHH) has received little attention for its role during hBMSC chondrogenesis despite being considered a master regulator of endochondral ossification. In this study, we investigated the role that endogenously produced IHH plays during hBMSC chondrogenesis. We began by analyzing the expression of IHH throughout differentiation using quantitative polymerase chain reaction and found that IHH expression was upregulated dramatically upon chondrogenic induction and peaked from days 9 to 12 of differentiation, which coincided with a concomitant increase in the expression of chondrogenesis- and hypertrophy-related markers, suggesting a potential role for endogenously produced IHH in driving hBMSC chondrogenesis. More importantly, pharmacological inhibition of Hedgehog signaling with cyclopamine or knockdown of IHH almost completely blocked TGFβ1-induced chondrogenesis in hBMSCs, demonstrating that endogenously produced IHH is necessary for hBMSC chondrogenesis. Furthermore, overexpression of IHH was sufficient to drive chondrogenic differentiation, even when TGFβ signaling was inhibited. Finally, stimulation with TGFβ1 induced a significant and sustained upregulation of IHH expression within 3 h that preceded an upregulation in all cartilage-related genes analyzed, and knockdown of IHH blocked the effects of TGFβ1 entirely, suggesting that the effects of TGFβ1 are being mediated through endogenously produced IHH. Together, our findings demonstrate that endogenously produced IHH is playing a critical role in regulating hBMSC chondrogenesis.

  2. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

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    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Nakagawa, Shin [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takamura, Naoki [Pharmaceutical Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka (Japan); Kato, Akiko [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takebayashi, Minoru [Department of Psychiatry, National Hospital Organization Kure Medical Center, Kure (Japan); Hisaoka-Nakashima, Kazue [Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima (Japan); Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan)

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  3. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    International Nuclear Information System (INIS)

    Boku, Shuken; Nakagawa, Shin; Takamura, Naoki; Kato, Akiko; Takebayashi, Minoru; Hisaoka-Nakashima, Kazue; Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro

    2013-01-01

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis

  4. Kinetics and comparison of δ-aminolevulinic-acid-induced endogenous protoporphyrin-IX in single cell by steady state and multiphoton fluorescence imaging

    Science.gov (United States)

    Ganesan, Singaravelu; Elangovan, Masilamani; Periasamy, Ammasi

    2001-04-01

    Photodynamic Therapy has emerged as a new modality in the treatment of various nonmalignant and malignant diseases. It involves the systemic administration of tumor specific photo-sensitizers with the subsequent application of visible light. This combination causes the generation of cytotoxic species, which damage sensitive targets, producing cell injury and tumor destruction. Although, photofrin is the only photosensitizer currently approved for PDT and tumor detection, its concomitant cutaneous photosensitization poses a significant problem. Hence, δ-aminoleuvulinic acid (δ-ALA) a precursor for the endogenous production of Protoporphyrin IX, through heme biosynthesis pathway, has gained significant importance in the Photodynamic Therapy. Though δ-ALA is present naturally in the cells, exogenous δ-ALA helps to synthesis more of PpIX in the tumor cells, as the fast growing tumor cells take up the administered δ-ALA more than the normal cells. Based on these facts, many invasive studies have been reported on the kinetics of δ-ALA at cellular level by chemical extraction of PpIX from the cells. In the present study we have studied the kinetics of δ-ALA induced PpIX fluorescence from Hela cells by perchloric/Methanol extraction method. However, the amount of PpIX synthesized in the cells at different point of incubation time by noninvasive methods has not been reported. Hence we have also used a noninvasive technique of measuring the kinetics δ-ALA induced PPIX fluorescence from Hela, an epithelial cell derived from human cervical cancer by both single photon (steady state) and multi photon excitation. From the studies it is observed that the δ-ALA induced PpIX is more at 2 hours incubation time for 2 mM of δ-ALA concentration. Further, it is observed that with steady state fluorescence imaging method, the excitation light itself cause the Photodynamic damage, due to the prolonged exposure of the cells than in multi photon excitation, leading to the rounding

  5. Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

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    Ilaria Roato

    Full Text Available BACKGROUND: Phenylketonuria (PKU is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF and receptor activator of NFκB ligand (RANKL. Flow cytometry was utilized to analyze osteoclast precursors (OCPs and T cell phenotype. Tumour necrosis factor α (TNF-α, RANKL and osteoprotegerin (OPG were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS. This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis

  6. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells.

    Science.gov (United States)

    Konigsberg, M; Pérez, V I; Ríos, C; Liu, Y; Lee, S; Shi, Y; Van Remmen, H

    2013-01-01

    In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1-5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3-4 m) and old (29-30 m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

  7. Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells

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    Brill Monika S

    2011-04-01

    Full Text Available Abstract Background While the diversity and spatio-temporal origin of olfactory bulb (OB GABAergic interneurons has been studied in detail, much less is known about the subtypes of glutamatergic OB interneurons. Results We studied the temporal generation and diversity of Neurog2-positive precursor progeny using an inducible genetic fate mapping approach. We show that all subtypes of glutamatergic neurons derive from Neurog2 positive progenitors during development of the OB. Projection neurons, that is, mitral and tufted cells, are produced at early embryonic stages, while a heterogeneous population of glutamatergic juxtaglomerular neurons are generated at later embryonic as well as at perinatal stages. While most juxtaglomerular neurons express the T-Box protein Tbr2, those generated later also express Tbr1. Based on morphological features, these juxtaglomerular cells can be identified as tufted interneurons and short axon cells, respectively. Finally, targeted electroporation experiments provide evidence that while the majority of OB glutamatergic neurons are generated from intrabulbar progenitors, a small portion of them originate from extrabulbar regions at perinatal ages. Conclusions We provide the first comprehensive analysis of the temporal and spatial generation of OB glutamatergic neurons and identify distinct populations of juxtaglomerular interneurons that differ in their antigenic properties and time of origin.

  8. Mobilization of endogenous bone marrow derived endothelial progenitor cells and therapeutic potential of parathyroid hormone after ischemic stroke in mice.

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    Li-Li Wang

    Full Text Available Stroke is a major neurovascular disorder threatening human life and health. Very limited clinical treatments are currently available for stroke patients. Stem cell transplantation has shown promising potential as a regenerative treatment after ischemic stroke. The present investigation explores a new concept of mobilizing endogenous stem cells/progenitor cells from the bone marrow using a parathyroid hormone (PTH therapy after ischemic stroke in adult mice. PTH 1-34 (80 µg/kg, i.p. was administered 1 hour after focal ischemia and then daily for 6 consecutive days. After 6 days of PTH treatment, there was a significant increase in bone marrow derived CD-34/Fetal liver kinase-1 (Flk-1 positive endothelial progenitor cells (EPCs in the peripheral blood. PTH treatment significantly increased the expression of trophic/regenerative factors including VEGF, SDF-1, BDNF and Tie-1 in the brain peri-infarct region. Angiogenesis, assessed by co-labeled Glut-1 and BrdU vessels, was significantly increased in PTH-treated ischemic brain compared to vehicle controls. PTH treatment also promoted neuroblast migration from the subventricular zone (SVZ and increased the number of newly formed neurons in the peri-infarct cortex. PTH-treated mice showed significantly better sensorimotor functional recovery compared to stroke controls. Our data suggests that PTH therapy improves endogenous repair mechanisms after ischemic stroke with functional benefits. Mobilizing endogenous bone marrow-derived stem cells/progenitor cells using PTH and other mobilizers appears an effective and feasible regenerative treatment after ischemic stroke.

  9. POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

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    M. M. França

    2015-12-01

    Full Text Available During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G and fasciculata/reticularis (F/R cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

  10. Induction and characterization of a replication competent cervid endogenous gammaretrovirus (CrERV) from mule deer cells.

    Science.gov (United States)

    Fábryová, Helena; Hron, Tomáš; Kabíčková, Hana; Poss, Mary; Elleder, Daniel

    2015-11-01

    Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation.

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    Erine H Budi

    2011-05-01

    Full Text Available The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.

  12. Acute modulation of cytokine gene expression in bovine peripheral blood mononuclear cells (PBMCs) by endogenous cortisol

    Science.gov (United States)

    Cortisol suppresses many aspects of immune function. However, recent publications suggest acute cortisol exposure may actually enhance immune function (Dhabhar. 2009. Neuroimmunomod. 16:300). The objective of this study was to determine the influence of acute increases in endogenous cortisol on expr...

  13. Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

    Science.gov (United States)

    Sourial, Mary; Doering, Laurie C

    2017-07-01

    The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  14. Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers

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    Kuzin Alexander

    2008-08-01

    Full Text Available Abstract Background The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers. Results Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS. Conclusion The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.

  15. Endogenous antipyretics.

    Science.gov (United States)

    Roth, Joachim

    2006-09-01

    The febrile increase of body temperature is regarded as a component of the complex host response to infection or inflammation that accompanies the activation of the immune system. Late phases of fever appear mediated by pro-inflammatory cytokines called endogenous pyrogens. The rise of body temperature is beneficial because it accelerates several components of the activated immune system. To prevent an excessive and dangerous rise of body temperature the febrile response is controlled, limited in strength and duration, and sometimes even prevented by the actions of endogenous antipyretic substances liberated systemically or within the brain during fever. In most cases the antipyretic effects are achieved by an inhibitory influence on the formation or action of endogenous pyrogens, or by effects on neuronal thermoregulatory circuits that are activated during fever. Endogenous antipyretic substances include steroid hormones, neuropeptides, cytokines and other molecules. It is the purpose of this review to consider the current state in the research on endogenous antipyretic systems.

  16. In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

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    F Di Meglio

    2009-08-01

    Full Text Available The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (a-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively inclines toward an increase in both a-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

  17. Human HMGA2 protein overexpressed in mice induces precursor T-cell lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Efanov, A; Zanesi, N; Coppola, V; Nuovo, G; Bolon, B; Wernicle-Jameson, D; Lagana, A; Hansjuerg, A; Pichiorri, F; Croce, C M

    2014-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a neoplasia of thymocytes characterized by the rapid accumulation of the precursors of T lymphocytes. HMGA2 (high-mobility group AT-hook 2) gene expression is extremely low in normal adult tissues, but it is overexpressed in many tumors. To identify the biological function of HMGA2, we generated transgenic mice carrying the human HMGA2 gene under control of the V H promoter/Eμ enhancer. Approximately 90% of Eμ-HMGA2 transgenic mice became visibly sick between 4 and 8 months due to the onset and progression of a T-ALL-like disease. Characteristic features included severe alopecia (30% of mice); enlarged lymph nodes and spleen; and profound immunological abnormalities (altered cytokine levels, hypoimmunoglobulinemia) leading to reduced immune responsiveness. Immunophenotyping showed accumulation of CD5+CD4+, CD5+CD8+ or CD5+CD8+CD4+ T-cell populations in the spleens and bone marrow of sick animals. These findings show that HMGA2-driven leukemia in mice closely resembles spontaneous human T-ALL, indicating that HMGA2 transgenic mice should serve as an important model for investigating basic mechanisms and potential new therapies of relevance to human T-ALL

  18. Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

    Science.gov (United States)

    Wang, Yaping; Cheng, Xiaoxin; He, Qian; Zheng, Yiyan; Kim, Dong H; Whittemore, Scott R; Cao, Qilin L

    2011-04-20

    Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

  19. Herpes simplex virus dances with amyloid precursor protein while exiting the cell.

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    Shi-Bin Cheng

    2011-03-01

    Full Text Available Herpes simplex type 1 (HSV1 replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP, a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/-6.7% and travel together with APP inside living cells (81.1+/-28.9%. This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/-0.2 to 0.3+/-0.1 µm/s and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile and velocity (from 0.3+/-0.1 to 0.4+/-0.1 µm/s of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic

  20. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice.

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    Liu, Yawei; Teige, Anna; Mondoc, Emma; Ibrahim, Saleh; Holmdahl, Rikard; Issazadeh-Navikas, Shohreh

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.

  1. Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets.

    Science.gov (United States)

    Crider, J Y; Xu, S X; Sharif, N A

    2001-01-01

    The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.

  2. Endogenous neural stem cells in central canal of adult rats acquired limited ability to differentiate into neurons following mild spinal cord injury

    Science.gov (United States)

    Liu, Yuan; Tan, Botao; Wang, Li; Long, Zaiyun; Li, Yingyu; Liao, Weihong; Wu, Yamin

    2015-01-01

    Endogenous neural stem cells in central canal of adult mammalian spinal cord exhibit stem cell properties following injury. In the present study, the endogenous neural stem cells were labeled with Dil to track the differentiation of cells after mild spinal cord injury (SCI). Compared with 1 and 14 days post mild injury, the number of endogenous neural stem cells significantly increased at the injured site of spinal cord on 3 and 7 days post-injury. Dil-labeled βIII-tublin and GFAP expressing cells could be detected on 7 days post-injury, which indicated that the endogenous neural stem cells in central canal of spinal cord differentiated into different type of neural cells, but there were more differentiated astrocytes than the neurons after injury. Furthermore, after injury the expression of inhibitory Notch1 and Hes1 mRNA began to increase at 6 hours and was evident at 12 and 24 hours, which maintained high levels up to 7 days post-injury. These results indicated that a mild SCI in rat is sufficient to induce endogenous neural stem cells proliferation and differentiation. However, the ability to differentiate into neurons is limited, which may be, at least in part, due to high expression of inhibitory Notch1 and Hes1 genes after injury. PMID:26097566

  3. Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

    Science.gov (United States)

    Wang, Xiaochen; Gong, Yuemin; Ema, Hideo

    2016-07-22

    Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult. Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses. The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs. Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.

  4. Bone morphogenetic protein signaling and olig1/2 interact to regulate the differentiation and maturation of adult oligodendrocyte precursor cells.

    Science.gov (United States)

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R; Cao, Qilin

    2007-12-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

  5. On the action of radioprotective agents on the endogenous serotonin content and radiosensitivity of isolated Ehrlich ascites tumour and E. coli B. cells

    International Nuclear Information System (INIS)

    Goncharenko, E.N.; Gorskaya, T.G.; Gusareva, Eh.V.; Konstantinova, M.M.; Panyushkina, N.V.; AN SSSR, Moscow. Inst. Biologii Razvitiya)

    1976-01-01

    At a higher radioresistance level attained by incubation of isolated Ehrlich ascites tumour and E. coli B. cells with MEA or noradrenaline, the endogenous serotonin content of these cells was found to increase. The extent of radioresistance increase and the serotonin content of the cells were interrelated, i.e. washing the protector off cells decreased both the radioresistance and the content of endogenous serotonin. It is concluded that radioresistance of cells is connected with the content of biologically active substances (serotonin) that possess radioprotective action

  6. The endogenous regenerative capacity of the damaged newborn brain: boosting neurogenesis with mesenchymal stem cell treatment

    OpenAIRE

    Donega, Vanessa; van Velthoven, Cindy TJ; Nijboer, Cora H; Kavelaars, Annemieke; Heijnen, Cobi J

    2013-01-01

    Neurogenesis continues throughout adulthood. The neurogenic capacity of the brain increases after injury by, e.g., hypoxia–ischemia. However, it is well known that in many cases brain damage does not resolve spontaneously, indicating that the endogenous regenerative capacity of the brain is insufficient. Neonatal encephalopathy leads to high mortality rates and long-term neurologic deficits in babies worldwide. Therefore, there is an urgent need to develop more efficient therapeutic strategie...

  7. Isolation of Mature (Peritoneum-Derived Mast Cells and Immature (Bone Marrow-Derived Mast Cell Precursors from Mice.

    Directory of Open Access Journals (Sweden)

    Steffen K Meurer

    Full Text Available Mast cells (MCs are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC or mucosal (MMC type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs and immature MC precursors from the bone marrow (BM. The latter are differentiated in vitro to yield BM-derived MCs (BMMC. These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

  8. Human fetuses do not register chromosome damage inflicted by radiation exposure in lymphoid precursor cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Ohtaki, K.; Kodama, Y.; Nakano, M.; Itoh, M.; Awa, A.A.; Cologne, J.B.

    2003-01-01

    Human fetuses are generally thought to be highly sensitive to radiation exposure since diagnostic, low-dose X rays (5-50 mSv) have been suggested to increase the risk of childhood leukemia by about 50%. In contrast, animal studies generally did not demonstrate a high radiosensitivity of fetuses and the underlying causes for the discrepancy are not understood. Here, we examined atomic-bomb survivors exposed in utero for translocation frequency in blood lymphocytes at 40 years of age. Contrary to our expectation of higher radiosensitivity in fetuses than in adults, the frequency did not increase with dose except for a small, but statistically significant increase (<1%) at doses below 0.1 Sv. Although an upward convex, humped dose response has been observed in other instances, the peak usually lies at doses above a few Gy, and few examples are known showing the peak response at such low doses. We interpret the results as indicating that fetal lymphoid and/or their precursor cells are sensitive to elimination through apoptosis when damaged. Our results provide a biological basis to resolve the long-standing controversy that substantial risk of childhood leukemia is implicated in human fetuses exposed to low-dose diagnostic X rays whereas animal studies composed mainly of exposures to higher doses consistently fail to confirm it

  9. PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation.

    Science.gov (United States)

    Baldassarro, Vito A; Marchesini, Alessandra; Giardino, Luciana; Calzà, Laura

    2017-07-01

    Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    International Nuclear Information System (INIS)

    Wilson, A.; Chen, W.-F.; Scollay, R.; Shortman, K.

    1982-01-01

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111 In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. (Auth.)

  11. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

  12. Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sun-Hyung Ha

    2016-04-01

    Full Text Available Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose polymerase, without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: β1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2 gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

  13. Honey can repairing damage of liver tissue due to protein energy malnutrition through induction of endogenous stem cells

    Directory of Open Access Journals (Sweden)

    R. Heru Prasetyo

    2017-06-01

    Full Text Available Aim: This study was to evaluate effect of honey in repairing damage of liver tissue due to energy protein malnutrition and in mobilization of endogenous stem cells. Materials and Methods: Male mice model of degenerative liver was obtained through food fasting but still have drinking water for 5 days. It caused energy protein malnutrition and damage of liver tissue. The administration of 50% (v/v honey was performed for 10 consecutive days, while the positive control group was fasted and not given honey and the negative control not fasted and without honey. Observations of regeneration the liver tissue based on histologically examination, observation of Hsp70 expression, and homing signal based on vascular endothelial growth factor-1 (VEGF-1 expression using immunohistochemistry technique. Observation on expression of CD34 and CD45 as the marker of auto mobilization of hematopoietic stem cells using flow cytometry technique. Results: There is regeneration of the liver tissue due to protein energy malnutrition, decrease of Hsp70 expression, increase of VEGF-1 expression, and high expression of CD34 and CD45. Conclusion: Honey can improve the liver tissue based on: (1 Mobilization of endogenous stem cells (CD34 and CD45; (2 Hsp70 and VEGF-1 expressions as regeneration marker of improvement, and (3 regeneration histologically of liver tissue.

  14. Honey can repairing damage of liver tissue due to protein energy malnutrition through induction of endogenous stem cells.

    Science.gov (United States)

    Prasetyo, R Heru; Hestianah, Eka Pramyrtha

    2017-06-01

    This study was to evaluate effect of honey in repairing damage of liver tissue due to energy protein malnutrition and in mobilization of endogenous stem cells. Male mice model of degenerative liver was obtained through food fasting but still have drinking water for 5 days. It caused energy protein malnutrition and damage of liver tissue. The administration of 50% (v/v) honey was performed for 10 consecutive days, while the positive control group was fasted and not given honey and the negative control not fasted and without honey. Observations of regeneration the liver tissue based on histologically examination, observation of Hsp70 expression, and homing signal based on vascular endothelial growth factor-1 (VEGF-1) expression using immunohistochemistry technique. Observation on expression of CD34 and CD45 as the marker of auto mobilization of hematopoietic stem cells using flow cytometry technique. There is regeneration of the liver tissue due to protein energy malnutrition, decrease of Hsp70 expression, increase of VEGF-1 expression, and high expression of CD34 and CD45. Honey can improve the liver tissue based on: (1) Mobilization of endogenous stem cells (CD34 and CD45); (2) Hsp70 and VEGF-1 expressions as regeneration marker of improvement, and (3) regeneration histologically of liver tissue.

  15. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

    Directory of Open Access Journals (Sweden)

    Isart Roca

    2015-01-01

    Full Text Available The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP made from induced pluripotent stem cells (iPSCs are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries.

  16. A transduced living hyaline cartilage graft releasing transgenic stromal cell-derived factor-1 inducing endogenous stem cell homing in vivo.

    Science.gov (United States)

    Zhang, Feng; Leong, Wenyan; Su, Kai; Fang, Yu; Wang, Dong-An

    2013-05-01

    Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4(+)(SDF-1 receptor) cell population, accompanied by upregulation of the CD34(+), CD44(+), and Sca-1(+) cell populations as well as a downregulation of the CD11b(+) cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.

  17. Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

    International Nuclear Information System (INIS)

    Winton, E.F.; Colenda, K.W.

    1987-01-01

    The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor cells in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors

  18. Activation of the aryl hydrocarbon receptor reduces the number of precursor and effector T cells, but preserves thymic CD4(+)CD25(+)Foxp3(+) regulatory T cells

    NARCIS (Netherlands)

    Schulz, V.J.; Smit, J.J.; Bol-Schoenmakers, M.; van Duursen, M.B.M.; van den Berg, M.; Pieters, R.H.H.

    2012-01-01

    Aryl hydrocarbon receptor (AhR) activation suppresses immune responses, including allergic sensitization, by increasing the percentage of regulatory (Treg) cells. Furthermore, AhR activation is known to affect thymic precursor T cells. However, the effect of AhR activation on intrathymic

  19. Effect of X-irradiation and vitamin C on DNA degradation and endogenous DNAase in embryonic chick lens cells

    International Nuclear Information System (INIS)

    Trevithick, J.R.; Chaudun, E.; Muel, A.S.; Courtois, Y.; Counis, M.F.

    1987-01-01

    The lens is an organ in which epithelial cells become elongated fibers. During this process, nuclei are transformed and the DNA is degraded. In previous studies, we described an autodigestion of the chromatin in isolated fiber nuclei but not in epithelial nuclei, but the level of DNAase activity was found to be identical in both epithelial and fiber nuclei of lenses at 11 days of development. In this study, we have investigated the possibility that x-irradiation might stimulate the nuclear endogenous activity responsible for chromatin breakdown or epithelial cells to a level comparable to that observed in fiber cells. We have observed that x-irradiation does not increase the nuclear epithelial DNAase activity. Conversely, vitamin C, suspected to prevent cataract formation by protecting DNA against free radical formation, has a damaging effect on the DNA of the lens of chick embryo in vitro. (author)

  20. EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

    Science.gov (United States)

    Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

    2010-01-01

    Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

  1. Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

    Science.gov (United States)

    Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

    2015-04-20

    During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Precursor T-cell acute lymphoblastic leukemia presenting with bone marrow necrosis: a case report

    Directory of Open Access Journals (Sweden)

    Khoshnaw Najmaddin SH

    2012-10-01

    Full Text Available Abstract Introduction Bone marrow necrosis is a clinicopathological condition diagnosed most often at postmortem examination, but it is also seen during the course of malignancy and is not always associated with a poor prognosis. The morphological features of bone marrow necrosis are disruption of the normal marrow architecture and necrosis of myeloid tissue and medullary stroma. Non-malignant conditions associated with bone marrow necrosis are sickle cell anemia, infections, drugs (sulfasalazine, interferon α, all-trans retinoic acid, granulocyte colony-stimulating factor and fludarabine, disseminated intravascular coagulation, antiphospholipid antibody syndrome and acute graft versus host diseases. The malignant causes are leukemia, lymphoma and metastatic carcinomas. Herein we report the case of a patient with precursor T-cell acute lymphoblastic leukemia and bone marrow necrosis at initial presentation. Case presentation A 10-year-old Kurdish boy was presented with generalized bone pain and fever of 1 month’s duration which was associated with sweating, easy fatigability, nose bleeding, breathlessness and severe weight loss. On examination, we observed pallor, tachypnea, tachycardia, low blood pressure, fever, petechial hemorrhage, ecchymoses, tortuous dilated veins over the chest and upper part of abdomen, multiple small cervical lymph node enlargements, mildly enlarged spleen, palpable liver and gross abdominal distention. Blood analysis revealed pancytopenia and elevated lactate dehydrogenase and erythrocyte sedimentation rate. Imaging results showed mediastinal widening on a planar chest X-ray and diffuse focal infiltration of the axial bone marrow on magnetic resonance imaging of the lumbosacral vertebrae. Bone marrow aspiration and biopsy examination showed extensive bone marrow necrosis. Immunophenotyping analysis of the bone marrow biopsy confirmed T-cell acute lymphoblastic leukemia, as CD3 and terminal deoxynucleotidyl

  3. Combination of exogenous cell transplantation and 5-HT4 receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model.

    Science.gov (United States)

    Yu, Hui; Zheng, Bai-Jun; Pan, Wei-Kang; Wang, Huai-Jie; Xie, Chong; Zhao, Yu-Ying; Chen, Xin-Lin; Liu, Yong; Gao, Ya

    2017-02-01

    Enteric neural crest-derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5-HT 4 receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5-HT 4 receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride-treated colons. At given days post-intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75 NTR and peripherin, respectively. eGFP-positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post-intervention, the number of peripherin-positive cells gradually increased in all groups. Significantly more peripherin-positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5-HT 4 receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. T cell dysfunction in the diabetes-prone BB rat. A role for thymic migrants that are not T cell precursors

    International Nuclear Information System (INIS)

    Georgiou, H.M.; Lagarde, A.C.; Bellgrau, D.

    1988-01-01

    Diabetes-prone BB (BB-DP) rats express several T cell dysfunctions which include poor proliferative and cytotoxic responses to alloantigen. The goal of this study was to determine the origin of these T cell dysfunctions. When BB-DP rats were thymectomized, T cell depleted, and transplanted with neonatal thymus tissue from diabetes-resistant and otherwise normal DA/BB F1 rats, the early restoration of T cell function proceeded normally on a cell-for-cell basis; i.e., peripheral T cells functioned like those from the thymus donor. Because the thymus in these experiments was subjected to gamma irradiation before transplantation and there was no evidence of F1 chimerism in the transplanted BB-DP rats, it appeared that the BB-DP T cell precursors could mature into normally functioning T cells if the maturation process occurred in a normal thymus. If the F1 thymus tissue was treated with dGua before transplantation, the T cells of these animals functioned poorly like those from untreated BB-DP rats. dGua poisons bone marrow-derived cells, including gamma radiation-resistant cells of the macrophage/dendritic cell lineages, while sparing the thymic epithelium. Therefore, the reversal of the T cell dysfunction depends on the presence in the F1 thymus of gamma radiation-resistant, dGua-sensitive F1 cells. Conversely, thymectomized and T cell-depleted F1 rats expressed T cell dysfunction when transplanted with gamma-irradiated BB thymus grafts. T cell responses were normal in animals transplanted with dGua-treated BB thymus grafts. With increasing time after thymus transplantation, T cells from all animals gradually expressed the functional phenotype of the bone marrow donor. Taken together these results suggest that BB-DP bone marrow-derived cells that are not T cell precursors influence the maturation environment in the thymus of otherwise normal BB-DP T cell precursors

  5. Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Shafarman, William N. [Univ. of Delaware, Newark, DE (United States)

    2015-10-12

    This project “Advanced Precursor Reaction Processing for Cu(InGa)(SeS)2 Solar Cells”, completed by the Institute of Energy Conversion (IEC) at the University of Delaware in collaboration with the Department of Chemical Engineering at the University of Florida, developed the fundamental understanding and technology to increase module efficiency and improve the manufacturability of Cu(InGa)(SeS)2 films using the precursor reaction approach currently being developed by a number of companies. Key results included: (1) development of a three-step H2Se/Ar/H2S reaction process to control Ga distribution through the film and minimizes back contact MoSe2 formation; (2) Ag-alloying to improve precursor homogeneity by avoiding In phase agglomeration, faster reaction and improved adhesion to allow wider reaction process window; (3) addition of Sb, Bi, and Te interlayers at the Mo/precursor junction to produce more uniform precursor morphology and improve adhesion with reduced void formation in reacted films; (4) a precursor structure containing Se and a reaction process to reduce processing time to 5 minutes and eliminate H2Se usage, thereby increasing throughput and reducing costs. All these results were supported by detailed characterization of the film growth, reaction pathways, thermodynamic assessment and device behavior.

  6. Scaffold-cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source.

    Science.gov (United States)

    Berner, A; Henkel, J; Woodruff, M A; Saifzadeh, S; Kirby, G; Zaiss, S; Gohlke, J; Reichert, J C; Nerlich, M; Schuetz, M A; Hutmacher, D W

    2017-07-01

    The properties of osteoblasts (OBs) isolated from the axial skeleton (tOBs) differ from OBs of the orofacial skeleton (mOBs) due to the different embryological origins of the bones. The aim of the study was to assess and compare the regenerative potential of allogenic bone marrow-derived mesenchymal progenitor cells with allogenic tOBs and allogenic mOBs in combination with a mPCL-TCP scaffold in critical-sized segmental bone defects in sheep tibiae. After 6 months, the tibiae were explanted and underwent biomechanical testing, micro-computed tomography (microCT) and histological and immunohistochemical analyses. Allogenic MPCs demonstrated a trend towards a better outcome in biomechanical testing and the mean values of newly formed bone. Biomechanical, microCT and histological analysis showed no significant differences in the bone regeneration potential of tOBs and mOBs in our in vitro study, as well as in the bone regeneration potential of different cell types in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

    Science.gov (United States)

    Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

    2009-05-01

    Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

  8. Polysialic acid modification of the synaptic cell adhesion molecule SynCAM 1 in human embryonic stem cell-derived oligodendrocyte precursor cells

    Directory of Open Access Journals (Sweden)

    Sebastian Werneburg

    2015-05-01

    Full Text Available Oligodendrocyte precursor cells (OPCs are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs.

  9. Case report of precursor B-cell lymphoblastic lymphoma presenting as syncope and cardiac mass in a nonimmunocompromised child.

    Science.gov (United States)

    Hahn, Barry; Rao, Sudha; Shah, Binita

    2007-08-01

    We report the case of a previously healthy, 10-year-old boy who presented to the emergency department with a syncopal episode. In the emergency department, the patient was diagnosed with a right atrial mass, later identified as a precursor B-cell lymphoblastic lymphoma (LL). Most causes of syncope in children are not life threatening. In most cases, it indicates a predisposition to vasovagal episodes. Lymphomas account for approximately 7% of malignancies among children younger than 20 years, are more common in white males and immunocompromised patients, and are predominantly tumors of T-cell origin. Children with non-Hodgkin lymphoma usually present with extranodal disease, most frequently involving the abdomen (31%), mediastinum (26%), or head and neck (29%). Our patient was unique in that he was a nonimmunocompromised, black boy, presenting with syncope in the setting of a large atrial mass identified as a precursor B-cell LL. To our knowledge, there are no reported cases of precursor B-cell LL presenting as syncope and a cardiac mass.

  10. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice

    DEFF Research Database (Denmark)

    Liu, Yawei; Teige, Anna; Mondoc, Emma

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition...... in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated...... with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells...

  11. Transplantation of Neural Precursor Cells Attenuates Chronic Immune Environment in Cervical Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Lennart Riemann

    2018-06-01

    Full Text Available Inflammation after traumatic spinal cord injury (SCI is non-resolving and thus still present in chronic injury stages. It plays a key role in the pathophysiology of SCI and has been associated with further neurodegeneration and development of neuropathic pain. Neural precursor cells (NPCs have been shown to reduce the acute and sub-acute inflammatory response after SCI. In the present study, we examined effects of NPC transplantation on the immune environment in chronic stages of SCI. SCI was induced in rats by clip-compression of the cervical spinal cord at the level C6-C7. NPCs were transplanted 10 days post-injury. The functional outcome was assessed weekly for 8 weeks using the Basso, Beattie, and Bresnahan scale, the CatWalk system, and the grid walk test. Afterwards, the rats were sacrificed, and spinal cord sections were examined for M1/M2 macrophages, T lymphocytes, astrogliosis, and apoptosis using immunofluorescence staining. Rats treated with NPCs had compared to the control group significantly fewer pro-inflammatory M1 macrophages and reduced immunodensity for inducible nitric oxide synthase (iNOS, their marker enzyme. Anti-inflammatory M2 macrophages were rarely present 8 weeks after the SCI. In this model, the sub-acute transplantation of NPCs did not support survival and proliferation of M2 macrophages. Post-traumatic apoptosis, however, was significantly reduced in the NPC group, which might be explained by the altered microenvironment following NPC transplantation. Corresponding to these findings, reactive astrogliosis was significantly reduced in NPC-transplanted animals. Furthermore, we could observe a trend toward smaller cavity sizes and functional improvement following NPC transplantation. Our data suggest that transplantation of NPCs following SCI might attenuate inflammation even in chronic injury stages. This might prevent further neurodegeneration and could also set a stage for improved neuroregeneration after SCI.

  12. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

    OpenAIRE

    Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

    2012-01-01

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

  13. Dentin and dental pulp regeneration by the patient’s endogenous cells

    OpenAIRE

    KIM, SAHNG G.; ZHENG, YING; ZHOU, JIAN; CHEN, MO; EMBREE, MILDRED C.; SONG, KAREN; JIANG, NAN; MAO, JEREMY J.

    2013-01-01

    The goal of regenerative endodontics is to restore the functions of the dental pulp–dentin complex. Two approaches are being applied toward dental pulp–dentin regeneration: cell transplantation and cell homing. The majority of previous approaches are based on cell transplantation by delivering ex vivo cultivated cells toward dental pulp or dentin regeneration. Many hurdles limit the clinical translation of cell transplantation such as the difficulty of acquiring and isolating viable cells, un...

  14. Endogenous interferon-β-inducible gene expression and interferon-β-treatment are associated with reduced T cell responses to myelin basic protein in multiple sclerosis

    DEFF Research Database (Denmark)

    Börnsen, Lars; Christensen, Jeppe Romme; Ratzer, Rikke

    2015-01-01

    Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for......-induced CD4+ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.......Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used...... for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels...

  15. Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors.

    Science.gov (United States)

    Costello, Alan; Lao, Nga; Clynes, Martin; Barron, Niall

    2017-01-01

    MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotype in the biotech industry. The use of constitutively expressed "miRNA sponge" vectors in which multiple, tandem miRNA binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for down regulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA-sponge that is in theory expressed only in the presence of an inducer.

  16. CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.

    Science.gov (United States)

    Baillat, David; Russell, William K; Wagner, Eric J

    2016-12-01

    The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

    Science.gov (United States)

    Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

    2013-01-01

    In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

  18. Morphological and immunological criteria of minimal residual disease detection in children with B-cell precursors acute lymphoblastic leukemia

    Science.gov (United States)

    Beznos, O. A.; Grivtsova, L. Yu; Popa, A. V.; Shervashidze, M. A.; Serebtyakova, I. N.; Tupitsyn, N. N.; Selchuk, V. U.; Grebennikova, O. P.; Titova, G. V.

    2018-01-01

    One of the key factors of prognosis and risk stratification in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is minimal residual disease (MRD). Identification of MRD on the day 15th is one of the most significant in prognosis of the disease. We compared data of a morphological and flow cytometry results of assessment of a bone marrow (BM) at the day 15th of induction chemotherapy in children with BCP-ALL.

  19. Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.

    Science.gov (United States)

    Chen, B P; Madrigal, A; Parham, P

    1990-09-01

    Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.

  20. L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

    Science.gov (United States)

    Huang, Hongbiao; Liu, Ningning; Guo, Haiping; Liao, Siyan; Li, Xiaofen; Yang, Changshan; Liu, Shouting; Song, Wenbin; Liu, Chunjiao; Guan, Lixia; Li, Bing; Xu, Li; Zhang, Change; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2012-01-01

    L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

  1. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  2. Survival and Functionality of Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes in a Nonhuman Primate Model for Multiple Sclerosis

    NARCIS (Netherlands)

    Thiruvalluvan, Arun; Czepiel, Marcin; Kap, Yolanda A.; Mantingh-Otter, Ietje; Vainchtein, Ilia; Kuipers, Jeroen; Bijlard, Marjolein; Baron, Wia; Giepmans, Ben; Brueck, Wolfgang; 'T Hart, Bert A.; Boddeke, Erik; Copray, Sjef

    2016-01-01

    : Fast remyelination by endogenous oligodendrocyte precursor cells (OPCs) is essential to prevent axonal and subsequent retrograde neuronal degeneration in demyelinating lesions in multiple sclerosis (MS). In chronic lesions, however, the remyelination capacity of OPCs becomes insufficient. Cell

  3. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells.

    Science.gov (United States)

    Gunthorpe, M J; Smith, G D; Davis, J B; Randall, A D

    2001-08-01

    Acid-sensing ion channels (ASICs) are a new and expanding family of proton-gated cation (Na+/Ca2+) channels that are widely expressed in sensory neurons and the central nervous system. Their distribution suggests that they may play a critical role in the sensation of the pain that accompanies tissue acidosis and may also be important in detecting the subtle pH variations that occur during neuronal signalling. Here, using whole-cell patch-clamp electrophysiology and reverse transcriptase-polymerase chain reaction (RT-PCR), we show that HEK293 cells, a commonly used cell line for the expression and characterisation of many ion channels, functionally express an endogenous proton-gated conductance attributable to the activity of human ASIC1a. These data therefore represent the first functional characterisation of hASIC1 and have many important implications for the use of HEK293 cells as a host cell system for the study of ASICs, vanilloid receptor-1 and any other proton-gated channel. With this latter point in mind we have devised a simple desensitisation strategy to selectively remove the contribution of hASIC1a from proton-gated currents recorded from HEK293 cells expressing vanilloid receptor-1.

  4. Induction of Apoptosis in Human Multiple Myeloma Cell Lines by Ebselen via Enhancing the Endogenous Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    2014-01-01

    Full Text Available Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

  5. Induction of apoptosis in human multiple myeloma cell lines by ebselen via enhancing the endogenous reactive oxygen species production.

    Science.gov (United States)

    Zhang, Liang; Zhou, Liwei; Du, Jia; Li, Mengxia; Qian, Chengyuan; Cheng, Yi; Peng, Yang; Xie, Jiayin; Wang, Dong

    2014-01-01

    Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM) cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS) accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC) completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

  6. IKZF1 DELETIONS ARE INDEPENDENT PROGNOSTIC FACTOR IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    G. A. Tsaur

    2016-01-01

    Full Text Available We assessed the prognostic significance of IKZF1 gene deletions in 141 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL  on Russian multicenter trial in pediatric clinics of Ekaterinburg and Orenburg. IKZF1 deletions were estimated by multiplex ligation-dependent probe amplification. IKZF1 deletions were revealed in 15 (10.6 % patients. IKZF1 deletions were associated with age older than 10 years (p = 0.007, initial white blood cell count higher than 30 × 109/l (p = 0.003, t(9;22(q34.q11 (p = 0.003 and delayed blast clearance: М3 status of bone marrow at day 15 of remission induction (p = 0.003, lack of hematological remission at day 36 (p < 0.001 and high levels of minimal residual disease at days 15, 36 and 85 (p = 0.014; p < 0.001; p = 0.001 correspondingly. Patients with IKZF1 deletions had significantly lower event-free survival (EFS (0.30 ± 0.15 vs 0.89 ± 0.03; p < 0.001 and overall survival (OS (0.44 ± 0.19 vs 0.93 ± 0.02; p < 0.001, while cumulative incidence of relapse was higher (0.67 ± 0.18 vs 0.07 ± 0.02; p < 0.001. In the multivariate analysis IKZF1 deletions were associated with decreased EFS (hazard ratio (HR 4.755; 95 % confidence interval (CI 1.856–12.185; p = 0.001, and OS (HR 4.208; 95 % CI 1.322–13.393; p = 0.015, but increased relapse risk (HR 9,083; 95 % CI 3.119–26.451; p < 0.001. IKZF1 deletions retained their prognostic significance in the intermediate risk group patients (p < 0.001, but not in standard or high-risk groups. Majority of IKZF1 deletions – 12 (80 % of 15 – were revealed in the “B-other” group (n = 83. In this cohort of patients IKZF1 deletions led to inferior EFS (HR 6.172; 95 % CI 1.834–20.767; p = 0.003 and higher relapse rate (HR 16.303; 95 % CI 3.324–79.965; p = 0.015. Thus, our results showed that IKZF1 deletions are independent risk factor in BCP-ALL patients.

  7. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

    Directory of Open Access Journals (Sweden)

    Budd A Tucker

    2011-04-01

    Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

  8. * Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

    Science.gov (United States)

    Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

    2017-08-01

    Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle

  9. Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

    Directory of Open Access Journals (Sweden)

    Manuela Fogli

    2008-07-01

    Full Text Available Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7. This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I molecules, HIV-1-infected p24(pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs and with the high frequency of the anergic CD56(neg/CD16(pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos blasts derived from primary T cells.

  10. Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens.

    Science.gov (United States)

    Shoura, Massa J; Gabdank, Idan; Hansen, Loren; Merker, Jason; Gotlib, Jason; Levene, Stephen D; Fire, Andrew Z

    2017-10-05

    Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples. Copyright © 2017 Shoura et al.

  11. Isolation of skin-derived precursors from human foreskin and their differentiation into neurons and glial cells

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    Bakhtiari M

    2010-12-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Skin-derived precursors (SKPs are a type of progenitor cells extracted from mammalian dermal tissue and can be differentiate to neural and mesodermal lineage in vitro. These cells can introduce an accessible autologos source of neural precursor cells for treatment of different neurodegenerative diseases. This research was done in order to set up isolation, culture, proliferation and differentiation of human skin derived precursors (hSKPs."n"nMethods: Human foreskin samples were cut into smaller pieces and cultured in proliferation medium after enzymatic digestion. To induce neural differentiation, cells were cultured in neural differentiation medium after fifth passage. We used immunocytochemistry and RT-PCR for characterization of the cells. Neuron and glial cell differentiation potential was assessed by immunofloresence using specific antibodies. The experiments were carried out in triplicate."n"nResults: After differentiation, βΙΙΙ- tubulin and neurofilament-M positive cells were observed that are specific markers for neurons. Moreover, glial fibrillary acid protein (GFAP and S100 positive cells were identified that are markers specifically express in glial cells. Detected neurons and glials were

  12. Stimulating endogenous cardiac regeneration

    Directory of Open Access Journals (Sweden)

    Amanda eFinan

    2015-09-01

    Full Text Available The healthy adult heart has a low turnover of cardiac myocytes. The renewal capacity, however, is augmented after cardiac injury. Participants in cardiac regeneration include cardiac myocytes themselves, cardiac progenitor cells, and peripheral stem cells, particularly from the bone marrow compartment. Cardiac progenitor cells and bone marrow stem cells are augmented after cardiac injury, migrate to the myocardium, and support regeneration. Depletion studies of these populations have demonstrated their necessary role in cardiac repair. However, the potential of these cells to completely regenerate the heart is limited. Efforts are now being focused on ways to augment these natural pathways to improve cardiac healing, primarily after ischemic injury but in other cardiac pathologies as well. Cell and gene therapy or pharmacological interventions are proposed mechanisms. Cell therapy has demonstrated modest results and has passed into clinical trials. However, the beneficial effects of cell therapy have primarily been their ability to produce paracrine effects on the cardiac tissue and recruit endogenous stem cell populations as opposed to direct cardiac regeneration. Gene therapy efforts have focused on prolonging or reactivating natural signaling pathways. Positive results have been demonstrated to activate the endogenous stem cell populations and are currently being tested in clinical trials. A potential new avenue may be to refine pharmacological treatments that are currently in place in the clinic. Evidence is mounting that drugs such as statins or beta blockers may alter endogenous stem cell activity. Understanding the effects of these drugs on stem cell repair while keeping in mind their primary function may strike a balance in myocardial healing. To maximize endogenous cardiac regeneration,a combination of these approaches couldameliorate the overall repair process to incorporate the participation ofmultiple cell players.

  13. An endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency.

    Science.gov (United States)

    Dorostkar, Rohollah; Bamdad, Taravat; Parsania, Masoud; Pouriayevali, Hassan

    2012-12-01

    Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-γ and IL-4 were evaluated. The obtained data showed that the production of IFN-γ from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups (pEL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups (p<0.001). The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination.

  14. Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

    Science.gov (United States)

    Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2016-05-31

    Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

  15. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Fendrick, J.L.; Iglewski, W.J. (Univ. of Rochester, NY (USA))

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  16. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    International Nuclear Information System (INIS)

    Fendrick, J.L.; Iglewski, W.J.

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [ 32 P]orthophosphate and 105 (vol/vol) fetal bovine serum. A 32 P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32 P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32 P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [ 32 P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32 P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [ 32 P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32 P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32 P label was lost from the EF-2 within 30 min

  17. Neurotensin is metabolized by endogenous proteases in prostate cancer cell lines.

    Science.gov (United States)

    Moody, T W; Mayr, C A; Gillespie, T J; Davis, T P

    1998-01-01

    The formation and processing of neurotensin (NT) by three prostate cancer cell lines was investigated. Neurotensin (NT) immunoreactivity was detected in conditioned media and extracts of LNCaP cells. Using HPLC techniques, the immunoreactivity extracted from LNCaP cells coeluted with synthetic NT standard. Metalloendopeptidase 3.4.24.15 activity was detected in PC-3, DU-145 and LNCaP cells, whereas high levels of neutral endopeptidase 3.4.24.1 1 activity was detected only in LNCaP cells. NT was relatively stable when incubated with PC-3 or D-145 cells but was rapidly degraded by LNCaP cells to NT1-11 and NT1-10. Phosphoramidon inhibited the metabolism of NT by LNCaP cells. These data suggest that NT is present in and metabolized by LNCaP cellular enzymes.

  18. 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.

  19. Nuclear entry of poliovirus protease-polymerase precursor 3CD: implications for host cell transcription shut-off

    International Nuclear Information System (INIS)

    Sharma, Rakhi; Raychaudhuri, Santanu; Dasgupta, Asim

    2004-01-01

    Host cell transcription mediated by all three RNA polymerases is rapidly inhibited after infection of mammalian cells with poliovirus (PV). Both genetic and biochemical studies have shown that the virus-encoded protease 3C cleaves the TATA-binding protein and other transcription factors at glutamine-glycine sites and is directly responsible for host cell transcription shut-off. PV replicates in the cytoplasm of infected cells. To shut-off host cell transcription, 3C or a precursor of 3C must enter the nucleus of infected cells. Although the 3C protease itself lacks a nuclear localization signal (NLS), amino acid sequence examination of 3D identified a potential single basic type NLS, KKKRD, spanning amino acids 125-129 within this polypeptide. Thus, a plausible scenario is that 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis ultimately leading to cleavage of transcription factors in the nucleus. Using transient transfection of enhanced green fluorescent protein (EGFP) fusion polypeptides, we demonstrate here that both 3CD and 3D are capable of entering the nucleus in PV-infected cells. However, both polypeptides remain in the cytoplasm in uninfected HeLa cells. Mutagenesis of the NLS sequence in 3D prevents nuclear entry of 3D and 3CD in PV-infected cells. We also demonstrate that 3CD can be detected in the nuclear fraction from PV-infected HeLa cells as early as 2 h postinfection. Significant amount of 3CD is found associated with the nuclear fraction by 3-4 h of infection. Taken together, these results suggest that both the 3D NLS and PV infection are required for the entry of 3CD into the nucleus and that this may constitute a means by which viral protease 3C is delivered into the nucleus leading to host cell transcription shut-off

  20. Activating receptor NKG2D targets RAE-1-expressing allogeneic neural precursor cells in a viral model of multiple sclerosis.

    Science.gov (United States)

    Weinger, Jason G; Plaisted, Warren C; Maciejewski, Sonia M; Lanier, Lewis L; Walsh, Craig M; Lane, Thomas E

    2014-10-01

    Transplantation of major histocompatibility complex-mismatched mouse neural precursor cells (NPCs) into mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in rapid rejection that is mediated, in part, by T cells. However, the contribution of the innate immune response to allograft rejection in a model of viral-induced neurological disease has not been well defined. Herein, we demonstrate that the natural killer (NK) cell-expressing-activating receptor NKG2D participates in transplanted allogeneic NPC rejection in mice persistently infected with JHMV. Cultured NPCs derived from C57BL/6 (H-2(b) ) mice express the NKG2D ligand retinoic acid early precursor transcript (RAE)-1 but expression was dramatically reduced upon differentiation into either glia or neurons. RAE-1(+) NPCs were susceptible to NK cell-mediated killing whereas RAE-1(-) cells were resistant to lysis. Transplantation of C57BL/6-derived NPCs into JHMV-infected BALB/c (H-2(d) ) mice resulted in infiltration of NKG2D(+) CD49b(+) NK cells and treatment with blocking antibody specific for NKG2D increased survival of allogeneic NPCs. Furthermore, transplantation of differentiated RAE-1(-) allogeneic NPCs into JHMV-infected BALB/c mice resulted in enhanced survival, highlighting a role for the NKG2D/RAE-1 signaling axis in allograft rejection. We also demonstrate that transplantation of allogeneic NPCs into JHMV-infected mice resulted in infection of the transplanted cells suggesting that these cells may be targets for infection. Viral infection of cultured cells increased RAE-1 expression, resulting in enhanced NK cell-mediated killing through NKG2D recognition. Collectively, these results show that in a viral-induced demyelination model, NK cells contribute to rejection of allogeneic NPCs through an NKG2D signaling pathway. © 2014 AlphaMed Press.

  1. The Endogenous Exposome

    Science.gov (United States)

    Nakamura, Jun; Mutlu, Esra; Sharma, Vyom; Collins, Leonard; Bodnar, Wanda; Yu, Rui; Lai, Yongquan; Moeller, Benjamin; Lu, Kun; Swenberg, James

    2014-01-01

    The concept of the Exposome, is a compilation of diseases and one’s lifetime exposure to chemicals, whether the exposure comes from environmental, dietary, or occupational exposures; or endogenous chemicals that are formed from normal metabolism, inflammation, oxidative stress, lipid peroxidation, infections, and other natural metabolic processes such as alteration of the gut microbiome. In this review, we have focused on the Endogenous Exposome, the DNA damage that arises from the production of endogenous electrophilic molecules in our cells. It provides quantitative data on endogenous DNA damage and its relationship to mutagenesis, with emphasis on when exogenous chemical exposures that produce identical DNA adducts to those arising from normal metabolism cause significant increases in total identical DNA adducts. We have utilized stable isotope labeled chemical exposures of animals and cells, so that accurate relationships between endogenous and exogenous exposures can be determined. Advances in mass spectrometry have vastly increased both the sensitivity and accuracy of such studies. Furthermore, we have clear evidence of which sources of exposure drive low dose biology that results in mutations and disease. These data provide much needed information to impact quantitative risk assessments, in the hope of moving towards the use of science, rather than default assumptions. PMID:24767943

  2. Combination of exogenous cell transplantation and 5-HT{sub 4} receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hui [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Zheng, Bai-Jun; Pan, Wei-Kang; Wang, Huai-Jie; Xie, Chong; Zhao, Yu-Ying [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Chen, Xin-Lin; Liu, Yong [Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Gao, Ya, E-mail: ygao@mail.xjtu.edu.cn [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China)

    2017-02-01

    Enteric neural crest-derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5-HT{sub 4} receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5-HT{sub 4} receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride-treated colons. At given days post-intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75{sup NTR} and peripherin, respectively. eGFP-positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post-intervention, the number of peripherin-positive cells gradually increased in all groups. Significantly more peripherin-positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5-HT{sub 4} receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model. - Highlights: • Survival and differentiation of exogenous ENCCs in treated colons. • With longer times post-intervention, the number of ENCCs and their progeny cells gradually increased. • Exogenous ENCCs combined with the 5-HT4 receptor agonist ffectively induced ENCCs proliferation and differentiation.

  3. Combination of exogenous cell transplantation and 5-HT4 receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model

    International Nuclear Information System (INIS)

    Yu, Hui; Zheng, Bai-Jun; Pan, Wei-Kang; Wang, Huai-Jie; Xie, Chong; Zhao, Yu-Ying; Chen, Xin-Lin; Liu, Yong; Gao, Ya

    2017-01-01

    Enteric neural crest-derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5-HT 4 receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5-HT 4 receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride-treated colons. At given days post-intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75 NTR and peripherin, respectively. eGFP-positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post-intervention, the number of peripherin-positive cells gradually increased in all groups. Significantly more peripherin-positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5-HT 4 receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model. - Highlights: • Survival and differentiation of exogenous ENCCs in treated colons. • With longer times post-intervention, the number of ENCCs and their progeny cells gradually increased. • Exogenous ENCCs combined with the 5-HT4 receptor agonist ffectively induced ENCCs proliferation and differentiation.

  4. Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma

    DEFF Research Database (Denmark)

    Jacobsen, Benedikte; Santoni-Rugiu, Eric; Illemann, Martin

    2012-01-01

    in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased...... in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier...... finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical...

  5. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

    Science.gov (United States)

    Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

    2012-09-10

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Antidiabetic actions of endogenous and exogenous GLP-1 in type 1 diabetic patients with and without residual ß-cell function

    DEFF Research Database (Denmark)

    Kielgast, Urd; Holst, Jens Juul; Madsbad, Sten

    2011-01-01

    To investigate the effect of exogenous as well as endogenous glucagon-like peptide 1 (GLP-1) on postprandial glucose excursions and to characterize the secretion of incretin hormones in type 1 diabetic patients with and without residual ß-cell function.......To investigate the effect of exogenous as well as endogenous glucagon-like peptide 1 (GLP-1) on postprandial glucose excursions and to characterize the secretion of incretin hormones in type 1 diabetic patients with and without residual ß-cell function....

  7. Hydroxyapatite coatings deposited by liquid precursor plasma spraying: controlled dense and porous microstructures and osteoblastic cell responses

    International Nuclear Information System (INIS)

    Huang Yi; Song Lei; Liu Xiaoguang; Xiao Yanfeng; Wu Yao; Chen Jiyong; Wu Fang; Gu Zhongwei

    2010-01-01

    Hydroxyapatite coatings were deposited on Ti-6Al-4V substrates by a novel plasma spraying process, the liquid precursor plasma spraying (LPPS) process. X-ray diffraction results showed that the coatings obtained by the LPPS process were mainly composed of hydroxyapatite. The LPPS process also showed excellent control on the coating microstructure, and both nearly fully dense and highly porous hydroxyapatite coatings were obtained by simply adjusting the solid content of the hydroxyapatite liquid precursor. Scanning electron microscope observations indicated that the porous hydroxyapatite coatings had pore size in the range of 10-200 μm and an average porosity of 48.26 ± 0.10%. The osteoblastic cell responses to the dense and porous hydroxyapatite coatings were evaluated with human osteoblastic cell MG-63, in respect of the cell morphology, proliferation and differentiation, with the hydroxyapatite coatings deposited by the atmospheric plasma spraying (APS) process as control. The cell experiment results indicated that the heat-treated LPPS coatings with a porous structure showed the best cell proliferation and differentiation among all the hydroxyapatite coatings. Our results suggest that the LPPS process is a promising plasma spraying technique for fabricating hydroxyapatite coatings with a controllable microstructure, which has great potential in bone repair and replacement applications.

  8. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

    Science.gov (United States)

    Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

    2013-01-01

    Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

  9. ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity.

    Science.gov (United States)

    Nugent, Lindsey F; Shi, Guangpu; Vistica, Barbara P; Ogbeifun, Osato; Hinshaw, Samuel J H; Gery, Igal

    2013-11-13

    Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 μg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.

  10. Interleukin-6 as an endogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral blood mononuclear cells.

    Science.gov (United States)

    Dinarello, C A; Cannon, J G; Mancilla, J; Bishai, I; Lees, J; Coceani, F

    1991-10-25

    Fever induced by endogenous as well as exogenous pyrogens is often prevented by cyclooxygenase inhibitors; endogenous pyrogens stimulate prostaglandin E2 (PGE2) in or near the thermoregulatory centers of the brain. The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), are two pyrogens which stimulate brain PGE2 formation during fever and also increase PGE2 synthesis in human mononuclear cells in vitro. In the present study, we examined whether interleukin-6 (IL-6) stimulates PGE2 formation in a manner similar to IL-1 and TNF. Both glycosylated and non-glycosylated forms of recombinant human IL-6 were tested. Following intravenous injection into rabbits, the glycosylated IL-6 was more pyrogenic than the non-glycosylated form and there was no evidence of synergy in the production of fever when IL-6 and IL-1 were given simultaneously. IL-6 fever was blocked by prior administration of the cyclooxygenase inhibitor ibuprofen. IL-6 was also pyrogenic in the cat by either the systemic or the intraventricular route. However, in both species, IL-6 was less effective than IL-1 beta. When given intraventricularly to cats, IL-6 produced an increase in PGE2 levels of the cerebrospinal fluid in parallel with the rise in body temperature. In the latter respect, IL-6 imitated IL-1 beta; however, IL-6 from 0.15-15 micrograms/ml did not increase mononuclear cell PGE2 production in vitro whereas IL-1 beta induced 20-30-fold increases in PGE2 at 100 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Data on endogenous bovine ovarian follicular cells peptides and small proteins obtained through Top-down High Resolution Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Valérie Labas

    2017-08-01

    Full Text Available The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017 http://dx.doi.org/10.1016/j.jprot.2017.03.027 [1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892. Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes.

  12. Endogenous GABA and Glutamate Finely Tune the Bursting of Olfactory Bulb External Tufted Cells

    Science.gov (United States)

    Hayar, Abdallah; Ennis, Matthew

    2008-01-01

    In rat olfactory bulb slices, external tufted (ET) cells spontaneously generate spike bursts. Although ET cell bursting is intrinsically generated, its strength and precise timing may be regulated by synaptic input. We tested this hypothesis by analyzing whether the burst properties are modulated by activation of ionotropic γ-aminobutyric acid (GABA) and glutamate receptors. Blocking GABAA receptors increased—whereas blocking ionotropic glutamate receptors decreased—the number of spikes/burst without changing the interburst frequency. The GABAA agonist (isoguvacine, 10 μM) completely inhibited bursting or reduced the number of spikes/burst, suggesting a shunting effect. These findings indicate that the properties of ET cell spontaneous bursting are differentially controlled by GABAergic and glutamatergic fast synaptic transmission. We suggest that ET cell excitatory and inhibitory inputs may be encoded as a change in the pattern of spike bursting in ET cells, which together with mitral/tufted cells constitute the output circuit of the olfactory bulb. PMID:17567771

  13. Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools

    DEFF Research Database (Denmark)

    Kelsen, Jesper; Larsen, Marianne; Sørensen, Jens Christian H.

    2010-01-01

    We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats ...

  14. Regulation and patterns of endogenous and exogenous gene expression during differentiation of embryonal carcinoma cells

    International Nuclear Information System (INIS)

    Astigiano, S.; Sherman, M.I.; Abarzua, P.

    1989-01-01

    Embryonal carcinoma (EC) cells offer an interesting model system for evaluating differentiation because the cells are pluripotent, thus resembling germ cells and embryonic stem cells, and because a number of agents have been defined that are capable of promoting the differentiation of these cells. This chapter examines how EC cells might be triggered to differentiate, with emphasis on retinoic acid because this compound is a potent, naturally occurring inducer that has been studied extensively in this system. The nature of alterations in gene expression during EC cell differentiation is reviewed from the perspective of evaluating whether these changes are likely to be responsible for, or a result of, the differentiation event. Finally, the authors consider in molecular terms why EC cells, but not their differentiated derivatives, are refractory to the expression of many viral genomes following infection. Based upon these studies, they propose that fundamental changes in gene expression that are observed when differentiation is triggered in EC cells are likely to be due to the disappearance or neutralization of strong repressor elements

  15. Endogene CGRP

    OpenAIRE

    Höfer, Martina

    2010-01-01

    Hintergrund und Ziele Die vorliegende tierexperimentelle Arbeit beschäftigt sich mit der Frage, welche Rolle endogenes Calcitonin-gene related peptide (CGRP) in der Niere spielt. Hierbei untersuchten wir die renale CGRP Freisetzung aus renalen Afferenzen in vitro anhand von gesunden Tieren und einem pathologischen Modell der Glomerulonephritis. Man weiß bereits, dass sowohl sympathische als auch primär sensorische Neuronen die Entzündung und die Immunantwort in der Peripherie regulieren (68)....

  16. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation.

    Directory of Open Access Journals (Sweden)

    Yuanfeng Wu

    Full Text Available Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512, whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs, but not by non-genotoxins (NGTXs. Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV. However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.

  17. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  18. Effect of α-bungarotoxin and etorphine on acetylcholine-evoked release of endogenous and radiolabeled catecholamines from primary culture of adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Kageyama, H.; Guidotti, A.

    1984-01-01

    Cell cultures of adrenal medulla have become an important research tool to study basic processes that regulate catecholamine storage, release and synthesis. Release has been studied either by labeling with [ 3 H]norepinephrine and measuring release of radioactivity or by measuring the endogenous catecholamines released with HPLC. Acetylcholine (5X10 -6 -10 -4 M) appears to release preferentially norepinephrine, although the cells store more epinephrine than norepinephrine. Etorphine and α-bungarotoxin antagonize the release of catecholamines elicited by acetylcholine. This inhibitory action appears to be greater when the measurement of endogenous catecholamines rather than radioactive norepinephrine is used to monitor the action of acetylcholine. The data suggest that the measurement of endogenous catecholamines by HPLC is preferable to the [ 3 H]NE loading and release technique, especially when analyzing the effects of low concentrations of drugs that are thought to affect nicotinic receptor function. (Auth.)

  19. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I.-Development of the in vivo culture and effects induced by the hyperthermia

    International Nuclear Information System (INIS)

    Bueren, J. A.; Nieto, M.

    1983-01-01

    The present report shows the agar diffusion chamber technique for culturing granulocyte- macrophage precursor cells, obtained from mice bone marrow. Diffusion chambers containing the bone marrow suspension are implanted intraperitoneally Into mice and constitute a compartment which avoids the migration of cells, but allows the transit of the mouse biological fluxes, necessary for the cellular proliferation. By means of this technique, we studied the lethal effects of the hyperthermia on the precursors and their capacity to repair sublethal damage. (Author) 129 refs

  20. Real-time measurements of endogenous CO production from vascular cells using an ultrasensitive laser sensor

    Science.gov (United States)

    Morimoto, Y.; Durante, W.; Lancaster, D. G.; Klattenhoff, J.; Tittel, F. K.

    2001-01-01

    Carbon monoxide (CO) has been implicated as a biological messenger molecule analogous to nitric oxide. A compact gas sensor based on a midinfrared laser absorption spectroscopy was developed for direct and real-time measurement of trace levels (in approximate pmol) of CO release by vascular cells. The midinfrared light is generated by difference frequency mixing of two nearinfrared lasers in a nonlinear optical crystal. A strong infrared absorption line of CO (4.61 microm) is chosen for convenient CO detection without interference from other gas species. The generation of CO from cultured vascular smooth muscle cells was detected every 20 s without any chemical modification to the CO. The sensitivity of the sensor reached 6.9 pmol CO. CO synthesis was measured from untreated control cells (0.25 nmol per 10(7) cells/h), sodium nitroprusside-treated cells (0.29 nmol per 10(7) cells/h), and hemin-treated cells (0.49 nmol per 10(7) cells/h). The sensor also detected decreases in CO production after the addition of the heme oxygenase (HO) inhibitor tin protoporphyrin-IX (from 0.49 to 0.02 nmol per 10(7) cells/h) and increases after the administration of the HO substrate hemin (from 0.27 to 0.64 nmol per 10(7) cells/h). These results demonstrate that midinfrared laser absorption spectroscopy is a useful technique for the noninvasive and real-time detection of trace levels of CO from biological tissues.

  1. Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Lu, Amy Q; Popova, Evgenya Y; Barnstable, Colin J

    2017-09-12

    In vitro differentiation of mouse embryonic stem cells (ESCs) into retinal fates can be used to study the roles of exogenous factors acting through multiple signaling pathways during retina development. Application of activin A during a specific time frame that corresponds to early embryonic retinogenesis caused increased generation of CRX + photoreceptor precursors and decreased PAX6 + retinal progenitor cells (RPCs). Following activin A treatment, SMAD2/3 was activated in RPCs and bound to promoter regions of key RPC and photoreceptor genes. The effect of activin on CRX expression was repressed by pharmacological inhibition of SMAD2/3 phosphorylation. Activin signaling through SMAD2/3 in RPCs regulates expression of transcription factors involved in cell type determination and promotes photoreceptor lineage specification. Our findings can contribute to the production of photoreceptors for cell replacement therapy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages.

    Directory of Open Access Journals (Sweden)

    Meret Cepero Malo

    Full Text Available Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers.

  3. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila (IP-Korea); (UPMC); (UMKC)

    2013-09-23

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

  4. Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

    DEFF Research Database (Denmark)

    Jensen, Pia; Gramsbergen, Jan-Bert; Zimmer, Jens

    2011-01-01

    Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation o...... enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells....... of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than......, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH...

  5. Cytokines as endogenous pyrogens.

    Science.gov (United States)

    Dinarello, C A

    1999-03-01

    Cytokines are pleiotropic molecules mediating several pathologic processes. Long before the discovery of cytokines as immune system growth factors or as bone marrow stimulants, investigators learned a great deal about cytokines when they studied them as the endogenous mediators of fever. The terms "granulocytic" or "endogenous pyrogen" were used to describe substances with the biologic property of fever induction. Today, we recognize that pyrogenicity is a fundamental biologic property of several cytokines and hence the clinically recognizeable property of fever links host perturbations during disease with fundamental perturbations in cell biology. In this review, the discoveries made on endogenous pyrogens are revisited, with insights into the importance of the earlier work to the present-day understanding of cytokines in health and in disease.

  6. Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells

    International Nuclear Information System (INIS)

    Denner, Joachim; Specke, Volker; Thiesen, Ulla; Karlas, Alexander; Kurth, Reinhard

    2003-01-01

    Human-tropic porcine endogenous retroviruses (PERV) such as PERV-A and PERV-B can infect human cells and are therefore a potential risk to recipients of xenotransplants. A similar risk is posed by recombinant viruses containing the receptor-binding site of PERV-A and large parts of the genome of the ecotropic PERV-C including its long terminal repeat (LTR). We describe here the unique organization of the PERV-C LTR and its changes during serial passage of recombinant virus in human cells. An increase in virus titer correlated with an increase in LTR length, caused by multiplication of 37-bp repeats containing nuclear factor Y binding sites. Luciferase dual reporter assays revealed a correlation between the number of repeats and the extent of expression. No alterations have been observed in the receptor-binding site, indicating that the increased titer is due to the changes in the LTR. These data indicate that recombinant PERVs generated during infection of human cells can adapt and subsequently replicate with greater efficiency

  7. Enhanced Performance of PbS-quantum-dot-sensitized Solar Cells via Optimizing Precursor Solution and Electrolytes

    Science.gov (United States)

    Tian, Jianjun; Shen, Ting; Liu, Xiaoguang; Fei, Chengbin; Lv, Lili; Cao, Guozhong

    2016-03-01

    This work reports a PbS-quantum-dot-sensitized solar cell (QDSC) with power conversion efficiency (PCE) of 4%. PbS quantum dots (QDs) were grown on mesoporous TiO2 film using a successive ion layer absorption and reaction (SILAR) method. The growth of QDs was found to be profoundly affected by the concentration of the precursor solution. At low concentrations, the rate-limiting factor of the crystal growth was the adsorption of the precursor ions, and the surface growth of the crystal became the limiting factor in the high concentration solution. The optimal concentration of precursor solution with respect to the quantity and size of synthesized QDs was 0.06 M. To further increase the performance of QDSCs, the 30% deionized water of polysulfide electrolyte was replaced with methanol to improve the wettability and permeability of electrolytes in the TiO2 film, which accelerated the redox couple diffusion in the electrolyte solution and improved charge transfer at the interfaces between photoanodes and electrolytes. The stability of PbS QDs in the electrolyte was also improved by methanol to reduce the charge recombination and prolong the electron lifetime. As a result, the PCE of QDSC was increased to 4.01%.

  8. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms

    DEFF Research Database (Denmark)

    Svingen, T; Jørgensen, Anne; Rajpert-De Meyts, E

    2014-01-01

    to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers......, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis...... and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further...

  9. Specific gut commensal flora locally alters T cell tuning to endogenous ligands.

    Science.gov (United States)

    Chappert, Pascal; Bouladoux, Nicolas; Naik, Shruti; Schwartz, Ronald H

    2013-06-27

    Differences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Drummond, Heather A

    2011-01-01

    The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. Copyright © 2011 S. Karger AG, Basel.

  11. Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors

    Science.gov (United States)

    Zuurbier, Linda; Gutierrez, Alejandro; Mullighan, Charles G.; Canté-Barrett, Kirsten; Gevaert, A. Olivier; de Rooi, Johan; Li, Yunlei; Smits, Willem K.; Buijs-Gladdines, Jessica G.C.A.M.; Sonneveld, Edwin; Look, A. Thomas; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

    2014-01-01

    Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients. PMID:23975177

  12. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  13. Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells

    International Nuclear Information System (INIS)

    Sharungbam, Geeta D; Schwager, Christian; Chiblak, Sara; Brons, Stephan; Hlatky, Lynn; Haberer, Thomas; Debus, Jürgen; Abdollahi, Amir

    2012-01-01

    Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more

  14. Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors

    DEFF Research Database (Denmark)

    Klein, Anders Bue; Nittegaard-Nielsen, Mia; Christensen, Julie T.

    2015-01-01

    the immortalized IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concentration-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol...

  15. Beneficial Effects of Melatonin Combined with Exercise on Endogenous Neural Stem/Progenitor Cells Proliferation after Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Youngjeon Lee

    2014-01-01

    Full Text Available Endogenous neural stem/progenitor cells (eNSPCs proliferate and differentiate into neurons and glial cells after spinal cord injury (SCI. We have previously shown that melatonin (MT plus exercise (Ex had a synergistic effect on functional recovery after SCI. Thus, we hypothesized that combined therapy including melatonin and exercise might exert a beneficial effect on eNSPCs after SCI. Melatonin was administered twice a day and exercise was performed on a treadmill for 15 min, six days per week for 3 weeks after SCI. Immunohistochemistry and RT-PCR analysis were used to determine cell population for late response, in conjunction with histological examination and motor function test. There was marked improvement in hindlimb function in SCI+MT+Ex group at day 14 and 21 after injury, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU-positive cells. Furthermore, MAP2 was increased in the injured thoracic segment, while GFAP was increased in the cervical segment, along with elevated numbers of BrdU-positive nestin-expressing eNSPCs in the SCI+MT+Ex group. The dendritic spine density was augmented markedly in SCI+MT and SCI+MT+Ex groups.These results suggest a synergistic effect of SCI+MT+Ex might create a microenvironment to facilitate proliferation of eNSPCs to effectively replace injured cells and to improve regeneration in SCI.

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

    Science.gov (United States)

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

    2011-01-01

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

  17. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers

    International Nuclear Information System (INIS)

    Bussink, Johan; Kaanders, Johannes H.A.M.; Kogel, Albert J. van der

    2003-01-01

    Background and purpose: Tumor oxygenation is recognized as an important determinant of the outcome of radiotherapy and possibly also of other treatment modalities in a number of tumor types and in particular in squamous cell carcinomas. The hypoxic status of various solid tumors has been related to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment. It has been demonstrated in head and neck cancer that hypoxic radioresistance can be successfully counteracted by hypoxia modifying approaches. The microregional distribution and the level of tumor hypoxia depend on oxygen consumption and temporal and spatial variations in blood supply. It is unclear if severely hypoxic cells can resume clonogenicity when O 2 and nutrients become available again as a result of (treatment related) changes in the tumor microenvironment. Non-terminally differentiated hypoxic cells that are capable of proliferation are important for outcome because of their resistance to radiotherapy and possibly other cytotoxic treatments. Various exogenous and endogenous markers for hypoxia are currently available and can be studied in relation to each other, the tumor architecture and the tumor microenvironment. Use of nitroimidazole markers with immunohistochemical detection allows studying tumor cell hypoxia at the microscopic level. Co-registration with other microenvironmental parameters, such as vascular architecture (vascular density), blood perfusion, tumor cell proliferation and apoptosis, offers the possibility to obtain a comprehensive functional image of tumor patho-physiology and to study the effects of different modalities of cancer treatment. Conclusion: A number of functional microregional parameters have emerged that are good candidates for future use as indicators of tumor aggressiveness and treatment response. The key question is whether these parameters can be used as tools for

  18. Beneficial effect of human induced pluripotent stem cell-derived neural precursors in spinal cord injury repair

    Czech Academy of Sciences Publication Activity Database

    Romanyuk, Nataliya; Amemori, Takashi; Turnovcová, Karolína; Procházka, Pavel; Onteniente, B.; Syková, Eva; Jendelová, Pavla

    2015-01-01

    Roč. 24, č. 9 (2015), s. 1781-1797 ISSN 0963-6897 R&D Projects: GA MŠk LH12024; GA ČR(CZ) GA13-00939S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : human induced pluripotent stem cells * neural precursors * spinal cord injury Subject RIV: FH - Neurology Impact factor: 3.427, year: 2015

  19. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    Czech Academy of Sciences Publication Activity Database

    Jiráková, Klára; Šeneklová, Monika; Jirák, D.; Turnovcová, Karolína; Vosmanská, M.; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    2016-01-01

    Roč. 11, č. 2016 (2016), s. 6267-6281 E-ISSN 1178-2013 R&D Projects: GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) 7F14057 Institutional support: RVO:68378041 ; RVO:61389013 ; RVO:68378271 Keywords : neural precursors * magnetic resonance imaging * cell differentiation Subject RIV: FH - Neurology; EB - Genetics ; Molecular Biology (FGU-C); CD - Macromolecular Chemistry (UMCH-V) Impact factor: 4.300, year: 2016

  20. Activation of autoreactive B cells by endogenous TLR7 and TLR3 RNA ligands.

    Science.gov (United States)

    Green, Nathaniel M; Moody, Krishna-Sulayman; Debatis, Michelle; Marshak-Rothstein, Ann

    2012-11-16

    The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.

  1. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Daniela Lehnen

    2017-10-01

    Full Text Available Human pluripotent stem cell (hPSC-derived mesencephalic dopaminergic (mesDA neurons can relieve motor deficits in animal models of Parkinson's disease (PD. Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

  2. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-10-10

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  3. Atomic layer deposition precursor step repetition and surface plasma pretreatment influence on semiconductor–insulator–semiconductor heterojunction solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Talkenberg, Florian, E-mail: florian.talkenberg@ipht-jena.de; Illhardt, Stefan; Schmidl, Gabriele; Schleusener, Alexander; Sivakov, Vladimir [Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Radnóczi, György Zoltán; Pécz, Béla [Centre for Energy Research, Institute of Technical Physics and Materials Science, Konkoly-Thege Miklós u. 29-33, H-1121 Budapest (Hungary); Dikhanbayev, Kadyrjan; Mussabek, Gauhar [Department of Physics and Engineering, al-Farabi Kazakh National University, 71 al-Farabi Ave., 050040 Almaty (Kazakhstan); Gudovskikh, Alexander [Nanotechnology Research and Education Centre, St. Petersburg Academic University, Russian Academy of Sciences, Hlopina Str. 8/3, 194021 St. Petersburg (Russian Federation)

    2015-07-15

    Semiconductor–insulator–semiconductor heterojunction solar cells were prepared using atomic layer deposition (ALD) technique. The silicon surface was treated with oxygen and hydrogen plasma in different orders before dielectric layer deposition. A plasma-enhanced ALD process was applied to deposit dielectric Al{sub 2}O{sub 3} on the plasma pretreated n-type Si(100) substrate. Aluminum doped zinc oxide (Al:ZnO or AZO) was deposited by thermal ALD and serves as transparent conductive oxide. Based on transmission electron microscopy studies the presence of thin silicon oxide (SiO{sub x}) layer was detected at the Si/Al{sub 2}O{sub 3} interface. The SiO{sub x} formation depends on the initial growth behavior of Al{sub 2}O{sub 3} and has significant influence on solar cell parameters. The authors demonstrate that a hydrogen plasma pretreatment and a precursor dose step repetition of a single precursor improve the initial growth behavior of Al{sub 2}O{sub 3} and avoid the SiO{sub x} generation. Furthermore, it improves the solar cell performance, which indicates a change of the Si/Al{sub 2}O{sub 3} interface states.

  4. Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin

    Directory of Open Access Journals (Sweden)

    Eisch Amelia

    2010-01-01

    Full Text Available Abstract Background Survivin is a unique member of the inhibitor of apoptosis protein (IAP family in that it exhibits antiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passenger protein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not been elucidated. Results To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ and the subgranular zone (SGZ. We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning. Conclusions The findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.

  5. Evaluation by mass fragmentography of metabolic pathways of endogenous and exogenous compounds in eukaryote cell cultures

    International Nuclear Information System (INIS)

    Padieu, P.; Maume, B.F.

    1977-01-01

    Carbon-14 labelled compounds in cell cultures are used to establish the interconnections between different metabolic pathways as well as the competitive action of effectors on these different pathways. Analysis was performed by the GC-MS combination. Identification was carried out by comparison with the mass spectra of d9-TMS, 35 Cl-TMS and 37 Cl-TMS derivatizations of the culture extracts. Examples are given of the metabolic study of hormonal steroids and of safrale, a carcinogenic compound, by differentiated eukaryotic cells in cultures from the rat

  6. Effect of PTTG on endogenous gene expression in HEK 293 cells

    Directory of Open Access Journals (Sweden)

    Panguluri Siva K

    2009-12-01

    Full Text Available Abstract Background Pituitary tumor transforming gene (PTTG, also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293 cells that do not express PTTG and analyzed the downstream genes using microarray analysis. Results A total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip™ array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad or adenovirus vector expressing GFP (AdGFP. Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of ≤ 0.05 and a fold change of ≥2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family. Conclusion PTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study

  7. Intravenous grafts of amniotic fluid-derived stem cells induce endogenous cell proliferation and attenuate behavioral deficits in ischemic stroke rats.

    Directory of Open Access Journals (Sweden)

    Naoki Tajiri

    Full Text Available We recently reported isolation of viable rat amniotic fluid-derived stem (AFS cells [1]. Here, we tested the therapeutic benefits of AFS cells in a rodent model of ischemic stroke. Adult male Sprague-Dawley rats received a 60-minute middle cerebral artery occlusion (MCAo. Thirty-five days later, animals exhibiting significant motor deficits received intravenous transplants of rat AFS cells or vehicle. At days 60-63 post-MCAo, significant recovery of motor and cognitive function was seen in stroke animals transplanted with AFS cells compared to vehicle-infused stroke animals. Infarct volume, as revealed by hematoxylin and eosin (H&E staining, was significantly reduced, coupled with significant increments in the cell proliferation marker, Ki67, and the neuronal marker, MAP2, in the dentate gyrus (DG [2] and the subventricular zone (SVZ of AFS cell-transplanted stroke animals compared to vehicle-infused stroke animals. A significantly higher number of double-labeled Ki67/MAP2-positive cells and a similar trend towards increased Ki67/MAP2 double-labeling were observed in the DG and SVZ of AFS cell-transplanted stroke animals, respectively, compared to vehicle-infused stroke animals. This study reports the therapeutic potential of AFS cell transplantation in stroke animals, possibly via enhancement of endogenous repair mechanisms.

  8. An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells.

    Science.gov (United States)

    Powell, Jonathan J; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E; Skepper, Jeremy N; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A; Gomez-Morilla, Inmaculada; Grime, Geoffrey W; Kirkby, Karen J; Mabbott, Neil A; Donaldson, David S; Williams, Ifor R; Rios, Daniel; Girardin, Stephen E; Haas, Carolin T; Bruggraber, Sylvaine F A; Laman, Jon D; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P H; Pele, Laetitia C

    2015-04-01

    In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

  9. An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

    Science.gov (United States)

    Powell, Jonathan J.; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E.; Skepper, Jeremy N.; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A.; Gomez-Morilla, Inmaculada; Grime, Geoffrey W.; Kirkby, Karen J.; Mabbott, Neil A.; Donaldson, David S.; Williams, Ifor R.; Rios, Daniel; Girardin, Stephen E.; Haas, Carolin T.; Bruggraber, Sylvaine F. A.; Laman, Jon D.; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P. H.; Pele, Laetitia C.

    2015-05-01

    In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1’, whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

  10. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    Science.gov (United States)

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPLW515L, MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but was not phosphorylated. We generated disulfide-bonded MPLW515L homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPLW515L with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPLW515L/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling. PMID:19261614

  11. An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Harikumar, A.; Edupuganti, R.R.; Sorek, M.; Azad, G.K.; Markoulaki, S.; Sehnalová, Petra; Legartová, Soňa; Bártová, Eva; Farkash-Amar, S.; Jaenish, R.; Alon, U.; Meshorer, E.

    2017-01-01

    Roč. 9, č. 4 (2017), s. 1304-1314 ISSN 2213-6711 Institutional support: RVO:68081707 Keywords : living human-cells * dynamic proteomics * rna-seq Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 7.338, year: 2016

  12. Selective Expression of an Endogenous Inhibitor of FAK Regulates Proliferation and Migration of Vascular Smooth Muscle Cells

    Science.gov (United States)

    Taylor, Joan M.; Mack, Christopher P.; Nolan, Kate; Regan, Christopher P.; Owens, Gary K.; Parsons, J. Thomas

    2001-01-01

    Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538–540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [3H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals. PMID:11238893

  13. Goat red blood cells as precursor for iron oxide nanocrystal synthesis to develop nuclear targeted lung cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sreevani, Vellingiri; Shanthi, Krishnamurthy; Kannan, Soundarapandian, E-mail: sk_protein@buc.edu.in

    2013-09-01

    Graphical abstract: - Highlights: • Molecular approach of synthesis of Fe{sub 2}O{sub 3}-NC using goat blood as a bio-precursor. • The method is simple, efficient and environment friendly. • Synthesized nanocrystals were characterized by UV–vis spectroscopy, XRD, SEM, TEM, DLS and EDS. • Nanocrystals exhibited potent cytotoxicity against A549 cancer cell. • Nuclear targeting with expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. - Abstract: In this study, we synthesised iron oxide nanocrystals (Fe{sub 2}O{sub 3}-NC) from goat blood (bio-precursor) using red blood cells (RBC) lysis method (a molecular level approach) for the first time. The formation of Fe{sub 2}O{sub 3}-NC was achieved through a single-phase chemical reduction method. The size distribution of Fe{sub 2}O{sub 3}-NC falls between 20–30 nm for pellet and 100–200 nm for lysate and were found to be crystalline. Fe{sub 2}O{sub 3}-NC demonstrated significant cytotoxicity on A549. We report the direct visualization of interactions between Fe{sub 2}O{sub 3}-NC and the cancer cell nucleus. The active transport of Fe{sub 2}O{sub 3}-NC to the nucleus induces major changes to nuclear phenotype via nuclear envelope invaginations. We further examined the root cause for the involvement of Fe{sub 2}O{sub 3}-NC on the expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. This functional proteomic analysis clearly implies that the lung cancer cell proliferation is perfectly targeted by the biosynthesised Fe{sub 2}O{sub 3}-NC which could provide new insight for nuclear-targeted cancer therapy.

  14. Chondrogenesis of Embryonic Stem Cell-Derived Mesenchymal Stem Cells Induced by TGFβ1 and BMP7 Through Increased TGFβ Receptor Expression and Endogenous TGFβ1 Production.

    Science.gov (United States)

    Lee, Patrick T; Li, Wan-Ju

    2017-01-01

    For decades stem cells have proven to be invaluable to the study of tissue development. More recently, mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) (ESC-MSCs) have emerged as a cell source with great potential for the future of biomedical research due to their enhanced proliferative capability compared to adult tissue-derived MSCs and effectiveness of musculoskeletal lineage-specific cell differentiation compared to ESCs. We have previously compared the properties and differentiation potential of ESC-MSCs to bone marrow-derived MSCs. In this study, we evaluated the potential of TGFβ1 and BMP7 to induce chondrogenic differentiation of ESC-MSCs compared to that of TGFβ1 alone and further investigated the cellular phenotype and intracellular signaling in response to these induction conditions. Our results showed that the expression of cartilage-associated markers in ESC-MSCs induced by the TGFβ1 and BMP7 combination was increased compared to induction with TGFβ1 alone. The TGFβ1 and BMP7 combination upregulated the expression of TGFβ receptor and the production of endogenous TGFβs compared to TGFβ1 induction. The growth factor combination also increasingly activated both of the TGF and BMP signaling pathways, and inhibition of the signaling pathways led to reduced chondrogenesis of ESC-MSCs. Our findings suggest that by adding BMP7 to TGFβ1-supplemented induction medium, ESC-MSC chondrogenesis is upregulated through increased production of endogenous TGFβ and activities of TGFβ and BMP signaling. J. Cell. Biochem. 118: 172-181, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. An endogenous aryl hydrocarbon receptor (AhR) ligand, ITE induces regulatory T cells (Tregs) and ameliorates experimental colitis.

    Science.gov (United States)

    Abron, Jessicca D; Singh, Narendra P; Mishra, Manoj K; Price, Robert L; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Singh, Udai P

    2018-04-19

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that affects millions of people with high morbidity and health-care cost. The precise etiology of IBD is unknown, but clear evidence suggests that intestinal inflammation is caused by an excessive immune response to mucosal antigens. Recent studies have shown that activation of the aryl hydrocarbon receptor (AhR) induces regulatory T cells (Tregs) and suppresses autoimmune diseases. In the current study, we investigated if nontoxic ligand of AhR, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), can attenuate dextran sodium sulphate (DSS)-induced colitis. Our studies demonstrated that in mice that received ITE treatment, in-vivo colitis pathogenesis, including a decrease in body weight, was significantly reversed along with the systemic and intestinal inflammatory cytokines. ITE increased the expression of Tregs in spleen, mesenteric lymph nodes (MLNs) and colon lamina propria lymphocytes (cLPL) of mice with colitis when compared to controls. This induction of Tregs was reversed by AhR antagonist treatment in-vitro. ITE treatment also increased dendritic cells (DCs; CD11c+) and decreased F4/80+ (macrophage) from the spleen, MLNs and cLPL in mice with colitis. ITE also reversed the systemic and intestinal frequency of CD4+T cells during colitis and suppressed inflammatory cytokines including IFN-γ, TNF-α, IL-17, IL-6 and IL-1 as well as induced IL-10 levels. These findings suggest that ITE attenuates colitis through induction of Tregs and reduction in inflammatory CD4+ T cells and cytokines. Thus, our work demonstrates that the nontoxic endogenous AhR ligand ITE, may serve as a therapeutic modality to treat IBD.

  16. Enhancing endogenous stem cells in the newbornvia delayed umbilical cord clamping

    Institute of Scientific and Technical Information of China (English)

    Christopher Lawton; Sandra Acosta; Nate Watson; Chiara Gonzales-Portillo; hTeo Diamandis; Naoki Tajiri; Yuji Kaneko; Paul R. Sanberg; Cesar V. Borlongan

    2015-01-01

    There is currently no consensus among clinicians and scientists over the appropriate or optimal timing for umbilical cord clamping. However, many clinical studies have suggested that delayed cord clamping is associated with various neonatal beneifts including increased blood volume, reduced need for blood transfusion, increased cerebral oxygenation in pre-term infants, and decreased frequency of iron deifciency anemia in term infants. Human umbilical cord blood con-tains signiifcant amounts of stem and progenitor cells and is currently used in the treatment of several life-threatening diseases. We propose that delayed cord clamping be encouraged as it en-hances blood lfow from the placenta to the neonate, which is accompanied by an increase supply of valuable stem and progenitor cells, as well as may improve blood oxygenation and increase blood volume, altogether reducing the infant’s susceptibility to both neonatal and age-related diseases.

  17. Enhancing endogenous stem cells in the newborn via delayed umbilical cord clamping

    Directory of Open Access Journals (Sweden)

    Christopher Lawton

    2015-01-01

    Full Text Available There is currently no consensus among clinicians and scientists over the appropriate or optimal timing for umbilical cord clamping. However, many clinical studies have suggested that delayed cord clamping is associated with various neonatal benefits including increased blood volume, reduced need for blood transfusion, increased cerebral oxygenation in pre-term infants, and decreased frequency of iron deficiency anemia in term infants. Human umbilical cord blood contains significant amounts of stem and progenitor cells and is currently used in the treatment of several life-threatening diseases. We propose that delayed cord clamping be encouraged as it enhances blood flow from the placenta to the neonate, which is accompanied by an increase supply of valuable stem and progenitor cells, as well as may improve blood oxygenation and increase blood volume, altogether reducing the infant′s susceptibility to both neonatal and age-related diseases.

  18. CHANGES IN ENDOGENOUS CYTOKININ CONCENTRATIONS IN CHLORELLA (CHLOROPHYCEAE) IN RELATION TO LIGHT AND THE CELL CYCLE

    Czech Academy of Sciences Publication Activity Database

    Stirk, W. A.; van Staden, J.; Novák, Ondřej; Doležal, Karel; Strnad, Miroslav; Dobrev, Petre; Sipos, G.; Oerdoeg, V.; Balint, P.

    2011-01-01

    Roč. 47, č. 2 (2011), s. 291-301 ISSN 0022-3646 R&D Projects: GA ČR GA301/08/1649; GA ČR GA206/09/1284 Institutional research plan: CEZ:AV0Z50380511 Keywords : cell cycle * Chlorella * cis-zeatin * cytokinins * N6-(2-isopentenyl)adenine Subject RIV: EF - Botanics Impact factor: 2.071, year: 2011

  19. Interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) cells and their precursors express the VGO1 antigen

    International Nuclear Information System (INIS)

    Denegri, J.F.; Peterson, J.; Tilley, P.

    1989-01-01

    Precursor and effector cells of recombinant interleukin-2 (r-IL-2)-induced lymphokine-activated killer (LAK) activity were investigated for their expression of VGO1. Peripheral blood lymphocytes (PBL) from normal donors were purified and separated in a FACS 420 into VGO1+- and VGO1- cell fractions before and after culture for 96 hr with 100 U/ml of r-IL-2. Their lytic activity against K 562 and Daudi cells was measured in a 51Cr release assay. The majority, if not all, of the LAK effector and precursor cells was VGO1+ lymphocytes. The expression of VGO1 by LAK precursor cells remained stable under the culture conditions used in our experiments. VGO1- lymphocytes cultured with r-IL-2 demonstrated neither LAK-induced activity nor expression of VGO1 antigen

  20. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. II. - X irradiation effects and influence of hyperthermia on the radiosensitivity

    International Nuclear Information System (INIS)

    Bueren, J.A.; Nieto, M.

    1983-01-01

    The effects of the X-irradiation on the viability of the granulocyte-macrophage precursors, has been determined by means of the agar diffusion chamber culture technique. The results show the high radiosensitivity of these cells, with survival parameter similar to those previously reported in the literature about different granulocyte-macrophage precursors. When a hyperthermic treatment is performed prior to the X-irradiation, a radiosensitization phenomenon is observed due to the synergism existent between hyperthermia and X rays on the lethality of the precursors. (Authors) 37 refs

  1. Signaling through three chemokine receptors triggers the migration of transplanted neural precursor cells in a model of multiple sclerosis.

    Science.gov (United States)

    Cohen, Mikhal E; Fainstein, Nina; Lavon, Iris; Ben-Hur, Tamir

    2014-09-01

    Multiple sclerosis (MS) is a multifocal disease, and precursor cells need to migrate into the multiple lesions in order to exert their therapeutic effects. Therefore, cell migration is a crucial element in regenerative processes in MS, dictating the route of delivery, when cell transplantation is considered. We have previously shown that inflammation triggers migration of multi-potential neural precursor cells (NPCs) into the white matter of experimental autoimmune encephalomyelitis (EAE) rodents, a widely used model of MS. Here we investigated the molecular basis of this attraction. NPCs were grown from E13 embryonic mouse brains and transplanted into the lateral cerebral ventricles of EAE mice. Transplanted NPC migration was directed by three tissue-derived chemokines. Stromal cell-derived factor-1α, monocyte chemo-attractant protein-1 and hepatocyte growth factor were expressed in the EAE brain and specifically in microglia and astrocytes. Their cognate receptors, CXCR4, CCR2 or c-Met were constitutively expressed on NPCs. Selective blockage of CXCR4, CCR2 or c-Met partially inhibited NPC migration in EAE brains. Blocking all three receptors had an additive effect and resulted in profound inhibition of NPC migration, as compared to extensive migration of control NPCs. The inflammation-triggered NPC migration into white matter tracts was dependent on a motile NPC phenotype. Specifically, depriving NPCs from epidermal growth factor (EGF) prevented the induction of glial commitment and a motile phenotype (as indicated by an in vitro motility assay), hampering their response to neuroinflammation. In conclusion, signaling via three chemokine systems accounts for most of the inflammation-induced, tissue-derived attraction of transplanted NPCs into white matter tracts during EAE. Copyright © 2014. Published by Elsevier B.V.

  2. Autoreactive effector/memory CD4+ and CD8+ T cells infiltrating grafted and endogenous islets in diabetic NOD mice exhibit similar T cell receptor usage.

    Directory of Open Access Journals (Sweden)

    Ramiro Diz

    Full Text Available Islet transplantation provides a "cure" for type 1 diabetes but is limited in part by recurrent autoimmunity mediated by β cell-specific CD4(+ and CD8(+ T cells. Insight into the T cell receptor (TCR repertoire of effector T cells driving recurrent autoimmunity would aid the development of immunotherapies to prevent islet graft rejection. Accordingly, we used a multi-parameter flow cytometry strategy to assess the TCR variable β (Vβ chain repertoires of T cell subsets involved in autoimmune-mediated rejection of islet grafts in diabetic NOD mouse recipients. Naïve CD4(+ and CD8(+ T cells exhibited a diverse TCR repertoire, which was similar in all tissues examined in NOD recipients including the pancreas and islet grafts. On the other hand, the effector/memory CD8(+ T cell repertoire in the islet graft was dominated by one to four TCR Vβ chains, and specific TCR Vβ chain usage varied from recipient to recipient. Similarly, islet graft- infiltrating effector/memory CD4(+ T cells expressed a limited number of prevalent TCR Vβ chains, although generally TCR repertoire diversity was increased compared to effector/memory CD8(+ T cells. Strikingly, the majority of NOD recipients showed an increase in TCR Vβ12-bearing effector/memory CD4(+ T cells in the islet graft, most of which were proliferating, indicating