WorldWideScience

Sample records for cell barrier alterations

  1. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Barrier cell sheath formation

    International Nuclear Information System (INIS)

    Kesner, J.

    1980-04-01

    The solution for electrostatic potential within a simply modeled tandem mirror thermal barrier is seen to exhibit a sheath at each edge of the cell. The formation of the sheath requires ion collisionality and the analysis assmes that the collisional trapping rate into the barrier is considerably slower than the barrier pump rate

  3. Alteration of blood-brain barrier integrity by retroviral infection.

    Directory of Open Access Journals (Sweden)

    Philippe V Afonso

    2008-11-01

    Full Text Available The blood-brain barrier (BBB, which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans, both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

  4. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  5. Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

    Science.gov (United States)

    Kim, Jin Hyoung; Hossain, Ferdaus Mohd Altaf; Patil, Ajit Mahadev; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Park, Sang-Youel; Lee, John-Hwa; Kim, Bumseok; Kim, Koanhoi; Eo, Seong Kug

    2016-10-01

    Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-β, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Can human activities alter the drowning fate of barrier islands?

    Science.gov (United States)

    Lorenzo-Trueba, J.; Ashton, A. D.; Jin, D.; Hoagland, P.; Kite-Powell, H.

    2012-12-01

    during landward migration. The model also demonstrates the potential for discontinuous shoreline retreat, with alternating periods of barrier stability and rapid migration, even for constant rates of sea-level rise. Anthropic activities can strongly interact with these behaviors. In particular, considering only cross-shore processes, beach nourishment activities widen the beach and can affect shoreface fluxes, and dune building, which curtails the overwash process, can potentially enhance barrier drowning by reducing overwash fluxes. Furthermore, coastal protection activities of adjacent communities or even individual property holders can be uncoordinated or coordinated, with their effects coupled along the coast through coastal reorientation and gradients in alongshore sediment transport. In the coordinated framework, owners act in concert to alter the barrier based upon community benefits, whereas in the non-coordinated framework owners alter only their own property. Another important role in management is the perception of future sea-level-rise-associated losses—communities manage their coast differently depending on their adopted forecast for sea-level rise. We find that coordinated behavior coupled with natural processes can substantially affect the drowning scenarios from the individual decision-making process.

  7. Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

    Czech Academy of Sciences Publication Activity Database

    Garbe, J.C.; Vrba, Lukáš; Sputova, K.; Fuchs, L.; Novák, Petr; Brothman, A.R.; Jackson, M.; Chin, K.; LaBarge, M.A.; Watts, G.; Futscher, B. W.; Stampfer, M.R.

    2014-01-01

    Roč. 13, č. 21 (2014), s. 3423-3435 ISSN 1538-4101 Institutional support: RVO:60077344 Keywords : genomic instability * human mammary epithelial cells * telomerase Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.565, year: 2014

  8. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar: Increased Abundance and Expression of a Calreticulin-Like Protein.

    Directory of Open Access Journals (Sweden)

    Giulia Micallef

    Full Text Available In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  9. Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells.

    Science.gov (United States)

    Roselli, Marianna; Finamore, Alberto; Hynönen, Ulla; Palva, Airi; Mengheri, Elena

    2016-09-29

    The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698 T is able to reduce the enterotoxigenic F4 + Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698 T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698 T cell wall components in the protection against F4 + ETEC infection using the intestinal Caco-2 cell line. Purified cell wall fragments (CWF) from L. amylovorus DSM 16698 T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4 + ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4 + ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4 + ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot. We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4 + ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4 + ETEC, inhibited membrane damage and prevented NF-kB activation. The results indicate that the various components of L. amylovorus DSM 16698 T cell wall may counteract the damage caused by F4 + ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4 + ETEC infection. U-CWF are

  10. Impedance-based cell monitoring: barrier properties and beyond

    Directory of Open Access Journals (Sweden)

    Benson Kathrin

    2013-01-01

    Full Text Available Abstract In multicellular organisms epithelial and endothelial cells form selective permeable interfaces between tissue compartments of different chemical compositions. Tight junctions which connect adjacent cells, control the passage of molecules across the barrier and, in addition, facilitate active transport processes. The cellular barriers are not static but can be deliberately modulated by exposure to specific external stimuli. In vitro models representing the essential absorption barriers of the body are nowadays available, thus allowing investigation of the parameters that control permeability as well as transport processes across those barriers. Independent of the origin of the barrier forming cells, techniques are needed to quantify their barrier integrity. One simple assay is to measure the permeability for given hydrophilic substrates possessing different molecular weights like sucrose or dextrans. However, this technique is time-consuming and labor-intensive. Moreover, radioactive or fluorescently-labeled substrates are needed to allow easy analytical detection. Finally, if transport processes are investigated, the standard permeant may interfere with the transport process under investigation or might even alter the barrier integrity by itself. Thus, independent, non-invasive techniques are needed to quantify the barrier integrity continuously during the experiment. Such techniques are available and are mainly based on the measurement of the transendothelial or transepithelial electrical resistance (TEER of barrier forming cells grown on porous membranes. Simple devices using two sets of electrodes (so-called Voltohmeters are widely used. In addition, an easy-to-use physical technique called impedance spectroscopy allows the continuous analysis of both the TEER and the electrical capacitance giving additional information about the barrier properties of cells grown on permeable membranes. This technique is useful as a quality control

  11. Electrochemical cell structure including an ionomeric barrier

    Science.gov (United States)

    Lambert, Timothy N.; Hibbs, Michael

    2017-06-20

    An apparatus includes an electrochemical half-cell comprising: an electrolyte, an anode; and an ionomeric barrier positioned between the electrolyte and the anode. The anode may comprise a multi-electron vanadium phosphorous alloy, such as VP.sub.x, wherein x is 1-5. The electrochemical half-cell is configured to oxidize the vanadium and phosphorous alloy to release electrons. A method of mitigating corrosion in an electrochemical cell includes disposing an ionomeric barrier in a path of electrolyte or ion flow to an anode and mitigating anion accumulation on the surface of the anode.

  12. Altered blood-brain barrier transport in neuro-inflammatory disorders.

    Science.gov (United States)

    Schenk, Geert J; de Vries, Helga E

    2016-06-01

    During neurodegenerative and neuroinflammatory disorders of the central nervous system (CNS), such as Alzheimer's disease (AD) and multiple sclerosis (MS), the protective function of the blood-brain barrier (BBB) may be severely impaired. The general neuro-inflammatory response, ranging from activation of glial cells to immune cell infiltration that is frequently associated with such brain diseases may underlie the loss of the integrity and function of the BBB. Consequentially, the delivery and disposition of drugs to the brain will be altered and may influence the treatment efficiency of such diseases. Altered BBB transport of drugs into the CNS during diseases may be the result of changes in both specific transport and non-specific transport pathways. Potential alterations in transport routes like adsorptive mediated endocytosis and receptor-mediated endocytosis may affect drug delivery to the brain. As such, drugs that normally are unable to traverse the BBB may reach their target in the diseased brain due to increased permeability. In contrast, the delivery of (targeted) drugs could be hampered during inflammatory conditions due to disturbed transport mechanisms. Therefore, the inventory of the neuro-inflammatory status of the neurovasculature (or recovery thereof) is of utmost importance in choosing and designing an adequate drug targeting strategy under disease conditions. Within this review we will briefly discuss how the function of the BBB can be affected during disease and how this may influence the delivery of drugs into the diseased CNS. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells.

    Science.gov (United States)

    Arnold, Kelly B; Burgener, Adam; Birse, Kenzie; Romas, Laura; Dunphy, Laura J; Shahabi, Kamnoosh; Abou, Max; Westmacott, Garrett R; McCorrister, Stuart; Kwatampora, Jessie; Nyanga, Billy; Kimani, Joshua; Masson, Lindi; Liebenberg, Lenine J; Abdool Karim, Salim S; Passmore, Jo-Ann S; Lauffenburger, Douglas A; Kaul, Rupert; McKinnon, Lyle R

    2016-01-01

    Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

  14. Microwave hyperthermia-induced blood-brain barrier alterations

    International Nuclear Information System (INIS)

    Lin, J.C.; Lin, M.F.

    1982-01-01

    We have studied the interaction of microwaves with the blood-brain barrier in Wistar rats. Indwelling catheters were placed in the femoral vein. Evans blue in isotonic saline was used as a visual indicator of barrier permeation. Irradiation with pulsed 2450-MHz microwaves for 20 min at average power densities of 0.5 to 2600 mW/cm 2 , which resulted in average specific absorption rages (SARs) of 0.04 to 200 mW/g in the brain, did not produce staining, except in regions that normally are highly permeable. When the incident power density was increased to 3000 mW/cm 2 (SAR of 240 mW/g), extravasation of Evans blue could be seen in the cortex, hippocampus, and midbrain. The rectal temperature, as monitored by a copper-constantan thermocouple, showed a maximum increase of less than 1.0/sup o/C. the brain temperature recorded in a similar group of animals using a non-field-perturbing thermistor exceeded 43/sup o/C. At the higher power density the extravasation depended on the irradition and euthanization times. In one series of experiments, rats were irradiated at 3000 mW/cm 2 for 5, 10, 15, and 20 min. Immediately after irradiation all except the 5-min animals exhibited increased permeability in some regions of the brain. Brains of rats euthanized 30 min after irradiation were free of Evans blue, while those euthanized 10 and 20 min postirradiation showed significant dye staining but with less intensity than those euthanized immediately after irradiation

  15. Clerics urge ban on altering germline cells.

    Science.gov (United States)

    Norman, C

    1983-06-24

    A resolution calling for a ban on genetic engineering of human reproductive cells has been signed by leaders of almost every major church group in the United States. Some of the religious leaders, while not certain that a total moratorium should be placed on altering germline cells, signed the statement in order to stimulate public debate on the issue. Legislation has recently been introduced in Congress to set up a committee to monitor genetic engineering and its human applications, but author Jeremy Rifkin, the impetus behind the church leaders' resolution, argues that such tampering threatens the gene pool and should be banned altogether.

  16. Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model.

    Directory of Open Access Journals (Sweden)

    Svitlana Garbuzova-Davis

    Full Text Available Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas.In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO, significant BBB alterations characterized by large Evans Blue (EB parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices.These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke.

  17. Cell proliferation alterations in Chlorella cells under stress conditions

    International Nuclear Information System (INIS)

    Rioboo, Carmen; O'Connor, Jose Enrique; Prado, Raquel; Herrero, Concepcion; Cid, Angeles

    2009-01-01

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  18. Cell proliferation alterations in Chlorella cells under stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: cid@udc.es [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)

    2009-09-14

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  19. Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

    Science.gov (United States)

    Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

    2017-09-01

    Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Molten carbonate fuel cell integral matrix tape and bubble barrier

    International Nuclear Information System (INIS)

    Reiser, C.A.; Maricle, D.L.

    1983-01-01

    A molten carbonate fuel cell matrix material is described made up of a matrix tape portion and a bubble barrier portion. The matrix tape portion comprises particles inert to molten carbonate electrolyte, ceramic particles and a polymeric binder, the matrix tape being flexible, pliable and having rubber-like compliance at room temperature. The bubble barrier is a solid material having fine porosity preferably being bonded to the matrix tape. In operation in a fuel cell, the polymer binder burns off leaving the matrix and bubble barrier providing superior sealing, stability and performance properties to the fuel cell stack

  1. Altered Blood-Brain Barrier Permeability in Patients With Systemic Lupus Erythematosus: A Novel Imaging Approach.

    Science.gov (United States)

    Gulati, Gaurav; Jones, Jordan T; Lee, Gregory; Altaye, Mekibib; Beebe, Dean W; Meyers-Eaton, Jamie; Wiley, Kasha; Brunner, Hermine I; DiFrancesco, Mark W

    2017-02-01

    To evaluate a safe, noninvasive magnetic resonance imaging (MRI) method to measure regional blood-brain barrier integrity and investigate its relationship with neurocognitive function and regional gray matter volume in juvenile-onset systemic lupus erythematosus (SLE). In this cross-sectional, case-control study, capillary permeability was measured as a marker of blood-brain barrier integrity in juvenile SLE patients and matched healthy controls, using a combination of arterial spin labeling and diffusion-weighted brain MRI. Regional gray matter volume was measured by voxel-based morphometry. Correlation analysis was done to investigate the relationship between regional capillary permeability and regional gray matter volume. Formal neurocognitive testing was completed (measuring attention, visuoconstructional ability, working memory, and psychomotor speed), and scores were regressed against regional blood-brain barrier integrity among juvenile SLE patients. Formal cognitive testing confirmed normal cognitive ability in all juvenile SLE subjects (n = 11) included in the analysis. Regional capillary permeability was negatively associated (P = 0.026) with neurocognitive performance concerning psychomotor speed in the juvenile SLE cohort. Compared with controls (n = 11), juvenile SLE patients had significantly greater capillary permeability involving Brodmann's areas 19, 28, 36, and 37 and caudate structures (P < 0.05 for all). There is imaging evidence of increased regional capillary permeability in juvenile SLE patients with normal cognitive performance using a novel noninvasive MRI technique. These blood-brain barrier outcomes appear consistent with functional neuronal network alterations and gray matter volume loss previously observed in juvenile SLE patients with overt neurocognitive deficits, supporting the notion that blood-brain barrier integrity loss precedes the loss of cognitive ability in juvenile SLE. Longitudinal studies are needed to

  2. Evaluation of a cyanoacrylate dressing to manage peristomal skin alterations under ostomy skin barrier wafers.

    Science.gov (United States)

    Milne, Catherine T; Saucier, Darlene; Trevellini, Chenel; Smith, Juliet

    2011-01-01

    Peristomal skin alterations under ostomy barrier wafers are a commonly reported problem. While a number of interventions to manage this issue have been reported, the use of a topically applied cyanoacrylate has received little attention. This case series describes the use of a topical cyanoacrylate for the management of peristomal skin alterations in persons living with an ostomy. Using a convenience sample, the topical cyanoacrylate dressing was applied to 11 patients with peristomal skin disruption under ostomy wafers in acute care and outpatient settings. The causes of barrier function interruption were also addressed to enhance outcomes. Patients were assessed for wound discomfort using a Likert Scale, time to healing, and number of appliance changes. Patient satisfaction was also examined. Average reported discomfort levels were 9.5 out of 10 at the initial peristomal irritation assessment visit decreased to 3.5 at the first wafer change and were absent by the second wafer change. Wafers had increasing wear time between changes in both settings with acute care patients responding faster. Epidermal resurfacing occurred within 10.2 days in outpatients and within 7 days in acute care patients. Because of the skin sealant action of this dressing, immediate adherence of the wafer was reported at all pouch changes.

  3. Functional and structural alterations of epithelial barrier properties of rat ileum following X-irradiation

    International Nuclear Information System (INIS)

    Dublineau, I.; Lebrun, F.; Grison, S.; Griffiths, N.M.

    2004-01-01

    Irradiation of the digestive system leads to alterations of the small intestine. We have characterized the disruption of the barrier integrity in rat ileum from 1 to 14 days following irradiation ranging from 6 to 12 Gy. The intestinal permeability to 14 C-mannitol and 3 H-dextran 70,000 was measured in vitro in Ussing chambers. In parallel to these functional studies, immunohistochemical analyses of junctional proteins (ZO-1 and β-catenin) of ileal epithelium were performed by confocal microscopy. Irradiation with 10 Gy induced a marked decrease in epithelial tissue resistance at three days and a fivefold increase in mannitol permeability, without modifications of dextran permeability. A disorganization of the localization for ZO-1 and β-catenin was also observed. At 7 days after irradiation, we observed a recovery of the organization of junctional proteins in parallel to a return of intestinal permeability to control value. In addition to these time-dependent effects, a gradual effect on epithelial integrity of the radiation doses was observed 3 days after irradiation. This study shows a disruption of the integrity of the intestinal barrier in rat ileum following abdominal X-irradiation, depending on the time postirradiation and on the delivered dose. The loss of barrier integrity was characterized by a disorganization of proteins of tight and adherent junctions, leading to increased intestinal permeability to mannitol. (author)

  4. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

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

  5. Alteration of intestinal barrier function during activity-based anorexia in mice.

    Science.gov (United States)

    Jésus, Pierre; Ouelaa, Wassila; François, Marie; Riachy, Lina; Guérin, Charlène; Aziz, Moutaz; Do Rego, Jean-Claude; Déchelotte, Pierre; Fetissov, Sergueï O; Coëffier, Moïse

    2014-12-01

    Anorexia nervosa is a severe eating disorder often leading to malnutrition and cachexia, but its pathophysiology is still poorly defined. Chronic food restriction during anorexia nervosa may induce gut barrier dysfunction, which may contribute to disease development and its complications. Here we have characterized intestinal barrier function in mice with activity-based anorexia (ABA), an animal model of anorexia nervosa. Male C57Bl/6 ABA or limited food access (LFA) mice were placed respectively in cages with or without activity wheel. After 5 days of acclimatization, both ABA and LFA mice had progressively limited access to food from 6 h/d at day 6 to 3 h/d at day 9 and until the end of experiment at day 17. A group of pair-fed mice (PF) was also compared to ABA. On day 17, food intake was lower in ABA than LFA mice (2.0 ± 0.18 g vs. 3.0 ± 0.14 g, p anorexia nervosa. The role of these alterations in the pathophysiology of anorexia nervosa should be further evaluated. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  6. Escherichia coli challenge and one type of smectite alter intestinal barrier of pigs.

    Science.gov (United States)

    Almeida, Juliana Abranches Soares; Liu, Yanhong; Song, Minho; Lee, Jeong Jae; Gaskins, H Rex; Maddox, Carol Wolfgang; Osuna, Orlando; Pettigrew, James Eugene

    2013-12-20

    An experiment was conducted to determine how an E. coli challenge and dietary clays affect the intestinal barrier of pigs. Two groups of 32 pigs (initial BW: 6.9 ± 1.0 kg) were distributed in a 2 × 4 factorial arrangement of a randomized complete block design (2 challenge treatments: sham or E. coli, and 4 dietary treatments: control, 0.3% smectite A, 0.3% smectite B and 0.3% zeolite), with 8 replicates total. Diarrhea score, growth performance, goblet cell size and number, bacterial translocation from intestinal lumen to lymph nodes, intestinal morphology, and relative amounts of sulfo and sialo mucins were measured. The E. coli challenge reduced performance, increased goblet cell size and number in the ileum, increased bacterial translocation from the intestinal lumen to the lymph nodes, and increased ileal crypt depth. One of the clays (smectite A) tended to increase goblet cell size in ileum, which may indicate enhanced protection. In conclusion, E. coli infection degrades intestinal barrier integrity but smectite A may enhance it.

  7. Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

    International Nuclear Information System (INIS)

    Ellison, M.D.B.

    1985-01-01

    The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study compares in rats, following acute hypertension, the cerebrovascular passage of 14 C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction

  8. Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

    Science.gov (United States)

    Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

    1995-06-01

    We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

  9. Physiological alterations in UV-irradiated cells: liquid holding recovery

    International Nuclear Information System (INIS)

    Aragao, B.R.

    1980-01-01

    The biochemical and physiological alterations that occur in ultraviolet irradiated cells, during liquid holding have been studied. Incubation in buffer acts not to interfer directly with the mechanic repairs but by promoting metabolic alterations that would block some irreversible and lethal physiological responses. (L.M.J.) [pt

  10. Electrolyte creepage barrier for liquid electrolyte fuel cells

    Science.gov (United States)

    Li, Jian [Alberta, CA; Farooque, Mohammad [Danbury, CT; Yuh, Chao-Yi [New Milford, CT

    2008-01-22

    A dielectric assembly for electrically insulating a manifold or other component from a liquid electrolyte fuel cell stack wherein the dielectric assembly includes a substantially impermeable dielectric member over which electrolyte is able to flow and a barrier adjacent the dielectric member and having a porosity of less than 50% and greater than 10% so that the barrier is able to measurably absorb and chemically react with the liquid electrolyte flowing on the dielectric member to form solid products which are stable in the liquid electrolyte. In this way, the barrier inhibits flow or creepage of electrolyte from the dielectric member to the manifold or component to be electrically insulated from the fuel cell stack by the dielectric assembly.

  11. Theoretical cell alteration model in the context of carcinogenesis

    International Nuclear Information System (INIS)

    Walsh, P.J.

    1976-01-01

    A model incorporating cell survival and alteration is used to discuss the general nature of cellular response to a toxic agent. Cell division and repair are discussed as regards their influence on dose-response relationships to bone-seeking radionuclides. The application of the model in its present form to specific biologic end points depends on the assumption that such end points are the result of some initial alteration

  12. InGaP Heterojunction Barrier Solar Cells

    Science.gov (United States)

    Welser, Roger E. (Inventor)

    2014-01-01

    A new solar cell structure called a heterojunction barrier solar cell is described. As with previously reported quantum-well and quantum-dot solar cell structures, a layer of narrow band-gap material, such as GaAs or indium-rich InGaP, is inserted into the depletion region of a wide band-gap PN junction. Rather than being thin, however, the layer of narrow band-gap material is about 400-430 nm wide and forms a single, ultrawide well in the depletion region. Thin (e.g., 20-50 nm), wide band-gap InGaP barrier layers in the depletion region reduce the diode dark current. Engineering the electric field and barrier profile of the absorber layer, barrier layer, and p-type layer of the PN junction maximizes photogenerated carrier escape. This new twist on nanostructured solar cell design allows the separate optimization of current and voltage to maximize conversion efficiency.

  13. CRIEPI's research results (2006-2011) and clarified future issues on alteration behavior of bentonite barrier by alkaline solutions

    International Nuclear Information System (INIS)

    Yokoyama, Shingo; Nakamura, Kunihiko; Tanaka, Yukihisa; Hironaga, Michihiko

    2013-01-01

    In radioactive waste disposal facilities, bentonite barrier would be altered by alkaline solutions which arise by leaching of cementitious materials. Consequently suitable properties of the bentonite barrier would be degraded for a long time period. In CRIEPI, the investigation on the alteration of the bentonite under alkaline conditions was started in 2006, and several CRIEPI reports have been published. Specifically, we have investigated the kinetics of montmorillonite dissolution, the mineralogical alteration of compacted bentonite (with high- and low-dry density) and the change of permeability of the compacted bentonite (with high- and low-dry density) during alteration under the alkaline conditions. Furthermore, stability of saponite, which has similar physical properties to the bentonite, under the alkaline conditions was also examined. In this report, we show the outline of those research results, and lay out the clarified future issues extracted from our results. Ten clarified future issues were divided three categories as follows: 1) the estimation of the alteration behavior of the bentonite by alkaline solutions, 2) the elucidation of the mechanism of physical properties (e.g., permeability, swelling properties and mechanistic properties) change of the compacted bentonites during alteration, and 3) the development of the model building and simulation technology concerning the change in physical properties during alteration under alkaline conditions. (author)

  14. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  15. Alteration of cell cycle progression by Sindbis virus infection

    International Nuclear Information System (INIS)

    Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

    2015-01-01

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G 1 phase preferred to proliferate during S/G 2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G 1 phase than in cells infected during S/G 2 phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases

  16. Altered blood-brain barrier permeability in rats with prehepatic portal hypertension turns to normal when portal pressure is lowered

    Science.gov (United States)

    Eizayaga, Francisco; Scorticati, Camila; Prestifilippo, Juan P; Romay, Salvador; Fernandez, Maria A; Castro, José L; Lemberg, Abraham; Perazzo, Juan C

    2006-01-01

    AIM: To study the blood-brain barrier integrity in prehepatic portal hypertensive rats induced by partial portal vein ligation, at 14 and 40 d after ligation when portal pressure is spontaneously normalized. METHODS: Adult male Wistar rats were divided into four groups: Group I: Sham14d , sham operated; Group II: PH14d , portal vein stenosis; (both groups were used 14 days after surgery); Group III: Sham40d, Sham operated and Group IV: PH40d Portal vein stenosis (Groups II and IV used 40 d after surgery). Plasma ammonia, plasma and cerebrospinal fluid protein and liver enzymes concentrations were determined. Trypan and Evans blue dyes, systemically injected, were investigated in hippocampus to study blood-brain barrier integrity. Portal pressure was periodically recorded. RESULTS: Forty days after stricture, portal pressure was normalized, plasma ammonia was moderately high, and both dyes were absent in central nervous system parenchyma. All other parameters were reestablished. When portal pressure was normalized and ammonia level was lowered, but not normal, the altered integrity of blood-brain barrier becomes reestablished. CONCLUSION: The impairment of blood-brain barrier and subsequent normalization could be a mechanism involved in hepatic encephalopathy reversibility. Hemodynamic changes and ammonia could trigger blood-brain barrier alterations and its reestablishment. PMID:16552803

  17. Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis.

    Science.gov (United States)

    Ciccia, Francesco; Guggino, Giuliana; Rizzo, Aroldo; Alessandro, Riccardo; Luchetti, Michele Maria; Milling, Simon; Saieva, Laura; Cypers, Heleen; Stampone, Tommaso; Di Benedetto, Paola; Gabrielli, Armando; Fasano, Alessio; Elewaut, Dirk; Triolo, Giovanni

    2017-06-01

    Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Ileal biopsies were obtained from 50 HLA-B27 + patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour. Published by the BMJ Publishing Group Limited. For permission to use

  18. Modeling the Response of Human Altered Natural Barrier Island Dynamics Along Assateague Island National Seashore to Climate Change

    Science.gov (United States)

    Carroll, A.; McNamara, D.; Schupp, C.

    2009-12-01

    Assateague Island National Seashore comprises a long barrier island located off the coasts of Maryland and Virginia. Geological evidence suggests that over recent centuries Assateague Island has steadily transgressed up the continental shelf in response to rising sea level. More recently, the natural barrier island dynamics governing Assateague’s evolution have been altered by human activity in three ways: the construction of a jetty and the subsequent interruption of alongshore sediment transport on the north end of Assateague and both the ongoing and abandoned maintenance of a continuous dune system along portions of Assateague with the concomitant modification to overwash dynamics. It is unclear how these varied human alterations to the natural barrier island dynamics will influence the response of Assateague to climate change induced shifts in forcing such as increased rates of sea level rise and changing storm patterns. We use LIDAR detected morphological data of Assateague Island as initial conditions in an alongshore extended model for barrier island dynamics including beach erosion, island overwash and inlet cutting during storms, and beach accretion, tidal delta growth and dune and vegetation growth between storms to explore the response of the various human altered segments of Assateague Island to forcing changes. Traditional models exploring barrier island evolution contain only cross-shore dynamics therefore lacking important alongshore-spatial dynamics in aeolian and surf zone sediment transport. Results show that including alongshore dynamics alter the steady state of Assateague relative to simulations that only include cross-shore dynamics. Results will also be presented exploring the potential for regime shifts in steady state behavior under various scenarios for the rate of sea level rise and storm climate and varying management strategies.

  19. Deciding Which Way to Go: How Do Insects alter Movements to Negotiate Barriers?

    Directory of Open Access Journals (Sweden)

    Roy E. Ritzmann

    2012-07-01

    Full Text Available Animals must routinely deal with barriers as they move through their natural environment. These challenges require directed changes in leg movements and posture performed in the context of ever changing internal and external conditions. In particular, cockroaches use a combination of tactile and visual information to evaluate objects in their path in order to effectively guide their movements in complex terrain. When encountering a large block, the insect uses its antennae to evaluate the object’s height then rears upward accordingly before climbing. A shelf presents a choice between climbing and tunneling that depends on how the antennae strike the shelf; tapping from above yields climbing, while tapping from below causes tunneling. However, ambient light conditions detected by the ocelli can bias that decision. Similarly, in a T-maze turning is determined by antennal contact but influenced by visual cues. These multi-sensory behaviors led us to look at the central complex as a center for sensori-motor integration within the insect brain. Visual and antennal tactile cues are processed within the central complex and, in tethered preparations, several central complex units changed firing rates in tandem with or prior to altered step frequency or turning, while stimulation through the implanted electrodes evoked these same behavioral changes. To further test for a central complex role in these decisions, we examined behavioral effects of brain lesions. Electrolytic lesions in restricted regions of the central complex generated site specific behavioral deficits. Similar changes were also found in reversible effects of procaine injections in the brain. Finally, we are examining these kinds of decisions made in a large arena that more closely matches the conditions under which cockroaches forage. Overall, our studies suggest that CC circuits may indeed influence the descending commands associated with navigational decisions, thereby making them

  20. Alterations in Factors Involved in Differentiation and Barrier Function in the Epithelium in Oral and Genital Lichen Planus.

    Science.gov (United States)

    Danielsson, Karin; Ebrahimi, Majid; Nylander, Elisabet; Wahlin, Ylva Britt; Nylander, Karin

    2017-02-08

    Lichen planus is a chronic recurrent inflammatory disease affecting both skin and mucosa, mainly in oral and/or genital regions. Keratinocytes go through a well-regulated process of proliferation and differentiation, alterations in which may result in defects in the protective epithelial barrier. Long-term barrier impairment might lead to chronic inflammation. In order to broaden our understanding of the differentiation process in mucosal lichen planus, we mapped the expression of 4 factors known to be involved in differentiation. Biopsies were collected from oral and genital lichen planus lesions and normal controls. Altered expression of all 4 factors in epithelium from lichen planus lesions was found, clearly indicating disturbed epithelial differentiation in lichen planus lesions.

  1. SURFACE-ALTERED ZEOLITES AS PERMEABLE BARRIERS FOR IN SITU TREATMENT OF CONTAMINATED GROUNDWATER

    International Nuclear Information System (INIS)

    Bowman, Robert S.; Li, Zhaohui; Roy, Stephen J.; Burt, Todd; Johnson, Timothy L.; Johnson, Richard L.

    1999-01-01

    The overall objective of this effort is to develop and test a zeolite-based permeable barrier system for containing and remediating contaminated groundwater. The projected product is an engineered and tested permeable barrier system that can be adopted by the commercial sector

  2. SURFACE-ALTERED ZEOLITES AS PERMEABLE BARRIERS FOR IN SITU TREATMENT OF CONTAMINATED GROUNDWATER

    Energy Technology Data Exchange (ETDEWEB)

    Robert S. Bowman; Zhaohui Li; Stephen J. Roy; Todd Burt; Timothy L. Johnson; Richard L. Johnson

    1999-08-30

    The overall objective of this effort is to develop and test a zeolite-based permeable barrier system for containing and remediating contaminated groundwater. The projected product is an engineered and tested permeable barrier system that can be adopted by the commercial sector.

  3. Cigarette smoke alters the secretome of lung epithelial cells.

    Science.gov (United States)

    Mossina, Alessandra; Lukas, Christina; Merl-Pham, Juliane; Uhl, Franziska E; Mutze, Kathrin; Schamberger, Andrea; Staab-Weijnitz, Claudia; Jia, Jie; Yildirim, Ali Ö; Königshoff, Melanie; Hauck, Stefanie M; Eickelberg, Oliver; Meiners, Silke

    2017-01-01

    Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    During the last decade, cell-penetrating peptides have been investigated for their ability to overcome the plasma membrane barrier of mammalian cells for the intracellular or transcellular delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides......, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell......-penetrating peptides as transmembrane drug delivery agents, according to the recent literature, and discusses critical issues and future challenges in relation to fully understanding the fundamental principles of the cell-penetrating peptide-mediated membrane translocation of cargoes and the exploitation...

  5. Small molecule alteration of RNA sequence in cells and animals.

    Science.gov (United States)

    Guan, Lirui; Luo, Yiling; Ja, William W; Disney, Matthew D

    2017-10-18

    RNA regulation and maintenance are critical for proper cell function. Small molecules that specifically alter RNA sequence would be exceptionally useful as probes of RNA structure and function or as potential therapeutics. Here, we demonstrate a photochemical approach for altering the trinucleotide expanded repeat causative of myotonic muscular dystrophy type 1 (DM1), r(CUG) exp . The small molecule, 2H-4-Ru, binds to r(CUG) exp and converts guanosine residues to 8-oxo-7,8-dihydroguanosine upon photochemical irradiation. We demonstrate targeted modification upon irradiation in cell culture and in Drosophila larvae provided a diet containing 2H-4-Ru. Our results highlight a general chemical biology approach for altering RNA sequence in vivo by using small molecules and photochemistry. Furthermore, these studies show that addition of 8-oxo-G lesions into RNA 3' untranslated regions does not affect its steady state levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Physicochemical and Geotechnical Alterations to MX-80 Bentonite at the Waste Canister Interface in an Engineered Barrier System

    Directory of Open Access Journals (Sweden)

    Christopher W. Davies

    2017-08-01

    Full Text Available The study investigated the basic geomechanical and mineralogical evolution of the bentonite barrier under various experimental boundary conditions which replicated the near-field Thermo-Hydro-Chemico (THC conditions in a repository. The relationships between the physicochemical alterations and changes in the geotechnical properties have seldom been studied, especially on a consistent dataset. This paper attempts to link the physicochemical properties of Na-bentonite (MX-80 to the macro-scale engineering functionality of the bentonite post THC exposure. Experiments investigated the impact of THC variables on the engineering and physicochemical functionality of the bentonite with respect to its application within a High-Level Waste (HLW engineered barrier system. Intrinsic alterations to the MX-80 bentonite under relatively short-term exposure to hydrothermal and chemical conditions were measured. Additionally, two long-term tests were conducted under ambient conditions to consider the impact of exposure duration. The intrinsic measurements were then related to the overall performance of the bentonite as a candidate barrier material for application in a UK geological disposal facility. Findings indicate that exposure to thermo-saline-corrosion conditions (i.e., corrosion products derived from structural grade 275 carbon steel inhibits the free swell capacity and plasticity of the bentonite. However, the measured values remained above the design limits set out for the Swedish multi-barrier concept, from which the UK concept may take a lead. Corrosion alone does not appear to significantly affect the geotechnical measurements compared with the influence of thermal loading and high saline pore water after relatively short-term exposure. Thermal and corrosion exposure displayed no impact on the intrinsic swelling of the smectite component, indicating that no significant structural alteration had occurred. However, when exploring more complex saline

  7. Radiation-induced motility alterations in medulloblastoma cells

    International Nuclear Information System (INIS)

    Rieken, Stefan; Rieber, Juliane; Brons, Stephan

    2015-01-01

    Photon irradiation has been repeatedly suspected of increasing tumor cell motility and promoting locoregional recurrence of disease. This study was set up to analyse possible mechanisms underlying the potentially radiation-altered motility in medulloblastoma cells. Medulloblastoma cell lines D425 and Med8A were analyzed in migration and adhesion experiments with and without photon and carbon ion irradiation. Expression of integrins was determined by quantitative FACS analysis. Matrix metalloproteinase concentrations within cell culture supernatants were investigated by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using Student's t-test. Both photon and carbon ion irradiation significantly reduced chemotactic medulloblastoma cell transmigration through 8-μm pore size membranes, while simultaneously increasing adherence to fibronectin- and collagen I- and IV-coated surfaces. Correspondingly, both photon and carbon ion irradiation downregulate soluble MMP9 concentrations, while upregulating cell surface expression of proadhesive extracellular matrix protein-binding integrin α 5 . The observed phenotype of radiation-altered motility is more pronounced following carbon ion than photon irradiation. Both photon and (even more so) carbon ion irradiation are effective in inhibiting medulloblastoma cell migration through downregulation of matrix metalloproteinase 9 and upregulation of proadhesive cell surface integrin α 5 , which lead to increased cell adherence to extracellular matrix proteins. (author)

  8. Cell elasticity with altered cytoskeletal architectures across multiple cell types.

    Science.gov (United States)

    Grady, Martha E; Composto, Russell J; Eckmann, David M

    2016-08-01

    The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function

    DEFF Research Database (Denmark)

    Bailey, D M; Evans, K A; James, P E

    2008-01-01

    We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood-brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia-mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O...... developed clinical AMS (AMS+) and were more hypoxaemic relative to subjects without AMS (AMS-). A more marked increase in the venous concentration of the ascorbate radical (A(*-)), lipid hydroperoxides (LOOH) and increased susceptibility of low-density lipoprotein (LDL) to oxidation was observed during...

  10. Biliary obstruction dissipates bioelectric sinusoidal-canalicular barrier without altering taurocholate uptake

    International Nuclear Information System (INIS)

    Cotting, J.; Zysset, T.; Reichen, J.

    1989-01-01

    To study immediate events during extrahepatic cholestasis, we investigated the effect of short-term biliary obstruction on the bioelectrical sinusoidal-canalicular barrier in the rat using molecular weight-matched uncharged and negatively charged inert solute pairs. The bioelectrical barrier averaged -22 +/- 5 and -18 +/- 4 mV (NS) using the pair carboxy-/methoxyinulin and ferrocyanide/sucrose, respectively. After a 20-min biliary obstruction both decreased by 61 and 11%, respectively, but only the large molecular weight pair (the inulins) returned to base line after release of the obstruction. Inert solute clearances were increased after short biliary obstruction depending on molecular size and negative charge (ferrocyanide greater than sucrose greater than carboxyinulin greater than inulin), suggesting that both permeability and bioelectrical barriers were affected by obstruction. The hepatic extraction in vivo of a passively transported drug not excreted into bile (D-propranolol) was not affected by obstruction, whereas that of an actively transported drug (glycocholate) decreased from 66 +/- 8 to 41 +/- 20% during biliary obstruction (P less than 0.01). Unidirectional transfer of glycocholate was not affected by short-term biliary obstruction in the situ perfused rat liver; however, 2 min after [14C]glycocholate administration, increased return was observed in hepatic venous effluent in obstructed animals. Our findings demonstrate a loss of the bioelectrical barrier immediately after short-term biliary obstruction. Decreased hepatic extraction in the view of unaltered sinusoidal uptake demonstrates regurgitation of bile into blood during short-term biliary obstruction

  11. TCDD alters medial epithelial cell differentiation during palatogenesis

    International Nuclear Information System (INIS)

    Abbott, B.D.; Birnbaum, L.S.

    1989-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of [3H]TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation

  12. E. coli O124 K72 alters the intestinal barrier and the tight junctions proteins of guinea pig intestine.

    Science.gov (United States)

    Ren, Xiaomeng; Zhu, Yanyan; Gamallat, Yaser; Ma, Shenhao; Chiwala, Gift; Meyiah, Abdo; Xin, Yi

    2017-10-01

    Our research group previously isolated and identified a strain of pathogenic Escherichia coli from clinical samples called E. coli O124 K72. The present study was aimed at determining the potential effects of E. coli O124 K72 on intestinal barrier functions and structural proteins integrity in guinea pig. Guinea pigs were grouped into three groups; control (CG); E. coli O124 K72 (E. coli); and probiotics Lactobacillus rhamnosus (LGG). Initially, we create intestinal dysbiosis by giving all animals Levofloxacin for 10days, but the control group (CG) received the same volume of saline. Then, the animals received either E. coli O124 K72 (E. coli) or Lactobacillus rhamnosus (LGG) according to their assigned group. E. coli O124 K72 treatment significantly affected colon morphology and distorted intestinal barrier function by up-regulating Claudin2 and down-regulating Occludin. In addition, E. coli upregulated the mRNA expression of MUC1, MUC2, MUC13 and MUC15. Furthermore, suspected tumor was found in the E. coli treated animals. Our results suggested that E. coli O124 K72 strain has adverse effects on intestinal barrier functions and is capable of altering integrity of structural proteins in guinea pig model while at same time it may have a role in colon carcinogenesis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Genetic alterations in head and neck squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    Nagai M.A.

    1999-01-01

    Full Text Available The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations and tumor suppressor gene inactivation (loss of function mutations, leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

  14. Alterations induced in Escherichia Coli cells by gamma radiation

    International Nuclear Information System (INIS)

    Kappke, J.; Schelin, H.R.; Paschuk, S.A.; Denyak, V.; Silva, E.R. da; Jesus, E.F.O. de; Lopes, R.T.; Carlin, N.; Toledo, E.S.

    2007-01-01

    Modifications occurred in Escherichia coli cells exposed to gamma radiation ( 60 Co source) were investigated. The irradiations were done at the LIN-COPPE laboratory of the UFRJ and the analysis at the Biology Department of the UTFPR. The E. coli cells were irradiated with 30, 60, 90, 120, 150, 180, 210, 240, 300, 480, 600 e 750 Gy doses. The samples were analyzed with Gram-stain, biochemical tests in EPM, MIO and Lysine Broth, Simmons Cytrate Medium and Rhamnose Broth, antibiogram and isolation of auxotrophic mutants. It was observed that for the received doses the E. coli did not show morphological alterations in the tests. Some E. Coli cells showed to be able to deaminade the L-tryptophan or they changed their sensibility for amoxillin and cephaloonine after the irradiation. The existence of aauxotrophic mutants after irradiation was also verified. (author)

  15. STAMP alters the growth of transformed and ovarian cancer cells

    International Nuclear Information System (INIS)

    He, Yuanzheng; Blackford, John A Jr; Kohn, Elise C; Simons, S Stoney Jr

    2010-01-01

    Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC 50 ) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear. The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines. Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation. This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This

  16. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

    2010-08-20

    Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  17. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    International Nuclear Information System (INIS)

    Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

    2010-01-01

    Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  18. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Energy Technology Data Exchange (ETDEWEB)

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.

    2017-05-23

    ABSTRACT

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

    IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

  19. Ureaplasma parvum infection alters filamin a dynamics in host cells

    Directory of Open Access Journals (Sweden)

    Brown Mary B

    2011-04-01

    Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

  20. Genetic alterations in B-cell non-Hodgkin's lymphoma

    Directory of Open Access Journals (Sweden)

    Magić Zvonko

    2005-01-01

    Full Text Available Background. Although the patients with diagnosed B-NHL are classified into the same disease stage on the basis of clinical, histopathological, and immunological parameters, they respond significantly different to the applied treatment. This points out the possibility that within the same group of lymphoma there are different diseases at molecular level. For that reason many studies deal with the detection of gene alterations in lymphomas to provide a better framework for diagnosis and treatment of these hematological malignancies. Aim. To define genetic alterations in the B-NHL with highest possibilities for diagnostic purposes and molecular detection of MRD. Methods. Formalin fixed and paraffin embedded lymph node tissues from 45 patients were examined by different PCR techniques for the presence of IgH and TCR γ gene rearrangement; K-ras and H-ras mutations; c-myc amplification and bcl-2 translocation. There were 34 cases of B-cell non-Hodgkin’s lymphoma (B-NHL, 5 cases of T-cell non-Hodgkin’s lymphoma (T-NHL and 6 cases of chronic lymphadenitis (CL. The mononuclear cell fraction of the peripheral blood of 12 patients with B-NHL was analyzed for the presence of monoclonality at the time of diagnosis and in 3 to 6 months time intervals after an autologous bone marrow transplantation (BMT. Results. The monoclonality of B-lymphocytes, as evidenced by DNA fragment length homogeneity, was detected in 88 % (30/34 of B-NHL, but never in CL, T-NHL, or in normal PBL. Bcl-2 translocation was detected in 7/31 (22.6% B-NHL specimens, c-myc amplification 9/31 (29%, all were more than doubled, K-ras mutations in 1/31 (3.23% and H-ras mutations in 2/31 (6.45% of the examined B-NHL samples. In the case of LC and normal PBL, however, these gene alterations were not detected. All the patients (12 with B-NHL had dominant clone of B-lymphocyte in the peripheral blood at the time of diagnosis while only in 2 of 12 patients MRD was detected 3 or 6 months after

  1. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  3. Alteration of non-metallic barriers and evolution of solution chemistry in salt formations in Germany

    International Nuclear Information System (INIS)

    Herbert, H.J.; Becker, D.; Hagemann, S.; Meyer, Th.; Noseck, U.; Rubel, A.; Mauke, R.; Wollrath, J.

    2005-01-01

    Different Engineered Barrier Systems (EBS) materials considered in Germany for the sealing of repositories in salt formations are presented. Their long term behaviour in terms of interactions with salt solutions is discussed and evaluated. The discussed EBS materials are crushed salt, self sealing salt backfill, bentonite and salt concrete. Whereas the knowledge concerning the geochemical, geomechanical, hydrological and thermal behavior of crushed salt and salt concrete is well advanced further research is needed for other EBS materials. The self healing salt backfill has also been investigated in depth recently. In order to fully qualify this material large scale in situ experiments are still needed. The present knowledge on compacted bentonites in a salt environment is not yet sufficient for reliable predictions of the long-term performance in salt formations. The sealing concept of the low- and intermediate-level Radioactive Waste Repository Morsleben (ERAM) in a former rock salt and potash mine is presented. This concept is based on cementitious materials, i.e. salt concrete. The geochemical stability of different salt concretes in contact with brines expected in ERAM is addressed. It is shown how the results from leaching experiments and geochemical modelling are used in the safety analyses and how the chemical boundary conditions prevailing in the EBS influence the development of the permeability of the sealing system and thus control the radionuclide release. As a result of modelling the behaviour of the seals in the safety assessment it is shown, that the seals are corroded within a time span of about 20 000 years. The influence of the uncertainty in the model parameters on the safety of the repository was assessed by a variation of the initial permeability of the seal. The maximum dose rate resulting from the radionuclide release from ERAM is nearly independent of the variation of the initial permeability within four orders of magnitude. (authors)

  4. Alteration of the chronic wasting disease species barrier by in vitro prion amplification

    Science.gov (United States)

    Kurt, Timothy D.; Seelig, Davis M.; Schneider, Jay R.; Johnson, Christopher J.; Telling, Glenn C.; Heisey, Dennis M.; Hoover, Edward A.

    2011-01-01

    Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrPRES) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrPRES deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.

  5. Side chain variations radically alter the diffusion of poly(2-alkyl-2-oxazoline) functionalised nanoparticles through a mucosal barrier.

    Science.gov (United States)

    Mansfield, Edward D H; de la Rosa, Victor R; Kowalczyk, Radoslaw M; Grillo, Isabelle; Hoogenboom, Richard; Sillence, Katy; Hole, Patrick; Williams, Adrian C; Khutoryanskiy, Vitaliy V

    2016-08-16

    Functionalised nanomaterials are gaining popularity for use as drug delivery vehicles and, in particular, mucus penetrating nanoparticles may improve drug bioavailability via the oral route. To date, few polymers have been investigated for their muco-penetration, and the effects of systematic structural changes to polymer architectures on the penetration and diffusion of functionalised nanomaterials through mucosal tissue have not been reported. We investigated the influence of poly(2-oxazoline) alkyl side chain length on nanoparticle diffusion; poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), and poly(2-n-propyl-2-oxazoline) were grafted onto the surface of thiolated silica nanoparticles and characterised by FT-IR, Raman and NMR spectroscopy, thermogravimetric analysis, and small angle neutron scattering. Diffusion coefficients were determined in water and in a mucin dispersion (using Nanoparticle Tracking Analysis), and penetration through a mucosal barrier was assessed using an ex vivo fluorescence technique. The addition of a single methylene group in the side chain significantly altered the penetration and diffusion of the materials in both mucin dispersions and mucosal tissue. Nanoparticles functionalised with poly(2-methyl-2-oxazoline) were significantly more diffusive than particles with poly(2-ethyl-2-oxazoline) while particles with poly(2-n-propyl-2-oxazoline) showed no significant increase compared to the unfunctionalised particles. These data show that variations in the polymer structure can radically alter their diffusive properties with clear implications for the future design of mucus penetrating systems.

  6. Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration.

    Science.gov (United States)

    Karki, Surya B; Yildirim-Ayan, Eda; Eisenmann, Kathryn M; Ayan, Halim

    2017-01-01

    Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy.

  7. Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration

    Directory of Open Access Journals (Sweden)

    Surya B. Karki

    2017-01-01

    Full Text Available Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy.

  8. Altered lipid homeostasis in Sertoli cells stressed by mild hyperthermia.

    Directory of Open Access Journals (Sweden)

    Ana S Vallés

    Full Text Available Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC. Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days of rat testes to 43 °C led to accumulation of neutral lipids. This SC-specific lipid function took 1-2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43 °C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin and microfilament (f-actin organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster.

  9. Alterations in integrin expression modulates invasion of pancreatic cancer cells.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2009-01-01

    BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

  10. Understanding barriers to the introduction of precision medicines in non-small cell lung cancer: A qualitative interview protocol.

    Science.gov (United States)

    Wright, Stuart; Daker-White, Gavin; Newman, William; Payne, Katherine

    2018-01-01

    Background: While precision medicines targeting genetic mutations and alterations in non-small cell lung cancer (NSCLC) have been available since 2010, their adoption into clinical practice has been slow. Evidence suggests that a number of barriers, such as insufficient clinician knowledge, a need for training of test providers, or a lack of specific clinical guidelines, may slow the implementation of precision in general. However, little attention has been given to the barriers to providing precision medicines in NSCLC. The purpose of this protocol is to outline the design for a qualitative interview study to identify the barriers and facilitators to the provision of precision medicines for NSCLC. Methods: This study will use semi-structured interviews with clinicians (n=10), test providers (n=10), and service commissioners (n=10) to identify the perceived barriers and facilitators to providing historical, current, and future precision medicines in NSCLC. Participants will be identified through mailing list advertisements and snowball sampling. Recruitment will continue until data saturation, indicated by no new themes arising from the data. Interviews will be conducted by telephone to facilitate geographical diversity. The qualitative data will be analysed using a framework analysis with themes anticipated to relate to; relevant barriers to providing precision medicines, the impact of different barriers on medicine provision, changes in the ability to provide precision medicines over time, and strategies to facilitate the provision of precision medicines. Ethics: This study has been approved by the University of Manchester Proportionate Review Research Ethics Committee (Reference number: 2017-1885-3619). Written consent will be obtained from all participants. Conclusion: This study is the first to explore the barriers and facilitators to providing precision medicines for NSCLC in the English NHS. The findings will inform strategies to improve the implementation

  11. Altering β-cell number through stable alteration of miR-21 and miR-34a expression

    DEFF Research Database (Denmark)

    Backe, Marie Balslev; Novotny, Guy Wayne; Christensen, Dan Ploug

    2014-01-01

    RNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed...

  12. Cerium oxide nanoparticles alter the salt stress tolerance of Brassica napus L. by modifying the formation of root apoplastic barriers.

    Science.gov (United States)

    Rossi, Lorenzo; Zhang, Weilan; Ma, Xingmao

    2017-10-01

    Rapidly growing global population adds significant strains on the fresh water resources. Consequently, saline water is increasingly tapped for crop irrigation. Meanwhile, rapid advancement of nanotechnology is introducing more and more engineered nanoparticles into the environment and in agricultural soils. While some negative effects of ENPs on plant health at very high concentrations have been reported, more beneficial effects of ENPs at relatively low concentrations are increasingly noticed, opening doors for potential applications of nanotechnology in agriculture. In particular, we found that cerium oxide nanoparticles (CeO 2 NPs) improved plant photosynthesis in salt stressed plants. Due to the close connections between salt stress tolerance and the root anatomical structures, we postulated that CeO 2 NPs could modify plant root anatomy and improve plant salt stress tolerance. This study aimed at testing the hypothesis with Brassica napus in the presence of CeO 2 NPs (0, 500 mg kg -1 dry sand) and/or NaCl (0, 50 mM) in a growth chamber. Free hand sections of fresh roots were taken every seven days for three weeks and the suberin lamellae development was examined under a fluorescence microscope. The results confirmed the hypothesis that CeO 2 NPs modified the formation of the apoplastic barriers in Brassica roots. In salt stressed plants, CeO 2 NPs shortened the root apoplastic barriers which allowed more Na + transport to shoots and less accumulation of Na + in plant roots. The altered Na + fluxes and transport led to better physiological performance of Brassica and may lead to new applications of nanotechnology in agriculture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice.

    Science.gov (United States)

    Cheng, Zhuo; Wang, Liping; Qu, Meijie; Liang, Huaibin; Li, Wanlu; Li, Yongfang; Deng, Lidong; Zhang, Zhijun; Yang, Guo-Yuan

    2018-05-03

    Ischemic stroke induced matrixmetallo-proteinase-9 (MMP-9) upregulation, which increased blood-brain barrier permeability. Studies demonstrated that mesenchymal stem cell therapy protected blood-brain barrier disruption from several cerebrovascular diseases. However, the underlying mechanism was largely unknown. We therefore hypothesized that mesenchymal stem cells reduced blood-brain barrier destruction by inhibiting matrixmetallo-proteinase-9 and it was related to intercellular adhesion molecule-1 (ICAM-1). Adult ICR male mice (n = 118) underwent 90-min middle cerebral artery occlusion and received 2 × 10 5 mesenchymal stem cell transplantation. Neurobehavioral outcome, infarct volume, and blood-brain barrier permeability were measured after ischemia. The relationship between myeloperoxidase (MPO) activity and ICAM-1 release was further determined. We found that intracranial injection of mesenchymal stem cells reduced infarct volume and improved behavioral function in experimental stroke models (p mesenchymal stem cell-treated mice compared to the control group following ischemia (p cells and myeloperoxidase activity were decreased in mesenchymal stem cell-treated mice (p mesenchymal stem cell therapy attenuated blood-brain barrier disruption in mice after ischemia. Mesenchymal stem cells attenuated the upward trend of MMP-9 and potentially via downregulating ICAM-1 in endothelial cells. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway may influence MMP-9 expression of neutrophils and resident cells, and ICAM-1 acted as a key factor in the paracrine actions of mesenchymal stem cell.

  14. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

  15. Cat retinal ganglion cell receptive-field alterations after 6-hydroxydopamine induced dopaminergic amacrine cell lesions

    International Nuclear Information System (INIS)

    Maguire, G.W.; Smith, E.L. III

    1985-01-01

    Optic tract single-unit recordings were used to study ganglion cell response functions of the intact cat eye after 6-hydroxydopamine (6-OHDA) lesioning of the dopaminergic amacrine cell (AC) population of the inner retina. The impairment of the dopaminergic AC was verified by high pressure-liquid chromatography with electrochemical detection of endogenous dopamine content and by [ 3 H]dopamine high-affinity uptake; the dopaminergic ACs of the treated eyes demonstrated reduced endogenous dopamine content and reduced [ 3 H]dopamine uptake compared with that of their matched controls. Normal appearing [ 3 H]GABA and [ 3 H]-glycine uptake in the treated retinas suggests the absence of any nonspecific action of the 6-OHDA on the neural retina. The impairment of the dopaminergic AC population was found to alter a number of response properties in off-center ganglion cells, but this impairment had only a modest effect on the on-center cells. An abnormally high proportion of the off-center ganglion cells in the 6-OHDA treated eyes possessed nonlinear, Y-type receptive fields. These cells also possessed shift-responses of greater than normal amplitude, altered intensity-response functions, reduced maintained activities, and more transient center responses. Of the on-center type cells, only the Y-type on-center cells were affected by 6-OHDA, possessing higher than normal maintained activities and altered intensity-response functions. The on-center X-cells were unaffected by 6-OHDA treatment. The dopaminergic AC of the photopically adapted cat retina therefore modulates a number of ganglion cell response properties and within the limits of this study is most prominent in off-center ganglion cell circuitry

  16. Analysis of infiltration through a clay radon barrier at an UMTRA disposal cell

    International Nuclear Information System (INIS)

    1991-01-01

    An infiltration study was initiated in January 1988 to assess the percent saturation in, and infiltration through, clay radon barriers of typical Uranium Mill Tailings Remedial Action (UMTRA) Project disposal cells. Predicting infiltration through the radon barrier is necessary to evaluate whether the disposal cell will comply with the proposed US Environmental Protection Agency (EPA) groundwater protection standards (40 CFR 192). The groundwater standards require demonstrating that tailings seepage will not cause background concentrations or maximum concentration limits (MCLs) to be exceeded at the downgradient edge of the disposal facility (the point of compliance, or POC). This demonstration generally consists of incorporating the predicted seepage flux and the concentration of the specific hazardous constituents into a contaminant transport model, and predicting the resultant concentrations at the POC. The infiltration study consisted of a field investigation to evaluate moisture conditions in the radon barrier of the completed Shiprock, New Mexico, UMTRA Project disposal cell and previously completed UMTRA Project disposal cells at Clive, Utah, and Burrell, Pennsylvania. Coring was conducted to measure percent saturation profiles in the radon barriers at these disposal cells. In addition, a detailed investigation of the Shiprock radon barrier was conducted to establish the effects of meteorological stresses on moisture conditions in the filter layer and radon barrier. The Shiprock infiltration study was also intended to characterize hydraulic gradients and operational unsaturated hydraulic conductivities in the radon barrier

  17. Ochratoxim A alters cell adhesion and gap junction intercellular communication in MDCK cells

    International Nuclear Information System (INIS)

    Mally, Angela; Decker, Martina; Bekteshi, Michaela; Dekant, Wolfgang

    2006-01-01

    Ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date, but the mechanism of tumor formation by ochratoxin A remains largely unknown. Cell adhesion and cell-cell communication participate in the regulation of signaling pathways involved in cell proliferation and growth control and it is therefore not surprising that modulation of cell-cell signaling has been implicated in cancer development. Several nephrotoxicants and renal carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercell communication (GJIC) and/or cell adhesion, and the aim of this study was to determine if disruption of cell-cell interactions occurs in kidney epithelial cells in response to OTA treatment. MDCK cells were treated with OTA (0-50 μM) for up to 24 h and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, expression and intracellular localization of Cx43, E-cadherin and β-catenin were determined by immunoblot and immunofluorescence analysis. A clear decrease in the distance of dye transfer was evident following treatment with OTA at concentrations/incubation times which did not affect cell viability. Consistent with the functional inhibition of GJIC, treatment with OTA resulted in a dose-dependent decrease in Cx43 expression. In contrast to Cx43, OTA did not alter total amount of the adherens junction proteins E-cadherin and β-catenin. Moreover, Western blot analysis of Triton X-100 soluble and insoluble protein fractions did not indicate translocation of cell adhesion molecules from the membrane to the cytoplasm. However, a ∼78 kDa fragment of β-catenin was detected in the detergent soluble fraction, indicating proteolytic cleavage of β-catenin. Immunofluorescence analysis also revealed changes in the pattern of both β-catenin and E-cadherin labeling, suggesting that OTA may alter cell-adhesion. Taken together, these data support the hypothesis that disruption of cell-cell

  18. Radiofrequency field emitted by mobile phones and alteration of the blood-brain barrier: how strong is the experimental evidence?

    International Nuclear Information System (INIS)

    Lagroye, I.; Haro, E.; Billaudel, B.; Ruffie, G.; Poulletier de Gannes, F.; Taxile, M.; Laclau, M.; Veyret, B.; Leveque, P.

    2006-01-01

    Full text of publication follows: It is known that high power, thermal radiofrequency radiation (RFR) can alter the blood-brain barrier (BBB) permeability with a brain averaged specific absorption rate (BASAR) threshold evaluated at around 100 W/kg (1). Mobile communication technologies are using RFR with exposure guidelines for public local exposure at 2 W/kg, far lower than the threshold previously mentioned. However, in a paper recently published (2) the occurrence of BBB leakage and brain damage (presence of dark neurons) has been reported 50 days after a single 2-hour exposure of rats to a GSM-900 signal. In that investigation however, bias could have occurred as, for instance, exposed animals were mixed in terms of age (12- to 26-week old) and gender, while those differences were not taken into account in the analysis. Moreover, other groups have published contradictory results (3). Our group undertook a confirmation study of the Salford experiments within an international collaborative programme including technical improvements. Our study includes the detection of dark neurons, alteration of the permeability of the BBB and apoptosis 14 or 50 days after GSM-900 exposure. The exposure setup was the loop antenna that allows for head-only exposure. Five groups of 16 Fisher 344 rats (14 -week old) were exposed to GSM-900 during 2 hours at various SAR levels (0, 0.14 and 2.0 W/kg), or were used as cage control or positive controls. Positive controls were treated with kainic acid (10 mg/kg) or by cold injury (dry ice during 5 minutes). After exposure, rats were kept alive during 14 or 50 days to study brain damages. Then, they were anesthetized with urethane (i.p. 1.5 mg/kg), perfused with PBS and fixed with paraformaldehyde 4% (PAF 4%). Brains were extracted and put in cold PAF 4% during the following night, then placed in cold sucrose 20% during 2-3 days, frozen with isopentane and placed at -80 deg. C. Coding was done on brains. Frozen brains were cut in 3

  19. Radiofrequency field emitted by mobile phones and alteration of the blood-brain barrier: how strong is the experimental evidence?

    Energy Technology Data Exchange (ETDEWEB)

    Lagroye, I.; Haro, E.; Billaudel, B.; Ruffie, G.; Poulletier de Gannes, F.; Taxile, M.; Laclau, M.; Veyret, B. [PIOM/UMR 5501 and Bioelectromagnetics laboratory/EPHE, ENSCPB, 33607 Pessac, (France); Leveque, P. [IRCOM, CNRS UMR 6615, Limoges (France)

    2006-07-01

    Full text of publication follows: It is known that high power, thermal radiofrequency radiation (RFR) can alter the blood-brain barrier (BBB) permeability with a brain averaged specific absorption rate (BASAR) threshold evaluated at around 100 W/kg (1). Mobile communication technologies are using RFR with exposure guidelines for public local exposure at 2 W/kg, far lower than the threshold previously mentioned. However, in a paper recently published (2) the occurrence of BBB leakage and brain damage (presence of dark neurons) has been reported 50 days after a single 2-hour exposure of rats to a GSM-900 signal. In that investigation however, bias could have occurred as, for instance, exposed animals were mixed in terms of age (12- to 26-week old) and gender, while those differences were not taken into account in the analysis. Moreover, other groups have published contradictory results (3). Our group undertook a confirmation study of the Salford experiments within an international collaborative programme including technical improvements. Our study includes the detection of dark neurons, alteration of the permeability of the BBB and apoptosis 14 or 50 days after GSM-900 exposure. The exposure setup was the loop antenna that allows for head-only exposure. Five groups of 16 Fisher 344 rats (14 -week old) were exposed to GSM-900 during 2 hours at various SAR levels (0, 0.14 and 2.0 W/kg), or were used as cage control or positive controls. Positive controls were treated with kainic acid (10 mg/kg) or by cold injury (dry ice during 5 minutes). After exposure, rats were kept alive during 14 or 50 days to study brain damages. Then, they were anesthetized with urethane (i.p. 1.5 mg/kg), perfused with PBS and fixed with paraformaldehyde 4% (PAF 4%). Brains were extracted and put in cold PAF 4% during the following night, then placed in cold sucrose 20% during 2-3 days, frozen with isopentane and placed at -80 deg. C. Coding was done on brains. Frozen brains were cut in 3

  20. Altered Function and Expression of ABC Transporters at the Blood–Brain Barrier and Increased Brain Distribution of Phenobarbital in Acute Liver Failure Mice

    Directory of Open Access Journals (Sweden)

    Li Liu

    2018-03-01

    Full Text Available This study investigated alterations in the function and expression of P-glycoprotein (P-GP, breast cancer resistance protein (BCRP, and multidrug resistance-associated protein 2 (MRP2 at the blood–brain barrier (BBB of acute liver failure (ALF mice and its clinical significance. ALF mice were developed using intraperitoneal injection of thioacetamide. P-GP, BCRP, and MRP2 functions were determined by measuring the ratios of brain-to-plasma concentration of rhodamine 123, prazosin, and dinitrophenyl-S-glutathione, respectively. The mRNA and proteins expression levels of P-GP, BCRP, and MRP2 were evaluated with quantitative real-time PCR and western blot, respectively. MDCK-MDR1 and HCMEC/D3 cells were used to document the effects of the abnormally altered components in serum of ALF mice on the function and expression of P-GP. The clinical significance of alteration in P-GP function and expression was investigated by determining the distribution of the P-GP substrate phenobarbital (60 mg/kg, intravenous administration in the brain and loss of righting reflex (LORR induced by the drug (100 mg/kg. The results showed that ALF significantly downregulated the function and expression of both P-GP and BCRP, but increased the function and expression of MRP2 in the brain of mice. Cell study showed that increased chenodeoxycholic acid may be a reason behind the downregulated P-GP function and expression. Compared with control mice, ALF mice showed a significantly higher brain concentration of phenobarbital and higher brain-to-plasma concentration ratios. In accordance, ALF mice showed a significantly larger duration of LORR and shorter latency time of LORR by phenobarbital, inferring the enhanced pharmacological effect of phenobarbital on the central nervous system (CNS. In conclusion, the function and expression of P-GP and BCRP decreased, while the function and expression of MRP2 increased in the brain of ALF mice. The attenuated function and expression

  1. Transplantation of islet cells across major histocompatibility barriers after total lymphoid irradiation and infusion of allogeneic bone marrow cells

    International Nuclear Information System (INIS)

    Britt, L.D.; Scharp, D.W.; Lacy, P.E.; Slavin, S.

    1982-01-01

    Diabetic Lewis rats (AgB1/L) were evaluated as recipients of allogeneic Wistar-Furth (AgB2/2) isolated adult islets without the use of standard recipient immunosuppression. One group was treated with fractionated total lymphoid irradiation (TLI) and Wistar-Furth bone marrow cell reconstitution to proven chimerism prior to islet transplantation. This group returned to a prediabetic state following Wistar-Furth islet transplantation without any evidence of rejection for 100 days posttransplant. A second group of Lewis rats received only TLI without bone marrow treatment. They gave a varying result following islet transplantation with one recipient showing evidence of prolonged islet survival. A third chimeric control group did not receive isolated islets and did not alter their diabetic state. A fourth group was not given TLI nor donor bone marrow cells and uniformly rejected their allogeneic islets by 7 days. Thus, allogeneic adult islets will survive across major rat histocompatibility barriers using TLI and donor bone marrow chimerism as the only form of immunosuppression

  2. A sphingolipid-dependent diffusion barrier confines ER stress to the yeast mother cell

    Science.gov (United States)

    Clay, Lori; Caudron, Fabrice; Denoth-Lippuner, Annina; Boettcher, Barbara; Buvelot Frei, Stéphanie; Snapp, Erik Lee; Barral, Yves

    2014-01-01

    In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging. DOI: http://dx.doi.org/10.7554/eLife.01883.001 PMID:24843009

  3. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  4. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells

  5. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses.

    Science.gov (United States)

    Lau, Colleen M; Nish, Simone A; Yogev, Nir; Waisman, Ari; Reiner, Steven L; Reizis, Boris

    2016-03-07

    A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3(ITD) knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3(ITD/ITD) mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8(+) cDCs and noncanonical CD8(+) cDCs were expanded and showed specific alterations in their expression profiles. Flt3(ITD) mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner. © 2016 Lau et al.

  6. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  7. Noise alters guinea pig's blood-labyrinth barrier ultrastructure and permeability along with a decrease of cochlear Claudin-5 and Occludin.

    Science.gov (United States)

    Wu, Yong-Xiang; Zhu, Guo-Xia; Liu, Xin-Qin; Sun, Fei; Zhou, Ke; Wang, Shuang; Wang, Chun-Mei; Jia, Jin-Wen; Song, Jian-Tao; Lu, Lian-Jun

    2014-12-24

    Noise exposure (NE) is a severe modern health hazard that induces hearing impairment. However, the noise-induced ultrastructural changes of blood-labyrinth barrier (BLB) and the potential involvements of tight junction proteins (TJP) remain inconclusive. We investigated the effects of NE on not only the ultrastructure of cochlea and permeability of BLB but also the expression of TJP within the guinea pig cochlea. Male albino guinea pigs were exposed to white noise for 4 h or 2 consecutive days (115 dB sound pressure level, 6 hours per day) and the hearing impairments and light microscopic change of BLB were evaluated with auditory brainstem responses (ABR) and the cochlear sensory epithelia surface preparation, respectively. The cochlear ultrastructure and BLB permeability after NE 2d were revealed with transmission electron microscope (TEM) and lanthanum nitrate-tracing techniques, respectively. The potential alterations of TJPs Claudin-5 and Occludin were quantified with immunohistochemistry and western blot. NE induced significant hearing impairment and NE 2d contributed to significant outer hair cell (OHC) loss that is most severe in the first row of outer hair cells. Furthermore, the loosen TJ and an obvious leakage of lanthanum nitrate particles beneath the basal lamina were revealed with TEM. Moreover, a dose-dependent decrease of Claudin-5 and Occludin was observed in the cochlea after NE. All these findings suggest that both decrease of Claudin-5 and Occludin and increased BLB permeability are involved in the pathologic process of noise-induced hearing impairment; however, the causal relationship and underlying mechanisms should be further investigated.

  8. A microfluidic cell culture device with integrated microelectrodes for barrier studies

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin; Dufva, Martin; Kutter, Jörg P.

    We present an eight cell culture microfluidic device fabricated using thiol-ene ‘click’ chemistry with embedded microelectrodes for evaluating barrier properties of human intestinal epithelial cells. The capability of the microelectrodes for trans-epithelial electrical resistance (TEER) measureme......) measurements was demonstrated by using confluent human colorectal epithelial cells (Caco-2) and rat fibroblast (CT 26) cells cultured in the microfluidic device....

  9. Eavesdropping on altered cell-to-cell signaling in cancer by secretome profiling.

    Science.gov (United States)

    Klinke, David J

    2016-01-01

    In the past decade, cumulative clinical experiences with molecular targeted therapies and immunotherapies for cancer have promoted a shift in our conceptual understanding of cancer. This view shifted from viewing solid tumors as a homogeneous mass of malignant cells to viewing tumors as heterogeneous structures that are dynamically shaped by intercellular interactions among the variety of stromal, immune, and malignant cells present within the tumor microenvironment. As in any dynamic system, identifying how cells communicate to maintain homeostasis and how this communication is altered during oncogenesis are key hurdles for developing therapies to restore normal tissue homeostasis. Here, I discuss tissues as dynamic systems, using the mammary gland as an example, and the evolutionary concepts applied to oncogenesis. Drawing from these concepts, I present 2 competing hypotheses for how intercellular communication might be altered during oncogenesis. As an initial test of these competing hypotheses, a recent secretome comparison between normal human mammary and HER2+ breast cancer cell lines suggested that the particular proteins secreted by the malignant cells reflect a convergent evolutionary path associated with oncogenesis in a specific anatomical niche, despite arising in different individuals. Overall, this study illustrates the emerging power of secretome proteomics to probe, in an unbiased way, how intercellular communication changes during oncogenesis.

  10. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    Science.gov (United States)

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  11. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    Directory of Open Access Journals (Sweden)

    Lefeng Wang

    2017-01-01

    Full Text Available Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC, leading to barrier dysfunction and acute respiratory distress syndrome (ARDS. Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix] of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

  12. Barriers to Mental Health Service Use among Hematopoietic Stem Cell Transplant Survivors

    OpenAIRE

    Mosher, Catherine E.; DuHamel, Katherine N.; Rini, Christine M.; Li, Yuelin; Isola, Luis; Labay, Larissa; Rowley, Scott; Papadopoulos, Esperanza; Moskowitz, Craig; Scigliano, Eileen; Grosskreutz, Celia; Redd, William H.

    2009-01-01

    Summary This study examined barriers to mental health service use and their demographic, medical, and psychosocial correlates among hematopoietic stem cell transplant (HSCT) survivors. A sample of 253 HSCT survivors who were 1- to 3-years post-transplant completed measures of demographic, physical, psychological, and social characteristics as well as a newly modified measure of barriers to mental health service use. Only 50% of distressed HSCT survivors had received mental health services. An...

  13. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Directory of Open Access Journals (Sweden)

    Mónica Díaz-Coránguez

    Full Text Available Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  14. Transmigration of neural stem cells across the blood brain barrier induced by glioma cells.

    Science.gov (United States)

    Díaz-Coránguez, Mónica; Segovia, José; López-Ornelas, Adolfo; Puerta-Guardo, Henry; Ludert, Juan; Chávez, Bibiana; Meraz-Cruz, Noemi; González-Mariscal, Lorenza

    2013-01-01

    Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

  15. Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

    Science.gov (United States)

    Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

    2014-01-01

    Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

  16. Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells.

    Directory of Open Access Journals (Sweden)

    Andrea E Tóth

    Full Text Available Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line treated with methylglyoxal.Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.

  17. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  18. Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

    Science.gov (United States)

    Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

    2016-01-01

    Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

  19. Heat-induced alterations in the cell nucleus

    International Nuclear Information System (INIS)

    Kampinga, H.H.

    1989-01-01

    Hyperthermia may kill eukaryotic cells and may also enhance the radiosensitivity of those cells that survive the heat treatment. Clinically, the possible use of hyperthermia as an adjuvant in the radiotherapeutic treatment of cancer needs the understanding of mechanisms that underlay heat-induced cell death and radiosensitization. By in vitro heating of established human (HeLaS3) and rodent (Ehrlich Ascites Tumor and LM fibroblast) cell lines, both killing and radiosensitization were investigated. (author). 1067 refs.; 76 figs.; 19 tabs

  20. Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

    Science.gov (United States)

    Shenoy, Anitha K; Lu, Jianrong

    2016-10-01

    Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

    Science.gov (United States)

    Slavin, Spencer A; Leonard, Antony; Grose, Valerie; Fazal, Fabeha; Rahman, Arshad

    2018-03-01

    Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

  2. Maternal exposure to fish oil primes offspring to harbor intestinal pathobionts associated with altered immune cell balance.

    Science.gov (United States)

    Gibson, D L; Gill, S K; Brown, K; Tasnim, N; Ghosh, S; Innis, S; Jacobson, K

    2015-01-01

    Our previous studies revealed that offspring from rat dams fed fish oil (at 8% and 18% energy), developed impaired intestinal barriers sensitizing the colon to exacerbated injury later in life. To discern the mechanism, we hypothesized that in utero exposure to fish oil, rich in n-3 polyunsaturated fatty acid (PUFA), caused abnormal intestinal reparative responses to mucosal injury through differences in intestinal microbiota and the presence of naïve immune cells. To identify such mechanisms, gut microbes and naïve immune cells were compared between rat pups born to dams fed either n-6 PUFA, n-3 PUFA or breeder chow. Maternal exposure to either of the PUFA rich diets altered the development of the intestinal microbiota with an overall reduction in microbial density. Using qPCR, we found that each type of PUFA differentially altered the major gut phyla; fish oil increased Bacteroidetes and safflower oil increased Firmicutes. Both PUFA diets reduced microbes known to dominate the infant gut like Enterobacteriaceae and Bifidobacteria spp. when compared to the chow group. Uniquely, maternal fish oil diets resulted in offspring showing blooms of opportunistic pathogens like Bilophila wadsworthia, Enterococcus faecium and Bacteroides fragilis in their gut microbiota. As well, fish oil groups showed a reduction in colonic CD8+ T cells, CD4+ Foxp3+ T cells and arginase+ M2 macrophages. In conclusion, fish oil supplementation in pharmacological excess, at 18% by energy as shown in this study, provides an example where excess dosing in utero can prime offspring to harbor intestinal pathobionts and alter immune cell homeostasis.

  3. Altered effector function of peripheral cytotoxic cells in COPD

    Directory of Open Access Journals (Sweden)

    Corne Jonathan M

    2009-06-01

    Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

  4. Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC is high. It is well known that the epithelial mesenchymal transition (EMT and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface

  5. Endothelial progenitor cells physiology and metabolic plasticity in brain angiogenesis and blood-brain barrier modeling

    Directory of Open Access Journals (Sweden)

    Natalia Malinovskaya

    2016-12-01

    Full Text Available Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons. Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies.

  6. Transplantation Dose Alters the Differentiation Program of Hematopoietic Stem Cells.

    Science.gov (United States)

    Brewer, Casey; Chu, Elizabeth; Chin, Mike; Lu, Rong

    2016-05-24

    Hematopoietic stem cell (HSC) transplantation is the most prevalent stem cell therapy, but it remains a risky procedure. To improve this treatment, it is important to understand how transplanted stem cells rebuild the blood and immune systems and how this process is impacted by transplantation variables such as the HSC dose. Here, we find that, in the long term following transplantation, 70%-80% of donor-HSC-derived clones do not produce all measured blood cell types. High HSC doses lead to more clones that exhibit balanced lymphocyte production, whereas low doses produce more T-cell-specialized clones. High HSC doses also produce significantly higher proportions of early-differentiating clones compared to low doses. These complex differentiation behaviors uncover the clonal-level regeneration dynamics of hematopoietic regeneration and suggest that transplantation dose can be exploited to improve stem cell therapy. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Profiling of altered metabolomic states in Nicotiana tabacum cells induced by priming agents

    CSIR Research Space (South Africa)

    Mhlongo, MI

    2016-10-01

    Full Text Available tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid...

  8. Mobile phone radiation alters proliferation of hepatocarcinoma cells.

    Science.gov (United States)

    Ozgur, Elcin; Guler, Goknur; Kismali, Gorkem; Seyhan, Nesrin

    2014-11-01

    This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4',6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure.

  9. Interface Engineering of Organic Schottky Barrier Solar Cells and Its Application in Enhancing Performances of Planar Heterojunction Solar Cells

    OpenAIRE

    Fangming Jin; Zisheng Su; Bei Chu; Pengfei Cheng; Junbo Wang; Haifeng Zhao; Yuan Gao; Xingwu Yan; Wenlian Li

    2016-01-01

    In this work, we describe the performance of organic Schottky barrier solar cells with the structure of ITO/molybdenum oxide (MoOx)/boron subphthalocyanine chloride (SubPc)/bathophenanthroline (BPhen)/Al. The SubPc-based Schottky barrier solar cells exhibited a short-circuit current density (Jsc) of 2.59?mA/cm2, an open-circuit voltage (Voc) of 1.06?V, and a power conversion efficiency (PCE) of 0.82% under simulated AM1.5?G solar illumination at 100?mW/cm2. Device performance was substantiall...

  10. Recombination barrier layers in solid-state quantum dot-sensitized solar cells

    KAUST Repository

    Roelofs, Katherine E.

    2012-06-01

    By replacing the dye in the dye-sensitized solar cell design with semiconductor quantum dots as the light-absorbing material, solid-state quantum dot-sensitized solar cells (ss-QDSSCs) were fabricated. Cadmium sulfide quantum dots (QDs) were grown in situ by successive ion layer adsorption and reaction (SILAR). Aluminum oxide recombination barrier layers were deposited by atomic layer deposition (ALD) at the TiO2/hole-conductor interface. For low numbers of ALD cycles, the Al2O3 barrier layer increased open circuit voltage, causing an increase in device efficiency. For thicker Al2O3 barrier layers, photocurrent decreased substantially, leading to a decrease in device efficiency. © 2012 IEEE.

  11. Microtubules Growth Rate Alteration in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Irina B. Alieva

    2010-01-01

    Full Text Available To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC and “fast” (three times as much growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  12. Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle

    International Nuclear Information System (INIS)

    Finaurini, Sara; Basilico, Nicoletta; Corbett, Yolanda; D’Alessandro, Sarah; Parapini, Silvia; Olliaro, Piero; Haynes, Richard K.; Taramelli, Donatella

    2012-01-01

    Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro-erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure–activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deoxyartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy-artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In particular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the γ-globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24 h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid.

  13. Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

    NARCIS (Netherlands)

    Baert, Yoni; Braye, Aude; Struijk, Robin B.; van Pelt, Ans M. M.; Goossens, Ellen

    2015-01-01

    To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of

  14. Magnetron sputtered gadolinia-doped ceria diffusion barriers for metal-supported solid oxide fuel cells

    DEFF Research Database (Denmark)

    Sønderby, Steffen; Klemensø, Trine; Christensen, Bjarke H.

    2014-01-01

    Gadolinia-doped ceria (GDC) thin films are deposited by reactive magnetron sputtering in an industrial-scale setup and implemented as barrier layers between the cathode and electrolyte in metal-based solid oxide fuel cells consisting of a metal support, an electrolyte of ZrO2 co-doped with Sc2O3...

  15. Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS): A Pilot Study.

    Science.gov (United States)

    Lindheim, Lisa; Bashir, Mina; Münzker, Julia; Trummer, Christian; Zachhuber, Verena; Leber, Bettina; Horvath, Angela; Pieber, Thomas R; Gorkiewicz, Gregor; Stadlbauer, Vanessa; Obermayer-Pietsch, Barbara

    2017-01-01

    Polycystic ovary syndrome (PCOS) is a common female endocrinopathy of unclear origin characterized by hyperandrogenism, oligo-/anovulation, and ovarian cysts. Women with PCOS frequently display overweight, insulin resistance, and systemic low-grade inflammation. We hypothesized that endotoxemia resulting from a leaky gut is associated with inflammation, insulin resistance, fat accumulation, and hyperandrogenemia in PCOS. In this pilot study, we compared the stool microbiome, gut permeability, and inflammatory status of women with PCOS and healthy controls. 16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analysis were conducted in mothur and QIIME using different relative abundance cut-offs. Gut barrier integrity, endotoxemia, and inflammatory status were evaluated using serum and stool markers and associations with reproductive, metabolic, and anthropometric parameters were investigated. The stool microbiome of PCOS patients showed a lower diversity and an altered phylogenetic composition compared to controls. We did not observe significant differences in any taxa with a relative abundance>1%. When looking at rare taxa, the relative abundance of bacteria from the phylum Tenericutes, the order ML615J-28 (phylum Tenericutes) and the family S24-7 (phylum Bacteroidetes) was significantly lower and associated with reproductive parameters in PCOS patients. Patients showed alterations in some, but not all markers of gut barrier function and endotoxemia. Patients with PCOS have a lower diversity and an altered phylogenetic profile in their stool microbiome, which is associated with clinical parameters. Gut barrier dysfunction and endotoxemia were not driving factors in this patient cohort, but may contribute to the clinical phenotype in certain PCOS patients.

  16. Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS: A Pilot Study.

    Directory of Open Access Journals (Sweden)

    Lisa Lindheim

    Full Text Available Polycystic ovary syndrome (PCOS is a common female endocrinopathy of unclear origin characterized by hyperandrogenism, oligo-/anovulation, and ovarian cysts. Women with PCOS frequently display overweight, insulin resistance, and systemic low-grade inflammation. We hypothesized that endotoxemia resulting from a leaky gut is associated with inflammation, insulin resistance, fat accumulation, and hyperandrogenemia in PCOS. In this pilot study, we compared the stool microbiome, gut permeability, and inflammatory status of women with PCOS and healthy controls.16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analysis were conducted in mothur and QIIME using different relative abundance cut-offs. Gut barrier integrity, endotoxemia, and inflammatory status were evaluated using serum and stool markers and associations with reproductive, metabolic, and anthropometric parameters were investigated.The stool microbiome of PCOS patients showed a lower diversity and an altered phylogenetic composition compared to controls. We did not observe significant differences in any taxa with a relative abundance>1%. When looking at rare taxa, the relative abundance of bacteria from the phylum Tenericutes, the order ML615J-28 (phylum Tenericutes and the family S24-7 (phylum Bacteroidetes was significantly lower and associated with reproductive parameters in PCOS patients. Patients showed alterations in some, but not all markers of gut barrier function and endotoxemia.Patients with PCOS have a lower diversity and an altered phylogenetic profile in their stool microbiome, which is associated with clinical parameters. Gut barrier dysfunction and endotoxemia were not driving factors in this patient cohort, but may contribute to the clinical phenotype in certain PCOS patients.

  17. Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

    Science.gov (United States)

    Smith, I M; Baker, A; Arneborg, N; Jespersen, L

    2015-11-01

    The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast

  18. HTLV-1 Alters T Cells for Viral Persistence and Transmission

    Directory of Open Access Journals (Sweden)

    Azusa Tanaka

    2018-03-01

    Full Text Available Human T-cell leukemia virus type 1 (HTLV-1 was the first retrovirus to be discovered as a causative agent of adult T-cell leukemia-lymphoma (ATL and chronic inflammatory diseases. Two viral factors, Tax and HTLV-1 bZIP factor (HBZ, are thought to be involved in the leukemogenesis of ATL. Tax expression is frequently lost due to DNA methylation in the promoter region, genetic changes to the tax gene, and deletion of the 5′ long terminal repeat (LTR in approximately half of all ATL cases. On the other hand, HBZ is expressed in all ATL cases. HBZ is known to function in both protein form and mRNA form, and both forms play an important role in the oncogenic process of HTLV-1. HBZ protein has a variety of functions, including the suppression of apoptosis, the promotion of proliferation, and the impairment of anti-viral activity, through the interaction with several host cellular proteins including p300/CBP, Foxp3, and Foxo3a. These functions dramatically modify the transcriptional profiling of host T cells. HBZ mRNA also promotes T cell proliferation and viability. HBZ changes infected T cells to CCR4+TIGIT+CD4+ effector/memory T cells. This unique immunophenotype enables T cells to migrate into various organs and tissues and to survive in vivo. In this review, we summarize how HBZ hijacks the transcriptional networks and immune systems of host T cells to contribute to HTLV-1 pathogenesis on the basis of recent new findings about HBZ and tax.

  19. Bacterial lipopolysaccharide-induced systemic inflammation alters perfusion of white matter-rich regions without altering flow in brain-irrigating arteries: Relationship to blood-brain barrier breakdown?

    Science.gov (United States)

    Dhaya, Ibtihel; Griton, Marion; Raffard, Gérard; Amri, Mohamed; Hiba, Bassem; Konsman, Jan Pieter

    2018-01-15

    To better understand brain dysfunction during sepsis, cerebral arterial blood flow was assessed with Phase Contrast Magnetic Resonance Imaging, perfusion with Arterial Spin Labeling and structure with diffusion-weighted Magnetic Resonance Imaging in rats after intraperitoneal administration of bacterial lipopolysaccharides. Although cerebral arterial flow was not altered, perfusion of the corpus callosum region and diffusion parallel to its fibers were higher after lipopolysaccharide administration as compared to saline injection. In parallel, lipopolysaccharide induced perivascular immunoglobulin-immunoreactivity in white matter. These findings indicate that systemic inflammation can result in increased perfusion, blood-brain barrier breakdown and altered water diffusion in white matter. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Arctigenin from Fructus Arctii (Seed of Burdock) Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

    Science.gov (United States)

    Shin, Hee Soon; Jung, Sun Young; Back, Su Yeon; Do, Jeong-Ryong; Shon, Dong-Hwa

    2015-01-01

    Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) and ovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function. PMID:26550018

  1. Arctigenin from Fructus Arctii (Seed of Burdock Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

    Directory of Open Access Journals (Sweden)

    Hee Soon Shin

    2015-01-01

    Full Text Available Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER value (as an index of barrier function and ovalbumin (OVA permeation (as an index of permeability to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  2. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Coisne Caroline

    2013-01-01

    Full Text Available Abstract Background The central nervous system (CNS is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB. Methods and design An in vitro mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the in vitro BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These in vitro and in vivo live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells crawl over long distances against the direction of

  3. γδ T cells in homeostasis and host defence of epithelial barrier tissues

    DEFF Research Database (Denmark)

    Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

    2017-01-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue...... homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body — namely, the epidermis and the intestine — and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity...

  4. Bactericidal Antibiotics Increase Hydroxyphenyl Fluorescein Signal by Altering Cell Morphology

    DEFF Research Database (Denmark)

    Paulander, Wilhelm; Wang, Ying; Folkesson, Sven Anders

    2014-01-01

    It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH center dot) formation. Flow cytometric detection of OH center dot by hydroxyphenyl fluorescein (HPF) probe oxidation was used...... to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS) concentration. Independently of antibiotics, increased fluorescence was seen...... for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP). Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin...

  5. TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Ricci J Haines

    Full Text Available Since inflammatory bowel diseases (IBD represent significant morbidity and mortality in the US, the need for defining novel drug targets and inflammatory mechanisms would be of considerable benefit. Although protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK has been primarily studied in an oncogenic context, it was noted that PTK6 null mice exhibited significantly enhanced colonic epithelial barrier function. Considering that the inflammatory functions of PTK6 have not yet been explored, we hypothesized that cytokines responsible for mediating IBD, such as TNFα/IFNγ, may solicit the action of PTK6 to alter barrier function. After first assessing critical mediators of TNFα/IFNγ driven epithelial barrier dysfunction, we further explored the possibility of PTK6 in this inflammatory context. In this report, we showed that PTK6 siRNA and PTK6 null young adult mouse colonic epithelial cells (YAMC exhibited significant attenuation of TNFα/IFNγ induced barrier dysfunction as measured by electric cell-substrate impedance sensing (ECIS assay and permeability assays. In addition, PTK6 null cells transfected with PTK6 cDNA displayed restored barrier dysfunction in response to TNFα/IFNγ, while the cells transfected with vector alone showed similar attenuation of barrier dysfunction. Furthermore, using subcellular fractionation and immunocytochemistry experiments, we found that PTK6 plays a role in FoxO1 nuclear accumulation leading to down-regulation of claudin-3, a tight junction protein. Moreover, we searched for relevant miRNA candidates putative for targeting PTK6 in order to identify and assess the impact of microRNA that target PTK6 with respect to TNFα/IFNγ induced barrier dysfunction. Subsequently, we assayed likely targets and determined their effectiveness in attenuating PTK6 expression as well as cytokine induced barrier dysfunction. Results showed that miR-93 reduced PTK6 expression and attenuated TNF

  6. Alterations of red blood cell metabolome in overhydrated hereditary stomatocytosis.

    NARCIS (Netherlands)

    Darghouth, D.; Koehl, B.; Heilier, J.F.; Madalinski, G.; Bovee, P.H.; Bosman, G.J.C.G.M.; Delaunay, J.; Junot, C.; Romeo, P.H.

    2011-01-01

    Overhydrated hereditary stomatocytosis, clinically characterized by hemolytic anemia, is a rare disorder of the erythrocyte membrane permeability to monovalent cations, associated with mutations in the Rh-associated glycoprotein gene. We assessed the red blood cell metabolome of 4 patients with this

  7. Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

    DEFF Research Database (Denmark)

    Müller, B K; Kabos, P; Belhage, B

    1996-01-01

    Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

  8. Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.

    Science.gov (United States)

    Hollmann, Emma K; Bailey, Amanda K; Potharazu, Archit V; Neely, M Diana; Bowman, Aaron B; Lippmann, Ethan S

    2017-04-13

    Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm 2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm 2 and fluorescein permeability below 1.95 × 10 -7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm 2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated

  9. B-cell subset alterations and correlated factors in HIV-1 infection.

    Science.gov (United States)

    Pensieroso, Simone; Galli, Laura; Nozza, Silvia; Ruffin, Nicolas; Castagna, Antonella; Tambussi, Giuseppe; Hejdeman, Bo; Misciagna, Donatella; Riva, Agostino; Malnati, Mauro; Chiodi, Francesca; Scarlatti, Gabriella

    2013-05-15

    During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. B-cell phenotype and correlating factors were evaluated. Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. Significant differences were observed among patients' groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4 T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. Our results indicate that viremia and nadir CD4 T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

  10. Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Johnson

    2012-02-01

    Full Text Available Helicobacter pylori lives within the mucus layer of the human stomach, in close proximity to gastric epithelial cells. While a great deal is known about the effects of H. pylori on human cells and the specific bacterial products that mediate these effects, relatively little work has been done to investigate alterations in H. pylori that may be triggered by bacterial contact with human cells. In this review, we discuss the spectrum of changes in bacterial physiology and morphology that occur when H. pylori is in contact with gastric epithelial cells. Several studies have reported that cell contact causes alterations in H. pylori gene transcription. In addition, H. pylori contact with gastric epithelial cells promotes the formation of pilus-like structures at the bacteria-host cell interface. The formation of these structures requires multiple genes in the cag pathogenicity island, and these structures are proposed to have an important role in the type IV secretion system-dependent process through which CagA enters host cells. Finally, H. pylori contact with epithelial cells can promote bacterial replication and the formation of microcolonies, phenomena that are facilitated by the acquisition of iron and other nutrients from infected cells. In summary, the gastric epithelial cell surface represents an important niche for H. pylori, and upon entry into this niche, the bacteria alter their behavior in a manner that optimizes bacterial proliferation and persistent colonization of the host.

  11. Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

    Science.gov (United States)

    Kho, Dan T; Johnson, Rebecca H; O'Carroll, Simon J; Angel, Catherine E; Graham, E Scott

    2017-09-21

    Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

  12. Comparative study of cell alterations in oral lichen planus and epidermoid carcinoma of the mouth mucosa.

    Science.gov (United States)

    Sousa, Fernando Augusto Cervantes Garcia de; Paradella, Thaís Cachuté; Brandão, Adriana Aigotti Haberbeck; Rosa, Luiz Eduardo Blumer

    2009-01-01

    Currently, much is discussed regarding the pre-malignant nature of mouth mucosa lichen planus. The present study aims at analyzing the alterations found in the epithelial cells present in the oral cavity lichen planus, comparing them to those found in epidermoid carcinoma. Histological cross-sections of oral lichen planus and epidermoid carcinoma, dyed by hematoxylineosin, were analyzed through light microscopy. The most frequently found alterations in oral lichen planus were: an increase in the nucleus/cytoplasm relation (93.33%), nucleus membrane thickness (86.67%) and bi-nucleus or multinucleous (86.67%). The Student t test (alpha=5%) revealed a statistically significant difference between the average number of cell alterations in oral lichen planus (5.87+/-1.57) and in epidermoid carcinoma (7.60+/-1.81). As to the types of alterations, the chi-squared test also revealed statistically significant differences among the lesions assessed in relation to the following cell alterations: nuclear excess chromatism, atypical mitoses, cellular pleomorphism and abnormal cell differentiation (poral lichen planus, the results obtained in this study show that the alterations present in oral lichen planus differ considerably from those seen in epidermoid carcinoma, thus showing how distinct these two diseases are.

  13. The effects of interfacial recombination and injection barrier on the electrical characteristics of perovskite solar cells

    Directory of Open Access Journals (Sweden)

    Lin Xing Shi

    2018-02-01

    Full Text Available Charge carrier recombination in the perovskite solar cells (PSCs has a deep influence on the electrical performance, such as open circuit voltage, short circuit current, fill factor and ultimately power conversion efficiency. The impacts of injection barrier, recombination channels, doping properties of carrier transport layers and light intensity on the performance of PSCs are theoretically investigated by drift-diffusion model in this work. The results indicate that due to the injection barrier at the interfaces of perovskite and carrier transport layer, the accumulated carriers modify the electric field distribution throughout the PSCs. Thus, a zero electric field is generated at a specific applied voltage, with greatly increases the interfacial recombination, resulting in a local kink of current density-voltage (J-V curve. This work provides an effective strategy to improve the efficiency of PSCs by pertinently reducing both the injection barrier and interfacial recombination.

  14. Methylation of the ATM promoter in glioma cells alters ionizing radiation sensitivity

    International Nuclear Information System (INIS)

    Roy, Kanaklata; Wang, Lilin; Makrigiorgos, G. Mike; Price, Brendan D.

    2006-01-01

    Glioblastomas are among the malignancies most resistant to radiation therapy. In contrast, cells lacking the ATM protein are highly sensitive to ionizing radiation. The relationship between ATM protein expression and radiosensitivity in 3 glioma cell lines was examined. T98G cells exhibited normal levels of ATM protein, whereas U118 and U87 cells had significantly lower levels of ATM and increased (>2-fold) sensitivity to ionizing radiation compared to T98G cells. The ATM promoter was methylated in U87 cells. Demethylation by azacytidine treatment increased ATM protein levels in the U87 cells and decreased their radiosensitivity. In contrast, the ATM promoter in U118 cells was not methylated. Further, expression of exogenous ATM did not significantly alter the radiosensitivity of U118 cells. ATM expression is therefore heterogeneous in the glioma cells examined. In conclusion, methylation of the ATM promoter may account for the variable radiosensitivity and heterogeneous ATM expression in a fraction of glioma cells

  15. Chronological alterations of diagnostic imaging of renal cell carcinoma

    International Nuclear Information System (INIS)

    Arima, Kiminobu; Sugimura, Yoshiki; Yanagawa, Makoto; Tochigi, Hiromi; Kawamura, Juichi

    1994-01-01

    A review of 156 cases of renal cell carcinoma diagnosed during a 20-year period demonstrated the changes of initial signs/symptoms and imaging modalities for detection and definition. According to the imaging modality used for diagnosing renal cell carcinoma, clinical pictures were chronologically examined over 4 periods: 1973 to 1979 (before CT era), 1980 to 1984 (early CT era), 1985 to 1987 (CT era) and 1988 to 1992 (CT/MRI era). With regards to initial signs or symptoms, the proportion of classical trials has gradually decreased, while that of tumors noted incidentally has increased. As for imaging modalities for detection, the proportion of IVP has gradually decreased and that of CT and US has increased over the periods. With regard to imaging modalities for definition, the proportion of angiography has decreased and that of CT has increased. From chronological changes in clinical pictures and imaging modalities, we suggested a decision tree of imaging modalities for detection and definition of renal cell carcinoma. (author)

  16. A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin; Trier, Sofie; Rahbek, Ulrik L

    2018-01-01

    with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional...... through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed...

  17. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

    Directory of Open Access Journals (Sweden)

    Lishi Xie

    Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

  18. Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

    Directory of Open Access Journals (Sweden)

    Valerie E. Ryman

    2016-01-01

    Full Text Available Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1 metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE, can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE, is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

  19. Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

    Directory of Open Access Journals (Sweden)

    Ariane Willems

    2010-11-01

    Full Text Available The observation that mice with a selective ablation of the androgen receptor (AR in Sertoli cells (SC (SCARKO mice display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin. Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2. It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

  20. Development of microfluidic cell culture devices towards an in vitro human intestinal barrier model

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin

    to enable real-time detection of cell responses, adjustment of cellular stimulation etc. leading to establishment of conditional experiments. In this project, microfluidic systems engineering was leveraged to develop an eight chamber multi-layer microchip for intestinal barrier studies. Sandwiched between...... the layers was a modified Teflon porous membrane for cell culture. The novelty lies in modifying the surface of the porous Teflon support membrane using thiol-ene ‘click’ chemistry, thus allowing the modified Teflon membrane to be bonded between the chip layers to form an enclosed microchip. Successful...... application of the multi-layer microchip was demonstrated by integrating the microchip to an existing cell culture fluidic system to culture the human intestinal epithelial cells, Caco-2, for long term studies. Under the continuous low flow conditions, the cells differentiated into columnar cells displaying...

  1. Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

    Science.gov (United States)

    Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

    2014-06-15

    Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Barrier potential design criteria in multiple-quantum-well-based solar-cell structures

    Science.gov (United States)

    Mohaidat, Jihad M.; Shum, Kai; Wang, W. B.; Alfano, R. R.

    1994-01-01

    The barrier potential design criteria in multiple-quantum-well (MQW)-based solar-cell structures is reported for the purpose of achieving maximum efficiency. The time-dependent short-circuit current density at the collector side of various MQW solar-cell structures under resonant condition was numerically calculated using the time-dependent Schroedinger equation. The energy efficiency of solar cells based on the InAs/Ga(y)In(1-y)As and GaAs/Al(x)Ga(1-x)As MQW structues were compared when carriers are excited at a particular solar-energy band. Using InAs/Ga(y)In(1-y)As MQW structures it is found that a maximum energy efficiency can be achieved if the structure is designed with barrier potential of about 450 meV. The efficiency is found to decline linearly as the barrier potential increases for GaAs/Al(x)Ga(1-x)As MQW-structure-based solar cells.

  3. Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

    International Nuclear Information System (INIS)

    Saathoff, Manuela; Blum, Barbara; Quast, Thomas; Kirfel, Gregor; Herzog, Volker

    2004-01-01

    The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier

  4. Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Maroschik, Belinda; Gürtler, Anne; Krämer, Anne; Rößler, Ute; Gomolka, Maria; Hornhardt, Sabine; Mörtl, Simone; Friedl, Anna A

    2014-01-01

    Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels

  5. Prenatal cadmium exposure alters postnatal immune cell development and function

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Miranda L.; Holásková, Ida; Elliott, Meenal; Brundage, Kathleen M.; Schafer, Rosana; Barnett, John B., E-mail: jbarnett@hsc.wvu.edu

    2012-06-01

    Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl{sub 2} (10 ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7 weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7 weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2 weeks of age. At 7 weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4{sup +}FoxP3{sup +}CD25{sup +} (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8{sup +}CD223{sup +} T cells were markedly decreased in the spleens in all offspring at 7 weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can

  6. Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

    Science.gov (United States)

    Webb, Tonya J; Carey, Gregory B; East, James E; Sun, Wenji; Bollino, Dominique R; Kimball, Amy S; Brutkiewicz, Randy R

    2016-08-01

    Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Understanding barriers to the introduction of precision medicines in non-small cell lung cancer: A qualitative interview protocol [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Stuart Wright

    2018-03-01

    Full Text Available Background: While precision medicines targeting genetic mutations and alterations in non-small cell lung cancer (NSCLC have been available since 2010, their adoption into clinical practice has been slow. Evidence suggests that a number of barriers, such as insufficient clinician knowledge, a need for training of test providers, or a lack of specific clinical guidelines, may slow the implementation of precision in general. However, little attention has been given to the barriers to providing precision medicines in NSCLC. The purpose of this protocol is to outline the design for a qualitative interview study to identify the barriers and facilitators to the provision of precision medicines for NSCLC.   Methods: This study will use semi-structured interviews with clinicians (n=10, test providers (n=10, and service commissioners (n=10 to identify the perceived barriers and facilitators to providing historical, current, and future precision medicines in NSCLC. Participants will be identified through mailing list advertisements and snowball sampling. Recruitment will continue until data saturation, indicated by no new themes arising from the data. Interviews will be conducted by telephone to facilitate geographical diversity. The qualitative data will be analysed using a framework analysis with themes anticipated to relate to; relevant barriers to providing precision medicines, the impact of different barriers on medicine provision, changes in the ability to provide precision medicines over time, and strategies to facilitate the provision of precision medicines.   Ethics: This study has been approved by the University of Manchester Proportionate Review Research Ethics Committee (Reference number: 2017-1885-3619. Written consent will be obtained from all participants.   Conclusion: This study is the first to explore the barriers and facilitators to providing precision medicines for NSCLC in the English NHS. The findings will inform strategies to

  8. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    Lohrer, H.

    1987-01-01

    The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.) [de

  9. Simulation of the long term alteration of clay minerals in engineered bentonite barriers: nucleation and growth of secondary clay particles

    International Nuclear Information System (INIS)

    Fritz, B.; Clement, A.; Zwingmann, H.; Noguera, C.

    2010-01-01

    Document available in extended abstract form only. The long term stability of clay rich rocks used as barriers to the migration of radionuclides in the environment of nuclear wastes has been intensively studied, looking at the geochemical interactions between clay minerals and aqueous solutions. These studies combine experimental approaches for the short term and numerical modellings for the long term extrapolations, in the frame of the research supported by ANDRA in the French design for High Level Waste (HLW) repository. The main objective of the geochemical numerical tools devoted to clay-solutions interaction processes was to predict the feed-back effects of mineralogical and chemical transformations of clay mineral, in repository conditions as defined by Andra, on their physical and transport properties (porosity, molecular diffusion, permeability). The 1D transport-reaction coupled simulation was done using the code KIRMAT, at 100 deg. C for 100000 years. The fluid considered is that of the Callovo-Oxfordian geological formation (COX) and assumed to diffuse into the clay barrier from one side. On the other side, ferrous iron, is provided by the steel overpack corrosion. Under these conditions, montmorillonite of the clay barrier is only partially transformed into illite, chlorite, and saponite. The simulation shows that only outer parts of the clay barrier is significantly modified, mainly at the interface with the geological environment. These modifications correspond to a closure of the porosity, followed by a decrease of mass transport by molecular diffusion. Near the COX, the swelling pressure of the clays from the barrier is predicted to decrease, but in its major part, the engineered barrier seems to keep its initial physical properties (porosity, molecular diffusion, permeability, swelling pressure). In this modelling approach, the very important role of secondary clay minerals has to be taken into account with relevant kinetic rate laws; particularly

  10. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  11. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    Science.gov (United States)

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  12. The B-Cell Follicle in HIV Infection: Barrier to a Cure.

    Science.gov (United States)

    Bronnimann, Matthew P; Skinner, Pamela J; Connick, Elizabeth

    2018-01-01

    The majority of HIV replication occurs in secondary lymphoid organs (SLOs) such as the spleen, lymph nodes, and gut-associated lymphoid tissue. Within SLOs, HIV RNA + cells are concentrated in the B-cell follicle during chronic untreated infection, and emerging data suggest that they are a major source of replication in treated disease as well. The concentration of HIV RNA + cells in the B-cell follicle is mediated by several factors. Follicular CD4 + T-cell subsets including T-follicular helper cells and T-follicular regulatory cells are significantly more permissive to HIV than extrafollicular subsets. The B cell follicle also contains a large reservoir of extracellular HIV virions, which accumulate on the surface of follicular dendritic cells (FDCs) in germinal centers. FDC-bound HIV virions remain infectious even in the presence of neutralizing antibodies and can persist for months or even years. Moreover, the B-cell follicle is semi-immune privileged from CTL control. Frequencies of HIV- and SIV-specific CTL are lower in B-cell follicles compared to extrafollicular regions as the majority of CTL do not express the follicular homing receptor CXCR5. Additionally, CTL in the B-cell follicle may be less functional than extrafollicular CTL as many exhibit the recently described CD8 T follicular regulatory phenotype. Other factors may also contribute to the follicular concentration of HIV RNA + cells. Notably, the contribution of NK cells and γδ T cells to control and/or persistence of HIV RNA + cells in secondary lymphoid tissue remains poorly characterized. As HIV research moves increasingly toward the development of cure strategies, a greater understanding of the barriers to control of HIV infection in B-cell follicles is critical. Although no strategy has as of yet proven to be effective, a range of novel therapies to address these barriers are currently being investigated including genetically engineered CTL or chimeric antigen receptor T cells that express

  13. The B-Cell Follicle in HIV Infection: Barrier to a Cure

    Directory of Open Access Journals (Sweden)

    Matthew P. Bronnimann

    2018-01-01

    Full Text Available The majority of HIV replication occurs in secondary lymphoid organs (SLOs such as the spleen, lymph nodes, and gut-associated lymphoid tissue. Within SLOs, HIV RNA+ cells are concentrated in the B-cell follicle during chronic untreated infection, and emerging data suggest that they are a major source of replication in treated disease as well. The concentration of HIV RNA+ cells in the B-cell follicle is mediated by several factors. Follicular CD4+ T-cell subsets including T-follicular helper cells and T-follicular regulatory cells are significantly more permissive to HIV than extrafollicular subsets. The B cell follicle also contains a large reservoir of extracellular HIV virions, which accumulate on the surface of follicular dendritic cells (FDCs in germinal centers. FDC-bound HIV virions remain infectious even in the presence of neutralizing antibodies and can persist for months or even years. Moreover, the B-cell follicle is semi-immune privileged from CTL control. Frequencies of HIV- and SIV-specific CTL are lower in B-cell follicles compared to extrafollicular regions as the majority of CTL do not express the follicular homing receptor CXCR5. Additionally, CTL in the B-cell follicle may be less functional than extrafollicular CTL as many exhibit the recently described CD8 T follicular regulatory phenotype. Other factors may also contribute to the follicular concentration of HIV RNA+ cells. Notably, the contribution of NK cells and γδ T cells to control and/or persistence of HIV RNA+ cells in secondary lymphoid tissue remains poorly characterized. As HIV research moves increasingly toward the development of cure strategies, a greater understanding of the barriers to control of HIV infection in B-cell follicles is critical. Although no strategy has as of yet proven to be effective, a range of novel therapies to address these barriers are currently being investigated including genetically engineered CTL or chimeric antigen receptor T cells

  14. Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

    Directory of Open Access Journals (Sweden)

    Sharma Kamal

    2008-12-01

    Full Text Available Abstract Background Effective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells. Methods Magnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging. Results We found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells. Conclusion The therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

  15. Spray pyrolysis of doped-ceria barrier layers for solid oxide fuel cells

    DEFF Research Database (Denmark)

    Szymczewska, Dagmara; Chrzan, Aleksander; Karczewski, Jakub

    2017-01-01

    Gadolinium doped ceria (Ce0.8Gd0.2O2 − x-CGO) layer fabricated by spray pyrolysis is investigated as the diffusion barrier for solid oxide fuel cell. It is deposited between the La0.6Sr0.4FeO3 − δ cathode and the yttria stabilized zirconia electrolyte to mitigate harmful interdiffusion...

  16. Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

    Directory of Open Access Journals (Sweden)

    Leavell Sarah

    2010-05-01

    Full Text Available Abstract Background Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control and then cats were vaginally challenged with cell-associated or cell-free FIV. Results Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls. Conclusions The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus can be modulated by mucosal exposure to uninfected heterologous cells.

  17. Effects of topographical and mechanical property alterations induced by oxygen plasma modification on stem cell behavior.

    Science.gov (United States)

    Yang, Yong; Kulangara, Karina; Lam, Ruby T S; Dharmawan, Rena; Leong, Kam W

    2012-10-23

    Polymeric substrates intended for cell culture and tissue engineering are often surface-modified to facilitate cell attachment of most anchorage-dependent cell types. The modification alters the surface chemistry and possibly topography. However, scant attention has been paid to other surface property alterations. In studying oxygen plasma treatment of polydimethylsiloxane (PDMS), we show that oxygen plasma treatment alters the surface chemistry and, consequently, the topography and elasticity of PDMS at the nanoscale level. The elasticity factor has the predominant effect, compared with the chemical and topographical factors, on cell adhesions of human mesenchymal stem cells (hMSCs). The enhanced focal adhesions favor cell spreading and osteogenesis of hMSCs. Given the prevalent use of PDMS in biomedical device construction and cell culture experiments, this study highlights the importance of understanding how oxygen plasma treatment would impact subsequent cell-substrate interactions. It helps explain inconsistency in the literature and guides preparation of PDMS-based biomedical devices in the future.

  18. Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki; Yoichiro; Tanabe, Kazumi; Umeki, Shigeko; Nakamura, Nori; Yamakido, Michio; Hamamoto, Kazuko.

    1990-04-01

    The T-cell receptor CD3 (TCR/CD3) complex plays a central role in antigen recognition and activation of mature T cells, and therefore abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of surface TCR/CD3 expression among human mature CD4 + T cells. The presence of variant CD4 + T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T-cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T-cell dysfunction. (author)

  19. γδ T cells in homeostasis and host defence of epithelial barrier tissues.

    Science.gov (United States)

    Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

    2017-12-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

  20. 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

  1. Acute exposure to ergot alkaloids from endophyte-infected tall fescue does not alter absorptive or barrier function of the isolated bovine ruminal epithelium.

    Science.gov (United States)

    Foote, A P; Penner, G B; Walpole, M E; Klotz, J L; Brown, K R; Bush, L P; Harmon, D L

    2014-07-01

    Ergot alkaloids in endophyte-infected (Neotyphodium coenophialum) tall fescue (Lolium arundinaceum) have been shown to cause a reduction in blood flow to the rumen epithelium as well as a decrease in volatile fatty acids (VFA) absorption from the washed rumen of steers. Previous data also indicates that incubating an extract of endophyte-infected tall fescue seed causes an increase in the amount of VFA absorbed per unit of blood flow, which could result from an alteration in the absorptive or barrier function of the rumen epithelium. An experiment was conducted to determine the acute effects of an endophyte-infected tall fescue seed extract (EXT) on total, passive or facilitated acetate and butyrate flux across the isolated bovine rumen as well as the barrier function measured by inulin flux and tissue conductance (G t ). Flux of ergovaline across the rumen epithelium was also evaluated. Rumen tissue from the caudal dorsal sac of Holstein steers (n=6), fed a common diet, was collected and isolated shortly after slaughter and mounted between two halves of Ussing chambers. In vitro treatments included vehicle control (80% methanol, 0.5% of total volume), Low EXT (50 ng ergovaline/ml) and High EXT (250 ng ergovaline/ml). Results indicate that there is no effect of acute exposure to ergot alkaloids on total, passive or facilitated flux of acetate or butyrate across the isolate bovine rumen epithelium (P>0.51). Inulin flux (P=0.16) and G t (P>0.17) were not affected by EXT treatment, indicating no alteration in barrier function due to acute ergot alkaloid exposure. Ergovaline was detected in the serosal buffer of the High EXT treatment indicating that the flux rate is ~0.25 to 0.44 ng/cm2 per hour. Data indicate that specific pathways for VFA absorption and barrier function of the rumen epithelium are not affected by acute exposure to ergot alkaloids from tall fescue at the concentrations tested. Ergovaline has the potential to be absorbed from the rumen of cattle that

  2. Altered expression of glycosaminoglycans in metastatic 13762NF rat mammary adenocarcinoma cells

    International Nuclear Information System (INIS)

    Steck, P.A.; Cheong, P.H.; Nakajima, M.; Yung, W.K.A.; Moser, R.P.; Nicolson, G.L.

    1987-01-01

    A difference in the expression and metabolism of [ 35 S]sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate. These results suggested that altered glycosaminoglycan expression and metabolism may be associated with the metastatic process in 13762NF rat mammary tumor cells

  3. Barriers to Liposomal Gene Delivery: from Application Site to the Target.

    Science.gov (United States)

    Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

    2016-01-01

    Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

  4. Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

    Science.gov (United States)

    Pollock, Abigail H; Tedla, Nicodemus; Hancock, Sarah E; Cornely, Rhea; Mitchell, Todd W; Yang, Zhengmin; Kockx, Maaike; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-05-15

    Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. Altered ganglioside GD3 in HeLa cells might influence the cytotoxic abilities of NK cells

    OpenAIRE

    Lee, Wen-Chi; Lee, Wen-Ling; Shyong, Wen-Yuann; Yang, Lin-Wei; Ko, Min-Chun; Yeh, Chang-Ching; Edmond Hsieh, Shie-Liang; Wang, Peng-Hui

    2012-01-01

    Objective: Previously, we found that altered sialidases in HeLa cells in a natural killer-HeLa (NK-HeLa) coculture system contributed to the decreased cytotoxic ability of NK cells. However, changes that occur in the glycosylation of the HeLa cells in the NK-HeLa coculture system remain unknown. Materials and Methods: An NK-HeLa coculture system was used to examine the changes that occur in the gangliosides of HeLa cells. Results: GD3 expression in HeLa cells was significantly increased...

  6. The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.

    Directory of Open Access Journals (Sweden)

    Christine S Schwaiger

    Full Text Available The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3(10-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure and the first intermediate state towards channel closing (C1, modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C1 model, and directly observe the previously suggested sliding 3(10-helix region in S4. Through systematic free energy calculations, we show that the C1 state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C1 states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.

  7. The healthy donor profile of immunoregulatory soluble mediators is altered by stem cell mobilization and apheresis.

    Science.gov (United States)

    Melve, Guro Kristin; Ersvaer, Elisabeth; Paulsen Rye, Kristin; Bushra Ahmed, Aymen; Kristoffersen, Einar K; Hervig, Tor; Reikvam, Håkon; Hatfield, Kimberley Joanne; Bruserud, Øystein

    2018-05-01

    Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and thereafter harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. Plasma levels of 38 soluble mediators (cytokines, soluble adhesion molecules, proteases, protease inhibitors) were analyzed in samples derived from healthy stem cell donors before G-CSF treatment and after 4 days, both immediately before and after leukapheresis. Donors could be classified into two main subsets based on their plasma mediator profile before G-CSF treatment. Seventeen of 36 detectable mediators were significantly altered by G-CSF; generally an increase in mediator levels was seen, including pro-inflammatory cytokines, soluble adhesion molecules and proteases. Several leukocyte- and platelet-released mediators were increased during apheresis. Both plasma and graft mediator profiles were thus altered and showed correlations to graft concentrations of leukocytes and platelets; these concentrations were influenced by the apheresis device used. Finally, the mediator profile of the allotransplant recipients was altered by graft infusion, and based on their day +1 post-transplantation plasma profile our recipients could be divided into two major subsets that differed in overall survival. G-CSF alters the short-term plasma mediator profile of healthy stem cell donors. These effects together with the leukocyte and platelet levels in the graft determine the mediator profile of the stem cell grafts. Graft infusion also alters the systemic mediator profile of the recipients, but further studies are required to clarify whether such graft-induced alterations have a prognostic impact. Copyright © 2018. Published by Elsevier Inc.

  8. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  9. An individually fitted physical barrier device as a tool to restrict the birds’ spatial access: can their use alter behavioral responses?

    DEFF Research Database (Denmark)

    Pellegrini, S.; Marin, R.H.; Guzman, Diego Alberto

    2015-01-01

    fabric bands around the wings’ base. To be useful, the IPB should allow natural movements and not affect the expression of behaviors (non-invasive). This study assessed whether the IPB may alter adult Japanese quail behavioral responses using 4 classical test situations: Open-Field, Runway, Time Budget...... in Home Box, and Mating Interactions. Open-field ambulatory behaviors were affected 1 h, but not 7 d, after IPB was fitted, suggesting that 7 d (or less) are required to habituate to the device. After that time period, IPB fitted birds showed no differences in any of the behaviors registered in the other......Social interactions have been extensively studied in poultry in a variety of environmental situations. Many studies allow full social contacts between birds, but there are others in which the interactions are tested through barriers (wire mesh or glass). Thus a situation where, according...

  10. Cytological and oncogene alterations in radiation-transformed Syrian hamster embryo cells

    International Nuclear Information System (INIS)

    Trutschler, K.; Hieber, L.; Kellerer, A.M.

    1991-01-01

    Syrian hamster embryo (SHE) cells were neoplastically transformed by different types of ionizing radiation (γ-rays, α-particles or carbon ions). Transformed and tumor cell lines (derived from nude mice tumors) were analysed for alterations of the oncogenes c-Ha-ras and c-myc, i.e. RFLPs, gene amplifications, activation by point mutation, gene expression, and for cytological changes. In addition, the chromosome number and the numbers of micronuclei per cell have been determined in a series of cell lines. (author)

  11. Interface Engineering of Organic Schottky Barrier Solar Cells and Its Application in Enhancing Performances of Planar Heterojunction Solar Cells

    Science.gov (United States)

    Jin, Fangming; Su, Zisheng; Chu, Bei; Cheng, Pengfei; Wang, Junbo; Zhao, Haifeng; Gao, Yuan; Yan, Xingwu; Li, Wenlian

    2016-05-01

    In this work, we describe the performance of organic Schottky barrier solar cells with the structure of ITO/molybdenum oxide (MoOx)/boron subphthalocyanine chloride (SubPc)/bathophenanthroline (BPhen)/Al. The SubPc-based Schottky barrier solar cells exhibited a short-circuit current density (Jsc) of 2.59 mA/cm2, an open-circuit voltage (Voc) of 1.06 V, and a power conversion efficiency (PCE) of 0.82% under simulated AM1.5 G solar illumination at 100 mW/cm2. Device performance was substantially enhanced by simply inserting thin organic hole transport material into the interface of MoOx and SubPc. The optimized devices realized a 180% increase in PCE of 2.30% and a peak Voc as high as 1.45 V was observed. We found that the improvement is due to the exciton and electron blocking effect of the interlayer and its thickness plays a vital role in balancing charge separation and suppressing quenching effect. Moreover, applying such interface engineering into MoOx/SubPc/C60 based planar heterojunction cells substantially enhanced the PCE of the device by 44%, from 3.48% to 5.03%. Finally, we also investigated the requirements of the interface material for Schottky barrier modification.

  12. Tumor-altered dendritic cell function: implications for anti-tumor immunity

    Directory of Open Access Journals (Sweden)

    Kristian Michael Hargadon

    2013-07-01

    Full Text Available Dendritic cells are key regulators of both innate and adaptive immunity, and the array of immunoregulatory functions exhibited by these cells is dictated by their differentiation, maturation, and activation status. Although a major role for these cells in the induction of immunity to pathogens has long been appreciated, data accumulated over the last several years has demonstrated that DC are also critical regulators of anti-tumor immune responses. However, despite the potential for stimulation of robust anti-tumor immunity by DC, tumor-altered DC function has been observed in many cancer patients and tumor-bearing animals and is often associated with tumor immune escape. Such dysfunction has significant implications for both the induction of natural anti-tumor immune responses as well as the efficacy of immunotherapeutic strategies that target endogenous DC in situ or that employ exogenous DC as part of anti-cancer immunization maneuvers. In this review, the major types of tumor-altered DC function will be described, with emphasis on recent insights into the mechanistic bases for the inhibition of DC differentiation from hematopoietic precursors, the altered programming of DC precursors to differentiate into myeloid-derived suppressor cells or tumor-associated macrophages, the suppression of DC maturation and activation, and the induction of immunoregulatory DC by tumors, tumor-derived factors, and tumor-associated cells within the milieu of the tumor microenvironment. The impact of these tumor-altered cells on the quality of the overall anti-tumor immune response will also be discussed. Finally, this review will also highlight questions concerning tumor-altered DC function that remain unanswered, and it will address factors that have limited advances in the study of this phenomenon in order to focus future research efforts in the field on identifying strategies for interfering with tumor-associated DC dysfunction and improving DC-mediated anti

  13. The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

    Science.gov (United States)

    de Vallière, Cheryl; Vidal, Solange; Clay, Ieuan; Jurisic, Giorgia; Tcymbarevich, Irina; Lang, Silvia; Ludwig, Marie-Gabrielle; Okoniewski, Michal; Eloranta, Jyrki J; Kullak-Ublick, Gerd A; Wagner, Carsten A; Rogler, Gerhard; Seuwen, Klaus

    2015-09-15

    The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms. Copyright © 2015 the American Physiological Society.

  14. Breaking down the barriers to commercialization of fuel cells in transportation through Government - industry R&D programs

    Energy Technology Data Exchange (ETDEWEB)

    Chalk, S.G. [Dept. of Energy, Washington, DC (United States); Venkateswaran, S.R. [Energetics, Inc., Columbia, MD (United States)

    1996-12-31

    PEM fuel cell technology is rapidly emerging as a viable propulsion alternative to the internal combustion engine. Fuel cells offer the advantages of low emissions, high efficiency, fuel flexibility, quiet and continuous operation, and modularity. Over the last decade, dramatic advances have been achieved in the performance and cost of PEM fuel cell technologies for automotive applications. However, significant technical barriers remain to making fuel cell propulsion systems viable alternatives to the internal combustion engine. This paper focuses on the progress achieved and remaining technical barriers while highlighting Government-industry R&D efforts that are accelerating fuel cell technology toward commercialization.

  15. Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Smith, Ida Mosbech; Baker, A; Arneborg, Nils

    2015-01-01

    distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase....... In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability......). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. SIGNIFICANCE AND IMPACT...

  16. Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

    NARCIS (Netherlands)

    Bergen en Henegouwen, P.M.P. van; Linnemans, W.A.M.

    The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce

  17. Altered development of NKT cells, γδ T cells, CD8 T cells and NK cells in a PLZF deficient patient.

    Directory of Open Access Journals (Sweden)

    Maggie Eidson

    Full Text Available In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease.

  18. Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

    Directory of Open Access Journals (Sweden)

    Benjamin Krämer

    2017-05-01

    Full Text Available Innate lymphocyte cells (ILCs, a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(- individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+ patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+ and HIV(- individuals.

  19. Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Grøn, B.; Mandel, U.

    1998-01-01

    Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate......-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both...... epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells....

  20. Active barrier films of PET for solar cell application: Processing and characterization

    International Nuclear Information System (INIS)

    Rossi, Gabriella; Scarfato, Paola; Incarnato, Loredana

    2014-01-01

    A preliminary investigation was carried out on the possibility to improve the protective action offered by the standard multilayer structures used to encapsulate photovoltaic devices. With this aim, a commercial active barrier PET-based material, able to absorb oxygen when activated by liquid water, was used to produce flexible and transparent active barrier films, by means of a lab-scale film production plant. The obtained film, tested in terms of thermal, optical and oxygen absorption properties, shows a slow oxygen absorption kinetics, an acceptable transparency and an easy roll-to-roll processability, so proving itself as a good candidate for the development of protective coating for solar cells against the atmospheric degradation agents like the rain

  1. A method of producing a multilayer barrier structure for a solid oxide fuel cell

    DEFF Research Database (Denmark)

    2010-01-01

    The present invention provides a method of producing a multilayer barrier structure for a solid oxide cell stack, comprising the steps of: - providing a metal interconnect, wherein the metal interconnect is a ferritic stainless steel layer; - applying a first metal oxide layer on said metal...... oxide; and - reacting the metal oxide in said first metal oxide layer with the metal of said metal interconnect during the SOC-stack initialisation, and a solid oxide stack comprising an anode contact layer and support structure, an anode layer, an electrolyte layer, a cathode layer, a cathode contact...... layer, a metallic interconnect, and a multilayer barrier structure which is obtainable by the above method and through an initialisation step, which is carried out under controlled conditions for atmosphere composition and current load, which depends on the layer composition facilitating the formation...

  2. Skin barrier disruption by sodium lauryl sulfate-exposure alters the expressions of involucrin, transglutaminase 1, profilaggrin, and kallikreins during the repair phase in human skin in vivo.

    Science.gov (United States)

    Törmä, Hans; Lindberg, Magnus; Berne, Berit

    2008-05-01

    Detergents are skin irritants affecting keratinocytes. In this study, healthy volunteers were exposed to water (vehicle) and 1% sodium lauryl sulfate (SLS) under occlusive patch tests for 24 hours. The messenger RNA (mRNA) expression of keratinocyte differentiation markers and of enzymes involved in corneodesmosome degradation was examined in skin biopsies (n=8) during the repair phase (6 hours to 7 days postexposure) using real-time reverse-transcription PCR. It was found that the expression of involucrin was increased at 6 hours, but then rapidly normalized. The expression of transglutaminase 1 exhibited a twofold increase after 24 hours in the SLS-exposed skin. Profilaggrin was decreased after 6 hours. Later (4-7 days), the expression in SLS-exposed areas was >50% above than in control areas. An increased and altered immunofluorescence pattern of involucrin, transglutaminase 1, and filaggrin was also found (n=4). At 6 hours post-SLS exposure, the mRNA expression of kallikrein-7 (KLK-7) and kallikrein-5 (KLK-5) was decreased by 50 and 75%, respectively, as compared with control and water-exposed areas. Thereafter, the expression pattern of KLK-7 and KLK-5 was normalized. Changes in protein expression of KLK-5 were also found. In conclusion, SLS-induced skin barrier defects induce altered mRNA expression of keratinocyte differentiation markers and enzymes degrading corneodesmosomes.

  3. Chromosome alterations in the X-ray-induced transformants of cultured mouse cell line

    International Nuclear Information System (INIS)

    Kodama, Seiji; Komatsu, Kenshi; Okumura, Yutaka; Sasaki, M.S.

    1989-01-01

    Mouse m5S cells were subjected to soft X-ray irradiation. Twenty-four transformants were separated as indicators of focus formation. Two clones, cl.4103 and cl.6310, were chosen for the analysis of chromosome alterations in transformants. A parent strain, m5S/1M, served as the control. Anchorage independence (AG) was not detected in the control strain, irrespective of culture conditions and population doubling number (PDN). In the case of transformants, the frequency of AG was increased with increasing PDN for cl.4103, and was constant for cl.6310, irrespective of PDN. Karyotype of m5S/1M was 42, X, -Y, +der (6) t(6;13), t(8;8), +8, +15. In addition, -13, der(10) and -der(6)t(6;13), der(5), +mar occurred as karyotype alterations for cl.4103 and cl. 6310, respectively. The present experiment indicated that chromosome alterations secondary to primary alterations occur in a high frequency in the transforming process of X-ray irradiated cells, and that the secondary chromosome alterations result in selective proliferation of transformed clones. (Namekawa, K)

  4. Altered Natural Killer Cell Function in HIV-Exposed Uninfected Infants

    Directory of Open Access Journals (Sweden)

    Christiana Smith

    2017-04-01

    Full Text Available ObjectivesHIV-exposed uninfected (HEU infants have higher rates of severe and fatal infections compared with HIV-unexposed (HUU infants, likely due to immune perturbations. We hypothesized that alterations in natural killer (NK cell activity might occur in HEU infants and predispose them to severe infections.DesignCase–control study using cryopreserved peripheral blood mononuclear cells (PBMCs at birth and 6 months from HEU infants enrolled from 2002 to 2009 and HUU infants enrolled from 2011 to 2013.MethodsNK cell phenotype and function were assessed by flow cytometry after 20-h incubation with and without K562 cells.ResultsThe proportion of NK cells among PBMCs was lower at birth in 12 HEU vs. 22 HUU (1.68 vs. 10.30%, p < 0.0001 and at 6 months in 52 HEU vs. 72 HUU (3.09 vs. 4.65%, p = 0.0005. At birth, HEU NK cells demonstrated increased killing of K562 target cells (p < 0.0001 and increased expression of CD107a (21.65 vs. 12.70%, p = 0.047, but these differences resolved by 6 months. Stimulated HEU NK cells produced less interferon (IFNγ at birth (0.77 vs. 2.64%, p = 0.008 and at 6 months (4.12 vs. 8.39%, p = 0.001, and showed reduced perforin staining at 6 months (66.95 vs. 77.30%, p = 0.0008. Analysis of cell culture supernatants indicated that lower NK cell activity in HEU was associated with reduced interleukin (IL-12, IL-15, and IL-18. Addition of recombinant human IL-12 to stimulated HEU PBMCs restored IFNγ production to that seen in stimulated HUU cultures.ConclusionNK cell proportion, phenotype, and function are altered in HEU infants. NK cell cytotoxicity and degranulation are increased in HEU at birth, but HEU NK cells have reduced IFNγ and perforin production, suggesting an adequate initial response, but decreased functional reserve. NK cell function improved with addition of exogenous IL-12, implicating impaired production of IL-12 by accessory cells. Alterations in NK cell and accessory

  5. Altered T cell memory and effector cell development in chronic lymphatic filarial infection that is independent of persistent parasite antigen.

    Directory of Open Access Journals (Sweden)

    Cathy Steel

    2011-04-01

    Full Text Available Chronic lymphatic filarial (LF infection is associated with suppression of parasite-specific T cell responses that persist even following elimination of infection. While several mechanisms have been implicated in mediating this T cell specific downregulation, a role for alterations in the homeostasis of T effector and memory cell populations has not been explored. Using multiparameter flow cytometry, we investigated the role of persistent filarial infection on the maintenance of T cell memory in patients from the filarial-endemic Cook Islands. Compared to filarial-uninfected endemic normals (EN, microfilaria (mf positive infected patients (Inf had a reduced CD4 central memory (T(CM compartment. In addition, Inf patients tended to have more effector memory cells (T(EM and fewer effector cells (T(EFF than did ENs giving significantly smaller T(EFF:T(EM ratios. These contracted T(CM and T(EFF populations were still evident in patients previously mf+ who had cleared their infection (CLInf. Moreover, the density of IL-7Rα, necessary for T memory cell maintenance (but decreased in T effector cells, was significantly higher on memory cells of Inf and CLInf patients, although there was no evidence for decreased IL-7 or increased soluble IL7-Rα, both possible mechanisms for signaling defects in memory cells. However, effector cells that were present in Inf and CLInf patients had lower percentages of HLA-DR suggesting impaired function. These changes in T cell populations appear to reflect chronicity of infection, as filarial-infected children, despite the presence of active infection, did not show alterations in the frequencies of these T cell phenotypes. These data indicate that filarial-infected patients have contracted T(CM compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children

  6. Alteration of skin hydration and its barrier function by vehicle and permeation enhancers: a study using TGA, FTIR, TEWL and drug permeation as markers.

    Science.gov (United States)

    Shah, D K; Khandavilli, S; Panchagnula, R

    2008-09-01

    Vehicles and permeation enhancers (PEs) used in transdermal drug delivery (TDD) of a drug can affect skin hydration, integrity and permeation of the solute administered. This investigation was designed to study the effect of the most commonly used vehicles and PEs on rat skin hydration, barrier function and permeation of an amphiphilic drug, imipramine hydrochloride (IMH). An array of well-established techniques were used to confirm the findings of the study. Thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy were used to determine changes in skin hydration. Alteration of the stratum corneum (SC) structure was investigated using FTIR studies. To monitor the barrier function alteration, transepidermal water loss (TEWL) measurement and permeation studies were performed. Our findings indicate that with hydration, there was an increase in the bound water content of the skin, and pseudoequilibrium of hydration (a drastic decrease in hydration rate) was achieved at around 12 h. Hydration increased the ratio between amide-I and amide-II peaks in FTIR and reduced the C-H stretching peak area. Both propylene glycol (PG) and ethanol (EtOH) dehydrated skin, with the latter showing a predominant effect. Furthermore, it was confirmed that PG and EtOH decreased the bound water content due to alteration in the protein domains and extraction of SC lipids, respectively. The effect of hydration on the SC was found to be similar to that reported for temperature. Permeation studies revealed that the dehydration caused by vehicles decreased IMH flux, whereas the flux was enhanced by PEs. The role of partition was predominant for the permeation of IMH through dehydrated skin. A synergistic effect was observed for PG and menthol in the enhancement of IMH. Further findings provided strong evidence that PG affects protein domains and EtOH extracts lipids from the bilayer. Both PG and EtOH, with or without PEs, increased TEWL. Initial TEWL was well

  7. Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

    Science.gov (United States)

    Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

    2018-03-05

    Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

  8. Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

    Science.gov (United States)

    Nandakumar, Vivek; Hansen, Nanna; Glenn, Honor L; Han, Jessica H; Helland, Stephanie; Hernandez, Kathryn; Senechal, Patti; Johnson, Roger H; Bussey, Kimberly J; Meldrum, Deirdre R

    2016-08-09

    The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat's differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

  9. Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

    DEFF Research Database (Denmark)

    Orfila, C.; Huisman, M.M.H.; Willats, William George Tycho

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic...... polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were...... that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained...

  10. Cell phone use and parotid salivary gland alterations: no molecular evidence.

    Science.gov (United States)

    de Souza, Fabrício T A; Correia-Silva, Jeane F; Ferreira, Efigênia F; Siqueira, Elisa C; Duarte, Alessandra P; Gomez, Marcus Vinícius; Gomez, Ricardo S; Gomes, Carolina C

    2014-07-01

    The association between cell phone use and the development of parotid tumors is controversial. Because there is unequivocal evidence that the microenvironment is important for tumor formation, we investigated in the parotid glands whether cell phone use alters the expression of gene products related to cellular stress. We used the saliva produced by the parotid glands of 62 individuals to assess molecular alterations compatible with cellular stress, comparing the saliva from the gland exposed to cell phone radiation (ipsilateral) to the saliva from the opposite, unexposed parotid gland (contralateral) of each individual. We compared salivary flow, total protein concentration, p53, p21, reactive oxygen species (ROS), and salivary levels of glutathione (GSH), heat shock proteins 27 and 70, and IgA between the ipsilateral and contralateral parotids. No difference was found for any of these parameters, even when grouping individuals by period of cell phone use in years or by monthly average calls in minutes. We provide molecular evidence that the exposure of parotid glands to cell phone use does not alter parotid salivary flow, protein concentration, or levels of proteins of genes that are directly or indirectly affected by heat-induced cellular stress. ©2014 American Association for Cancer Research.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  12. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    Directory of Open Access Journals (Sweden)

    Abbas Jafari

    2017-02-01

    Full Text Available Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  13. Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

    Science.gov (United States)

    Komaki, Yukako; Mariñas, Benito J; Plewa, Michael J

    2014-10-07

    Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of

  14. Dynamic gene expression response to altered gravity in human T cells.

    Science.gov (United States)

    Thiel, Cora S; Hauschild, Swantje; Huge, Andreas; Tauber, Svantje; Lauber, Beatrice A; Polzer, Jennifer; Paulsen, Katrin; Lier, Hartwin; Engelmann, Frank; Schmitz, Burkhard; Schütte, Andreas; Layer, Liliana E; Ullrich, Oliver

    2017-07-12

    We investigated the dynamics of immediate and initial gene expression response to different gravitational environments in human Jurkat T lymphocytic cells and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. We used the Affymetrix GeneChip® Human Transcriptome Array 2.0 containing 44,699 protein coding genes and 22,829 non-protein coding genes and performed the experiments during a parabolic flight and a suborbital ballistic rocket mission to cross-validate gravity-regulated gene expression through independent research platforms and different sets of control experiments to exclude other factors than alteration of gravity. We found that gene expression in human T cells rapidly responded to altered gravity in the time frame of 20 s and 5 min. The initial response to microgravity involved mostly regulatory RNAs. We identified three gravity-regulated genes which could be cross-validated in both completely independent experiment missions: ATP6V1A/D, a vacuolar H + -ATPase (V-ATPase) responsible for acidification during bone resorption, IGHD3-3/IGHD3-10, diversity genes of the immunoglobulin heavy-chain locus participating in V(D)J recombination, and LINC00837, a long intergenic non-protein coding RNA. Due to the extensive and rapid alteration of gene expression associated with regulatory RNAs, we conclude that human cells are equipped with a robust and efficient adaptation potential when challenged with altered gravitational environments.

  15. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Jing Wang

    2014-10-01

    Full Text Available AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2 on untransfected human corneal endothelial cells (HCECs in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01 and the length of F-actin, reduced the mean optical density (P<0.01 of the collagen type IV in extracellular matrix (ECM and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  16. Cultured cells of the blood-brain barrier from apolipoprotein B-100 transgenic mice: effects of oxidized low-density lipoprotein treatment.

    Science.gov (United States)

    Lénárt, Nikolett; Walter, Fruzsina R; Bocsik, Alexandra; Sántha, Petra; Tóth, Melinda E; Harazin, András; Tóth, Andrea E; Vizler, Csaba; Török, Zsolt; Pilbat, Ana-Maria; Vígh, László; Puskás, László G; Sántha, Miklós; Deli, Mária A

    2015-07-17

    The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood-brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-14

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

  18. CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

    International Nuclear Information System (INIS)

    Desai, Amar; Webb, Bryan; Gerson, Stanton L.

    2014-01-01

    Background: Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. Materials and methods: A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes. Results: A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. Conclusions: CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs

  19. Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Kucerova, Lucia; Skolekova, Svetlana; Matuskova, Miroslava; Bohac, Martin; Kozovska, Zuzana

    2013-01-01

    Mesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3. The tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed. The AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil. Our work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses

  20. Three cell flying capacitor inverter for dielectric barrier discharge plasma applications

    International Nuclear Information System (INIS)

    Flores-Fuentes, A.; Lopez-Callejas, A.; Piedad-Beneitez, A. de la; Pena-Eguiluz, R.; Mercado-Cabrera, A.; Valencia-Alvarado, R.; Barocio, S.R.

    2009-01-01

    It is reported the design, construction and initial tests of a three cell flying capacitor inverter (TCFCI) in a half-bridge configuration. The device operates at a 200 k Hz frequency which leads to a voltage output at 12.5 k Hz presenting an acceptable response in an open-loop configuration. These features outdo those reported in the current multilevel converter literature. The TCFCI is driven by pulse width modulation, following a phase shift (PS-PWM) control strategy, in order to generate a steady AC voltage signal. This inverter is used to excite a dielectric barrier discharge cell (DBDC) intended for cold plasma generation at room pressure. Some results obtained for two different kinds of atmosphere, helium and argon, are presented. All the system having been tested, early recorded voltage and current waveforms, are included. Finally, three methods for calculating the related electric efficiency of the discharge cell are discussed. (author)

  1. Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition.

    Science.gov (United States)

    Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena

    2017-01-01

    Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO 3 - ) and ammonium (NH 4 + ). However, the composition of the N source is important, because excess of NH 4 + promotes morphological disorders. Plants cultured on NH 4 + as the sole N source exhibit serious growth inhibition, commonly referred to as "ammonium toxicity syndrome." NH 4 + -mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH 4 + nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH 4 + as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH 4 + toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH 4 + -mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia , a receptor-like kinase involved in the control of cell wall extension.

  2. Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

    Science.gov (United States)

    Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

    2013-01-01

    Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme. PMID:24204595

  3. Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

    Directory of Open Access Journals (Sweden)

    Philip Klepeisz

    Full Text Available Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs have concentrated on alterations induced in hepatocytes (HCs. A potential role of non-parenchymal liver cells (NPCs in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

  4. Pulsed Magnetic Field Improves the Transport of Iron Oxide Nanoparticles through Cell Barriers

    Science.gov (United States)

    Min, Kyoung Ah; Shin, Meong Cheol; Yu, Faquan; Yang, Meizhu; David, Allan E.; Yang, Victor C.; Rosania, Gus R.

    2013-01-01

    Understanding how a magnetic field affects the interaction of magnetic nanoparticles (MNPs) with cells is fundamental to any potential downstream applications of MNPs as gene and drug delivery vehicles. Here, we present a quantitative analysis of how a pulsed magnetic field influences the manner in which MNPs interact with, and penetrate across a cell monolayer. Relative to a constant magnetic field, the rate of MNP uptake and transport across cell monolayers was enhanced by a pulsed magnetic field. MNP transport across cells was significantly inhibited at low temperature under both constant and pulsed magnetic field conditions, consistent with an active mechanism (i.e. endocytosis) mediating MNP transport. Microscopic observations and biochemical analysis indicated that, in a constant magnetic field, transport of MNPs across the cells was inhibited due to the formation of large (>2 μm) magnetically-induced MNP aggregates, which exceeded the size of endocytic vesicles. Thus, a pulsed magnetic field enhances the cellular uptake and transport of MNPs across cell barriers relative to a constant magnetic field by promoting accumulation while minimizing magnetically-induced MNP aggregates at the cell surface. PMID:23373613

  5. Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth

    Directory of Open Access Journals (Sweden)

    Jonathan M. Page

    2015-09-01

    Full Text Available The contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2, poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

  6. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure. © 2013 Wiley Periodicals, Inc.

  7. Barriers to hydroxyurea adherence and health-related quality of life in adolescents and young adults with sickle cell disease.

    Science.gov (United States)

    Badawy, Sherif M; Thompson, Alexis A; Penedo, Frank J; Lai, Jin-Shei; Rychlik, Karen; Liem, Robert I

    2017-06-01

    To identify barriers to hydroxyurea adherence (negative beliefs, access, and/or recall barriers), and their relationship to adherence rates and health-related quality of life (HRQOL) among adolescents and young adults (AYA) with sickle cell disease (SCD). A cross-sectional survey was administered to 34 AYAs (12-22 years old) in SCD clinics from January to December 2015. Study measures included Brief Medication Questionnaire, Modified Morisky Adherence Scale 8-items, visual analog scale, and Patient Reported Outcomes Measurement Information System. Participants (59% male; 91% Black) had a median age of 13.5 years (IQR 12-18). Participants reported negative beliefs (32%), recall barriers (44%), and access barriers (32%). Participants with recall barriers reported worse pain (P=.02), fatigue (P=.05), and depression (P=.05). The number of adherence barriers inversely correlated with adherence level using ©MMAS-8 (r s =-.38, P=.02) and VAS dose (r s =-.25, P=.14) as well as MCV (r s =-.45, P=.01) and HbF% (r s =-.36, P=.05), suggesting higher hydroxyurea adherence in patients with fewer barriers. Patients with fewer barriers to hydroxyurea adherence were more likely to have higher adherence rates and better HRQOL scores. Routine assessment of hydroxyurea adherence and its related barriers could provide actionable information to improve adherence rates, HRQOL, and other clinical outcomes. © 2017 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.

  8. Understanding the free energy barrier and multiple timescale dynamics of charge separation in organic photovoltaic cells.

    Science.gov (United States)

    Yan, Yaming; Song, Linze; Shi, Qiang

    2018-02-28

    By employing several lattice model systems, we investigate the free energy barrier and real-time dynamics of charge separation in organic photovoltaic (OPV) cells. It is found that the combined effects of the external electric field, entropy, and charge delocalization reduce the free energy barrier significantly. The dynamic disorder reduces charge carrier delocalization and results in the increased charge separation barrier, while the effect of static disorder is more complicated. Simulation of the real-time dynamics indicates that the free charge generation process involves multiple time scales, including an ultrafast component within hundreds of femtoseconds, an intermediate component related to the relaxation of the hot charge transfer (CT) state, and a slow component on the time scale of tens of picoseconds from the thermally equilibrated CT state. Effects of hot exciton dissociation as well as its dependence on the energy offset between the Frenkel exciton and the CT state are also analyzed. The current results indicate that only a small energy offset between the band gap and the lowest energy CT state is needed to achieve efficient free charge generation in OPV devices, which agrees with recent experimental findings.

  9. Understanding the free energy barrier and multiple timescale dynamics of charge separation in organic photovoltaic cells

    Science.gov (United States)

    Yan, Yaming; Song, Linze; Shi, Qiang

    2018-02-01

    By employing several lattice model systems, we investigate the free energy barrier and real-time dynamics of charge separation in organic photovoltaic (OPV) cells. It is found that the combined effects of the external electric field, entropy, and charge delocalization reduce the free energy barrier significantly. The dynamic disorder reduces charge carrier delocalization and results in the increased charge separation barrier, while the effect of static disorder is more complicated. Simulation of the real-time dynamics indicates that the free charge generation process involves multiple time scales, including an ultrafast component within hundreds of femtoseconds, an intermediate component related to the relaxation of the hot charge transfer (CT) state, and a slow component on the time scale of tens of picoseconds from the thermally equilibrated CT state. Effects of hot exciton dissociation as well as its dependence on the energy offset between the Frenkel exciton and the CT state are also analyzed. The current results indicate that only a small energy offset between the band gap and the lowest energy CT state is needed to achieve efficient free charge generation in OPV devices, which agrees with recent experimental findings.

  10. Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants

    Directory of Open Access Journals (Sweden)

    Pankaj Dhonukshe

    2012-01-01

    Full Text Available Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration.

  11. Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells1

    Science.gov (United States)

    Sprenger, Cynthia C T; Drivdahl, Rolf H; Woodke, Lillie B; Eyman, Daniel; Reed, May J; Carter, William G; Plymate, Stephen R

    2008-01-01

    Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs) are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer. PMID:19048114

  12. Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Cynthia C.T. Sprenger

    2008-12-01

    Full Text Available Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer.

  13. Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

    Science.gov (United States)

    Mendes de Aguiar, Claudia B N; Garcez, Ricardo Castilho; Alvarez-Silva, Marcio; Trentin, Andréa Gonçalves

    2002-11-01

    Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

  14. Schottky barrier enhancement on n-InP solar cell applications

    DEFF Research Database (Denmark)

    Clausen, Thomas; Leistiko, Otto

    1994-01-01

    It is demonstrated that the Schottky barrier height on n-type InP can be enhanced to values close to the energy bandgap (1.35 eV) by employing a AuZnCr metallization. The process is simple and requires only mild and fast annealing sequences with temperatures not exceeding 500°C. Also, no critical...... epitaxial growth step of junctions is needed, making the process fairly cheap. Thus, prospects for an efficient and simple solar cell device structure for space application purposes based on highly radiant-resistant InP are greatly improved...

  15. WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wen Jiang

    Full Text Available Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.

  16. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

    Science.gov (United States)

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam; Neil, James C

    2017-03-01

    The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV. IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most

  17. Pancreatic Beta-Cell Purification by Altering FAD and NAD(PH Metabolism

    Directory of Open Access Journals (Sweden)

    P. de Vos

    2008-07-01

    Full Text Available Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD and nicotinamide-adenine dinucleotide phosphate (NAD(PH autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(PH fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(PH contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(PH fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

  18. Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

    International Nuclear Information System (INIS)

    Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

    2009-01-01

    The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress

  19. Herbicide effects on freshwater benthic diatoms: Induction of nucleus alterations and silica cell wall abnormalities

    International Nuclear Information System (INIS)

    Debenest, T.; Silvestre, J.; Coste, M.; Delmas, F.; Pinelli, E.

    2008-01-01

    Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5 x 10 -6 , 10 -6 , 10 -7 M) for 6 h followed by a 24 h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per mille ), S.D. = 4.23) and the highest concentrations (29.40 per mille , S.D. = 8.49 for 10 -6 M and 35.96 per mille , S.D. = 3.71 for 5 x 10 -6 M) was statistically significant (Tukey test, P -6 and 5 x 10 -6 M; Tukey test, P < 0.05). These two parameters tended to increase together (Pearson correlation = 0.702, P < 0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic effects of MH. The alterations observed could be related to the effects of MH on the synthesis of the proteins involved in frustule formation or in the regulation of the cytoskeleton of the diatom cells

  20. Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells.

    Directory of Open Access Journals (Sweden)

    Mehrnaz Nouri

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE, the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers. These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms and at 14 days (i.e., at the stage of paralysis after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.

  1. Intestinal Barrier Dysfunction Develops at the Onset of Experimental Autoimmune Encephalomyelitis, and Can Be Induced by Adoptive Transfer of Auto-Reactive T Cells

    Science.gov (United States)

    Nouri, Mehrnaz; Bredberg, Anders; Weström, Björn; Lavasani, Shahram

    2014-01-01

    Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies. PMID:25184418

  2. Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells.

    Science.gov (United States)

    Koohestani, Faezeh; Braundmeier, Andrea G; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A

    2013-01-01

    Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

  3. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

    Science.gov (United States)

    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  4. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  5. Alterations in the nuclear proteome of HIV-1 infected T-cells

    International Nuclear Information System (INIS)

    DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

    2014-01-01

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

  6. Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

    Directory of Open Access Journals (Sweden)

    Wang Ting

    2012-08-01

    Full Text Available Abstract Background Exposure to particulate matter (PM is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. Objectives We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC barrier integrity and enhanced cardiopulmonary dysfunction. Methods Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm. Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. Results PM exposure induced tight junction protein Zona occludens-1 (ZO-1 relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin. N-acetyl-cysteine (NAC, 5 mM reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2, in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. Conclusions These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

  7. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-01-01

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [ 3 H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [ 3 H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  8. Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells

    DEFF Research Database (Denmark)

    Del Vecchio, Giovanna; Tscheik, Christian; Tenz, Kareen

    2012-01-01

    Claudin-5 is a tight junction (TJ) protein which limits the diffusion of small hydrophilic molecules. Thus, it represents a potential pharmacological target to improve drug delivery to the tissues protected by claudin-5-dependent barriers. Sodium caprate is known as an absorption enhancer which...... opens the paracellular space acting on TJ proteins and actin cytoskeleton. Its action on claudin-5 is not understood so far. Epithelial and endothelial systems were used to evaluate the effect of caprate on claudin-5 in TJ-free cells and on claudin-5 fully integrated in TJ. To this aim, confocal...... of endothelial and epithelial cells. In conclusion, the study further elucidates the cellular effects of caprate at the tight junctions....

  9. Tryps and trips: cell trafficking across the 100-year-old blood-brain barrier.

    Science.gov (United States)

    Bentivoglio, Marina; Kristensson, Krister

    2014-06-01

    One hundred years ago, Edwin E. Goldmann discovered the blood-brain barrier (BBB) using trypan dyes. These dyes were developed and named by Paul Ehrlich during his search for drugs to kill African trypanosomes (extracellular parasites that cause sleeping sickness) while sparing host cells. For Ehrlich, this was the first strategy based on the 'chemotherapy' concept he had introduced. The discovery of the BBB revealed, however, the difficulties in drug delivery to the brain. Mechanisms by which parasites enter, dwell, and exit the brain currently provide novel views on cell trafficking across the BBB. These mechanisms also highlight the role of pericytes and endocytosis regulation in BBB functioning and in disrupted BBB gating, which may be involved in the pathogenesis of neurodegeneration. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Altered 67Ga citrate distribution in patients with multiple red blood cell transfusions

    International Nuclear Information System (INIS)

    Engelstad, B.; Luk, S.S.; Hattner, R.S.

    1982-01-01

    Gallium-67 citrate studies from four patients who received multiple red blood cell transfusions were reviewed. Increased kidney, bladder, or bone localization was associated with decreased liver and colon activity. The findings suggest altered distribution due to competition with iron for receptor binding. Identification of inflammatory disease in two patients was possible. However, the effect of transfusions on detection of inflammatory or neoplastic diseases requires further evaluation

  11. Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver

    Directory of Open Access Journals (Sweden)

    Saura C. Sahu

    Full Text Available Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 μg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver. Keywords: Nanosilver, Silver nanoparticles, Nanoparticles, Toxicogenomics, DNA microarray, Global gene expression profiles, Caco2 cells

  12. Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

    Science.gov (United States)

    Chen, Richard J.; Zhang, Guofeng; Garfield, Susan H.; Shi, Yi-Jun; Chen, Kevin G.; Robey, Pamela G.; Leapman, Richard D.

    2015-01-01

    Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions. PMID:26565809

  13. Mutant p53 transfection of astrocytic cells results in altered cell cycle control, radiation sensitivity, and tumorigenicity

    International Nuclear Information System (INIS)

    Kanady, Kirk E.; Mei Su; Proulx, Gary; Malkin, David M.; Pardo, Francisco S.

    1995-01-01

    Introduction: Alterations in the p53 tumor suppressor gene are one of the most frequent genetic alterations in malignant gliomas. An understanding of the molecular genetic events leading to glial tumor progression would aid in designing therapeutic vectors for controlling these challenging tumor types. We investigated whether mutations in coding exons of the p53 gene result in functional changes altering cell cycle 'checkpoint' control and the intrinsic radiation sensitivity of glial cells. Methods: An astrocytic cell line was derived from a low grade astrocytoma and characterized to be of human karyotype and GFAP positivity. Additionally, the cellular population has never formed tumors in immune-deficient mice. At early passage ( 2 as parameters. Cell kinetic analyses after 2, 5, and 10 Gy of ionizing radiation were conducted using propidium iodide FACS analyses. Results: Overall levels of p53 expression were increased 5-10 fold in the transfected cellular populations. Astrocytic cellular populations transfected with mutant p53 revealed a statistically significant increase in levels of resistance to ionizing radiation in vitro (2-tailed test, SF2, MID). Astrocytic cellular populations transfected with mutant p53, unlike the parental cells, were tumorigenic in SCID mice. Cell kinetic analyses indicated that the untransfected cell line demonstrated dose dependent G1 and G2 arrests. Following transfection, however, the resultant cellular population demonstrated a predominant G2 arrest. Conclusions: Astrocytic cellular populations derived from low grade astrocytomas, are relatively radiation sensitive, non-tumorigenic, and have intact cell cycle ''checkpoints.'' Cellular populations resulting upon transfection of parental cells with a dominant negative p53 mutation, are relatively radiation resistant, when compared to both parental and mock-transfected cells. Transfected cells demonstrate abnormalities of cell cycle control at the G1/S checkpoint, increases in levels

  14. Alterations of the Blood-Brain Barrier and Regional Perfusion in Tumor Development: MRI Insights from a Rat C6 Glioma Model.

    Science.gov (United States)

    Huhndorf, Monika; Moussavi, Amir; Kramann, Nadine; Will, Olga; Hattermann, Kirsten; Stadelmann, Christine; Jansen, Olav; Boretius, Susann

    2016-01-01

    Angiogenesis and anti-angiogenetic medications play an important role in progression and therapy of glioblastoma. In this context, in vivo characterization of the blood-brain-barrier and tumor vascularization may be important for individual prognosis and therapy optimization. We analyzed perfusion and capillary permeability of C6-gliomas in rats at different stages of tumor-growth by contrast enhanced MRI and dynamic susceptibility contrast (DSC) MRI at 7 Tesla. The analyses included maps of relative cerebral blood volume (CBV) and signal recovery derived from DSC data over a time period of up to 35 days after tumor cell injections. In all rats tumor progression was accompanied by temporal and spatial changes in CBV and capillary permeability. A leakage of the blood-brain barrier (slow contrast enhancement) was observed as soon as the tumor became detectable on T2-weighted images. Interestingly, areas of strong capillary permeability (fast signal enhancement) were predominantly localized in the center of the tumor. In contrast, the tumor rim was dominated by an increased CBV and showed the highest vessel density compared to the tumor center and the contralateral hemisphere as confirmed by histology. Substantial regional differences in the tumor highlight the importance of parameter maps in contrast or in addition to region-of-interest analyses. The data vividly illustrate how MRI including contrast-enhanced and DSC-MRI may contribute to a better understanding of tumor development.

  15. Transporter-Guided Delivery of Nanoparticles to Improve Drug Permeation across Cellular Barriers and Drug Exposure to Selective Cell Types

    Directory of Open Access Journals (Sweden)

    Longfa Kou

    2018-01-01

    Full Text Available Targeted nano-drug delivery systems conjugated with specific ligands to target selective cell-surface receptors or transporters could enhance the efficacy of drug delivery and therapy. Transporters are expressed differentially on the cell-surface of different cell types, and also specific transporters are expressed at higher than normal levels in selective cell types under pathological conditions. They also play a key role in intestinal absorption, delivery via non-oral routes (e.g., pulmonary route and nasal route, and transfer across biological barriers (e.g., blood–brain barrier and blood–retinal barrier. As such, the cell-surface transporters represent ideal targets for nano-drug delivery systems to facilitate drug delivery to selective cell types under normal or pathological conditions and also to avoid off-target adverse side effects of the drugs. There is increasing evidence in recent years supporting the utility of cell-surface transporters in the field of nano-drug delivery to increase oral bioavailability, to improve transfer across the blood–brain barrier, and to enhance delivery of therapeutics in a cell-type selective manner in disease states. Here we provide a comprehensive review of recent advancements in this interesting and important area. We also highlight certain key aspects that need to be taken into account for optimal development of transporter-assisted nano-drug delivery systems.

  16. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  17. Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion

    Directory of Open Access Journals (Sweden)

    Lorène J. Lebrun

    2017-10-01

    Full Text Available Summary: Glucagon-like peptide 1 (GLP-1 is a hormone released from enteroendocrine L cells. Although first described as a glucoregulatory incretin hormone, GLP-1 also suppresses inflammation and promotes mucosal integrity. Here, we demonstrate that plasma GLP-1 levels are rapidly increased by lipopolysaccharide (LPS administration in mice via a Toll-like receptor 4 (TLR4-dependent mechanism. Experimental manipulation of gut barrier integrity after dextran sodium sulfate treatment, or via ischemia/reperfusion experiments in mice, triggered a rapid rise in circulating GLP-1. This phenomenon was detected prior to measurable changes in inflammatory status and plasma cytokine and LPS levels. In human subjects, LPS administration also induced GLP-1 secretion. Furthermore, GLP-1 levels were rapidly increased following the induction of ischemia in the human intestine. These findings expand traditional concepts of enteroendocrine L cell biology to encompass the sensing of inflammatory stimuli and compromised mucosal integrity, linking glucagon-like peptide secretion to gut inflammation. : Lebrun et al. demonstrate that enteroendocrine L cells sense lipopolysaccharides (pro-inflammatory bacterial compounds after gut injury and respond by secreting glucagon-like peptide 1. These findings expand concepts of L cell function to include roles as both a nutrient and pathogen sensor, linking glucagon-like peptide secretion to gut inflammation. Keywords: glucagon-like peptide 1, lipopolysaccharides, enteroendocrine cells, TLR4, gut injury, intestinal ischemia, inflammation

  18. Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation

    International Nuclear Information System (INIS)

    Willner, Jochen; Vordermark, Dirk; Schmidt, Michael; Gassel, Andreamaria; Flentje, Michael; Wirtz, Hubert

    2003-01-01

    Purpose: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo. Methods and Materials: Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using 3 H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index. Results: During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G 2 /M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls. Conclusion: In contrast to literature data, we observed no direct

  19. Alterations of proliferation and differentiation of hippocampal cells in prenatally stressed rats.

    Science.gov (United States)

    Sun, Hongli; Su, Qian; Zhang, Huifang; Liu, Weimin; Zhang, Huiping; Ding, Ding; Zhu, Zhongliang; Li, Hui

    2015-06-01

    To clarify the alterations of proliferation and differentiation of hippocampal cells in prenatally stressed rats. We investigated the impact of prenatal restraint stress on the hipocampal cell proliferation in the progeny with 5-bromo-2'-deoxyuridine (BrdU), which is a marker of proliferating cells and their progeny. In addition, we observed the differentiation of neural stem cells (NSCs) with double labeling of BrdU/neurofilament (NF), BrdU/glial fibrillary acidic protein (GFAP) in the hipocampus. Prenatal stress (PS) increased cell proliferation in the dentate gyrus (DG) only in female and neuron differentiation of newly divided cells in the DG and CA4 in both male and female. Moreover, the NF and GFAP-positive cells, but not the BrdU-positive cells, BrdU/NF and BrdU/GFAP-positive cells, were found frequently in the CA3 and CA1 in the offspring of each group. These results possibly suggest a compensatory adaptive response to neuronal damage or loss in hippocampus induced by PS. Copyright © 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  20. Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

    Science.gov (United States)

    Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

    2017-08-01

    Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.

  1. Initial Attempts of Development and Characterization of an In Vitro Blood Brain Barrier Model Derived from Human Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Hall, Vanessa Jane

    The human blood brain barrier has yet to be successfully replicated as an in vitro model. One of the more promising approaches has been to develop an in vitro model derived from human pluripotent stem cells. However, as promising as this model may be, a successful replication of the differentiation...... method on different kinds of pluripotent stem cell lines have yet to be accomplished. We try to approach the promising method as described by Stebbins et al. (2015) to differentiate human pluripotent stem cells into brain like endothelial cells (BECs). Five different human pluripotent stem cell lines...... configurations (mono culture, non-contact co-culture and contact co-culture) with primary rat astrocytes to induce barrier-like properties. Endothelial cell media supplemented with retinoic acid were then applied to the cells to ensure selective expansion of BECs. The different culture configurations were...

  2. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  3. Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J.; Anderson, Charles T.

    2015-11-02

    Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.

  4. Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

    Science.gov (United States)

    Navarro-Moreno, L G; Quintanar-Escorza, M A; González, S; Mondragón, R; Cerbón-Solorzáno, J; Valdés, J; Calderón-Salinas, J V

    2009-10-01

    Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

  5. Alterations in the extracellular matrix organization associated with the reexpression of tumorigenicity in human cell hybrids.

    Science.gov (United States)

    Der, C J; Stanbridge, E J

    1980-10-15

    The expression of fibronectin on the cell surface was evaluated on a series of intraspecific human cell hybrids formed between HeLa and normal fibroblast strains. Although these hybrids continued to express many of the in vitro transformation properties of their corresponding tumorigenic HeLa parent, they were now unable to form tumors when inoculated into athymic nude mice. From these suppressed hybrid populations, rare tumorigenic segregant subpopulations arose which had regained their tumorigenic capacity. A comparison of the expression of fibronectin on the cell surface was made between these tumorigenic segregant cell lines and their corresponding non-tumorigenic HeLa/fibroblast hybrid. Following specific immunofluorescent staining for fibronectin, a striking alteration in the cell surface organization was observed to correspond with the reexpression of tumorigenicity in these hybrids. Tumorigenic HeLa/fibroblast hybrids were also significantly altered in both their cellular and colonial morphology. Double immunofluorescent staining to simultaneously visualize both surface fibronectin and collagen revealed that these two extracellular matrix proteins displayed an extensive degree of codistribution and expressed a coordinate shift in organization which correlated with the appearance of tumorigenic segregant hybrid populations. These observations are in agreement with the apparently close structural association between fibronectin and collagen and suggest that the organization of these two components in the extracellular matrix may be an important determinant for in vivo growth potential.

  6. Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

    Science.gov (United States)

    Ramesh, Govindarajan; Wu, Honglu

    2012-01-01

    Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

  7. Reduction of microhemorrhages in the spinal cord of symptomatic ALS mice after intravenous human bone marrow stem cell transplantation accompanies repair of the blood-spinal cord barrier

    Science.gov (United States)

    Eve, David J.; Steiner, George; Mahendrasah, Ajay; Sanberg, Paul R.; Kurien, Crupa; Thomson, Avery; Borlongan, Cesar V.; Garbuzova-Davis, Svitlana

    2018-01-01

    Blood-spinal cord barrier (BSCB) alterations, including capillary rupture, have been demonstrated in animal models of amyotrophic lateral sclerosis (ALS) and ALS patients. To date, treatment to restore BSCB in ALS is underexplored. Here, we evaluated whether intravenous transplantation of human bone marrow CD34+ (hBM34+) cells into symptomatic ALS mice leads to restoration of capillary integrity in the spinal cord as determined by detection of microhemorrhages. Three different doses of hBM34+ cells (5 × 104, 5 × 105 or 1 × 106) or media were intravenously injected into symptomatic G93A SOD1 mice at 13 weeks of age. Microhemorrhages were determined in the cervical and lumbar spinal cords of mice at 4 weeks post-treatment, as revealed by Perls’ Prussian blue staining for ferric iron. Numerous microhemorrhages were observed in the gray and white matter of the spinal cords in media-treated mice, with a greater number of capillary ruptures within the ventral horn of both segments. In cell-treated mice, microhemorrhage numbers in the cervical and lumbar spinal cords were inversely related to administered cell doses. In particular, the pervasive microvascular ruptures determined in the spinal cords in late symptomatic ALS mice were significantly decreased by the highest cell dose, suggestive of BSCB repair by grafted hBM34+ cells. The study results provide translational outcomes supporting transplantation of hBM34+ cells at an optimal dose as a potential therapeutic strategy for BSCB repair in ALS patients. PMID:29535831

  8. B cell subset distribution is altered in patients with severe periodontitis.

    Science.gov (United States)

    Demoersman, Julien; Pochard, Pierre; Framery, Camille; Simon, Quentin; Boisramé, Sylvie; Soueidan, Assem; Pers, Jacques-Olivier

    2018-01-01

    Several studies have recently highlighted the implication of B cells in physiopathogenesis of periodontal disease by showing that a B cell deficiency leads to improved periodontal parameters. However, the detailed profiles of circulating B cell subsets have not yet been investigated in patients with severe periodontitis (SP). We hypothesised that an abnormal distribution of B cell subsets could be detected in the blood of patients with severe periodontal lesions, as already reported for patients with chronic inflammatory diseases as systemic autoimmune diseases. Fifteen subjects with SP and 13 subjects without periodontitis, according to the definition proposed by the CDC periodontal disease surveillance work group, were enrolled in this pilot observational study. Two flow cytometry panels were designed to analyse the circulating B and B1 cell subset distribution in association with the RANKL expression. A significantly higher percentage of CD27+ memory B cells was observed in patients with SP. Among these CD27+ B cells, the proportion of the switched memory subset was significantly higher. At the same time, human B1 cells, which were previously associated with a regulatory function (CD20+CD69-CD43+CD27+CD11b+), decreased in SP patients. The RANKL expression increased in every B cell subset from the SP patients and was significantly greater in activated B cells than in the subjects without periodontitis. These preliminary results demonstrate the altered distribution of B cells in the context of severe periodontitis. Further investigations with a larger cohort of patients can elucidate if the analysis of the B cell compartment distribution can reflect the periodontal disease activity and be a reliable marker for its prognosis (clinical trial registration number: NCT02833285, B cell functions in periodontitis).

  9. B cell subset distribution is altered in patients with severe periodontitis

    Science.gov (United States)

    Demoersman, Julien; Pochard, Pierre; Framery, Camille; Simon, Quentin; Boisramé, Sylvie; Soueidan, Assem

    2018-01-01

    Several studies have recently highlighted the implication of B cells in physiopathogenesis of periodontal disease by showing that a B cell deficiency leads to improved periodontal parameters. However, the detailed profiles of circulating B cell subsets have not yet been investigated in patients with severe periodontitis (SP). We hypothesised that an abnormal distribution of B cell subsets could be detected in the blood of patients with severe periodontal lesions, as already reported for patients with chronic inflammatory diseases as systemic autoimmune diseases. Fifteen subjects with SP and 13 subjects without periodontitis, according to the definition proposed by the CDC periodontal disease surveillance work group, were enrolled in this pilot observational study. Two flow cytometry panels were designed to analyse the circulating B and B1 cell subset distribution in association with the RANKL expression. A significantly higher percentage of CD27+ memory B cells was observed in patients with SP. Among these CD27+ B cells, the proportion of the switched memory subset was significantly higher. At the same time, human B1 cells, which were previously associated with a regulatory function (CD20+CD69-CD43+CD27+CD11b+), decreased in SP patients. The RANKL expression increased in every B cell subset from the SP patients and was significantly greater in activated B cells than in the subjects without periodontitis. These preliminary results demonstrate the altered distribution of B cells in the context of severe periodontitis. Further investigations with a larger cohort of patients can elucidate if the analysis of the B cell compartment distribution can reflect the periodontal disease activity and be a reliable marker for its prognosis (clinical trial registration number: NCT02833285, B cell functions in periodontitis). PMID:29447240

  10. Evidence for alteration of the membrane-bound ribosomes in Micrococcus luteus cells exposed to lead

    Energy Technology Data Exchange (ETDEWEB)

    Barrow, W; Himmel, M; Squire, P G; Tornabene, T G

    1978-01-01

    Micrococcus luteus cells exposed to Pb(NO/sub 3/)/sub 2/ contained cytosol ribosomal particles and disaggregated membranal ribosomal particles as determined by ultracentrifugation and spectral studies. Approximately 60% of the membrane ribosome fraction from lead exposed cells had a sedimentation value of 8.4S. Cytosol ribosome from lead exposed cells as well as membranal and cytosol ribosomes from control cells were comparable by their contents of predominantly the 70S type with the 50S and 100S present in relatively small amounts. The lead content of the 8.4S components was more than 200 times higher than the components with higher sedimentation coefficients from lead exposed cells and approximately 650 times more than that of control cell ribosomes. The cells exposed to lead, however, showed no adverse effects from the lead in respect to their growth rates and cellular yields. These results indicate that lead is interacting only at specific sites of the membrane and is inducing events initiated only in strategic cellular regions. These data further substantiate that subtle changes do occur in lead exposed cells that show no obvious effects. It is assumed that these minor alterations are, in toto, biologically significant. 24 references, 2 figures, 1 table.

  11. Response to Dengue virus infections altered by cytokine-like substances from mosquito cell cultures

    Directory of Open Access Journals (Sweden)

    Laosutthipong Chaowanee

    2010-11-01

    Full Text Available Abstract Background With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells. Results Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2 and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects. Conclusions This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.

  12. Proteome alteration induced by hTERT transfection of human fibroblast cells.

    Science.gov (United States)

    Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

    2008-04-17

    Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

  13. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  14. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

    International Nuclear Information System (INIS)

    Carter, Charleata A.; Hamm, Jonathan T.

    2009-01-01

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-κB (NF-κB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-α and NF-κB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30 μg/ml CSC significantly activated PKC-α, while CSC doses above 20 μg/ml CSC significantly activated NF-κB. As NF-κB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30 μg/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30 μg/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-α, NF-κB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  15. Alterations in gene expression profiles between radioresistant and radiosensitive cell lines

    International Nuclear Information System (INIS)

    Zhou Fuxiang; Zhou Yunfeng; Xie Conghua; Dai Jing; Cao Zhen; Yu Haijun; Liao Zhengkai; Luo Zhiguo

    2007-01-01

    Objective: To study the-difference of gene expressions by the contrastive model including the cells with same pathological origin and genetic background, but definitely different radioresponse, and to find the main molecular targets related to radiosensitivity. Methods: Human larynx squamous carcinoma cell, Hep -2 was irradiated with dose of 637 cGy repeatedly to establish a radioresistant daughter cell line. The radiobiology characteristics were obtained using clone forming assay. The difference of gene expression between parent and daughter cells was detected by cDNA microarray using two different arrays including 14000 genes respectively. Results: A radioresistant cell strain Hep-2R was isolated from its parental strain Hep-2 cell. The SF 2 , D 0 , α, β for Hep-2R cell line were 0.6798, 3.24, 0.2951 and 0.0363, respectively, while 0.4148, 2.06, 0.1074 and 0.0405 for Hep-2, respectively (for SF 2 , χ 2 =63.957, P<0.001). Compared with Hep-2 cells, the expressions of 41 genes were significantly altered in the radioresistant Hep-2R cells, including 22 genes up-regulated and 19 genes down-regulated, which were involved in DNA repair, regulation of the cell cycle, cell proliferation, cytoskeleton, protein synthesis, cellular metabolism and especially apoptosis which is responsible for the different radiosensitivity between these two larynx cancer cells. The telomere protection protein gene, POT1, was the mostly up-regulated by 3.348 times. Conclusions: There is difference of gene expression between the radioresistant contrastive models. POT1 gene may be the target of radiosensitization. (authors)

  16. Integrative analysis of copy number alteration and gene expression profiling in ovarian clear cell adenocarcinoma.

    Science.gov (United States)

    Sung, Chang Ohk; Choi, Chel Hun; Ko, Young-Hyeh; Ju, Hyunjeong; Choi, Yoon-La; Kim, Nyunsu; Kang, So Young; Ha, Sang Yun; Choi, Kyusam; Bae, Duk-Soo; Lee, Jeong-Won; Kim, Tae-Joong; Song, Sang Yong; Kim, Byoung-Gie

    2013-05-01

    Ovarian clear cell adenocarcinoma (Ov-CCA) is a distinctive subtype of ovarian epithelial carcinoma. In this study, we performed array comparative genomic hybridization (aCGH) and paired gene expression microarray of 19 fresh-frozen samples and conducted integrative analysis. For the copy number alterations, significantly amplified regions (false discovery rate [FDR] q genes demonstrating frequent copy number alterations (>25% of samples) that correlated with gene expression (FDR genes were mainly located on 8p11.21, 8p21.2-p21.3, 8q22.1, 8q24.3, 17q23.2-q23.3, 19p13.3, and 19p13.11. Among the regions, 8q24.3 was found to contain the most genes (30 of 94 genes) including PTK2. The 8q24.3 region was indicated as the most significant region, as supported by copy number, GISTIC, and integrative analysis. Pathway analysis using differentially expressed genes on 8q24.3 revealed several major nodes, including PTK2. In conclusion, we identified a set of 94 candidate genes with frequent copy number alterations that correlated with gene expression. Specific chromosomal alterations, such as the 8q24.3 gain containing PTK2, could be a therapeutic target in a subset of Ov-CCAs. Copyright © 2013. Published by Elsevier Inc.

  17. Environmentally induced epigenetic transgenerational inheritance of altered Sertoli cell transcriptome and epigenome: molecular etiology of male infertility.

    Directory of Open Access Journals (Sweden)

    Carlos Guerrero-Bosagna

    Full Text Available Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell that influences the onset of a specific disease (male infertility. A gestating female rat (F0 generation was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell epigenome and transcriptome that correlates with adult onset disease (male infertility. The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility.

  18. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

    Science.gov (United States)

    Máthé, Csaba; M-Hamvas, Márta; Vasas, Gábor

    2013-01-01

    Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization. PMID:24084787

  19. Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

    Directory of Open Access Journals (Sweden)

    Gábor Vasas

    2013-09-01

    Full Text Available Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs, a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN, an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization.

  20. Ultrastructural alterations in hypoxic EMT-6/RO cells treated with misonidazole

    International Nuclear Information System (INIS)

    Wilbur, D.C.; Mulcahy, R.T.

    1984-01-01

    Ultrastructural alterations in hypoxic EMT-6 tumor cells were quantitatively analyzed as a function of time in the presence and absence of 1.0mM MISO. Control and MISO-treated monolayer cultures were maintained in hypoxic chambers at 37 0 C. At intervals after initiation of hypoxia, the cells were fixed and prepared for electron microscopy. The major ultrastructural alterations observed in untreated and MISO-treated hypoxic cells included mitochondrial swelling and accumulation of cytoplasmic lipid vacuoles. Mean mitochondrial area and relative cytoplasmic area occupied by lipid vacuoles were determined morphometrically. Mitochondrial damage was also scored qualitatively based on distortions in configuration. In the absence of MISO both parameters of mitochondrial injury increased over a period of two hours, after which little further change was noted. A progressive increase in lipid vacuolization was also seen. In the presence of MISO, mitochondrial swelling and lipid vacuole formation were significantly increased. The proportion of irreversibly damaged mitochondria was markedly enhanced. MISO treatment also accelerated the expression of these changes. The accelerated expression of hypoxic-related injury in MISO treated cells suggests that cytotoxicity is related to accentuation of hypoxic injury, perhaps by inhibition of glycolysis

  1. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    Science.gov (United States)

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  2. Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.

    Science.gov (United States)

    Pai, Vaibhav P; Marshall, Aaron M; Hernandez, Laura L; Buckley, Arthur R; Horseman, Nelson D

    2009-01-01

    The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs

  3. Colchicine affects cell motility, pattern formation and stalk cell differentiation in Dictyostelium by altering calcium signaling.

    Science.gov (United States)

    Poloz, Yekaterina; O'Day, Danton H

    2012-04-01

    Previous work, verified here, showed that colchicine affects Dictyostelium pattern formation, disrupts morphogenesis, inhibits spore differentiation and induces terminal stalk cell differentiation. Here we show that colchicine specifically induces ecmB expression and enhances accumulation of ecmB-expressing cells at the posterior end of multicellular structures. Colchicine did not induce a nuclear translocation of DimB, a DIF-1 responsive transcription factor in vitro. It also induced terminal stalk cell differentiation in a mutant strain that does not produce DIF-1 (dmtA-) and after the treatment of cells with DIF-1 synthesis inhibitor cerulenin (100 μM). This suggests that colchicine induces the differentiation of ecmB-expressing cells independent of DIF-1 production and likely through a signaling pathway that is distinct from the one that is utilized by DIF-1. Depending on concentration, colchicine enhanced random cell motility, but not chemotaxis, by 3-5 fold (10-50 mM colchicine, respectively) through a Ca(2+)-mediated signaling pathway involving phospholipase C, calmodulin and heterotrimeric G proteins. Colchicine's effects were not due to microtubule depolymerization as other microtubule-depolymerizing agents did not have these effects. Finally normal morphogenesis and stalk and spore cell differentiation of cells treated with 10 mM colchicine were rescued through chelation of Ca2+ by BAPTA-AM and EDTA and calmodulin antagonism by W-7 but not PLC inhibition by U-73122. Morphogenesis or spore cell differentiation of cells treated with 50 mM colchicine could not be rescued by the above treatments but terminal stalk cell differentiation was inhibited by BAPTA-AM, EDTA and W-7, but not U-73122. Thus colchicine disrupts morphogenesis and induces stalk cell differentiation through a Ca(2+)-mediated signaling pathway involving specific changes in gene expression and cell motility. Copyright © 2011 International Society of Differentiation. Published by Elsevier B

  4. Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

    Science.gov (United States)

    Albert, Ina; Hefti, Martin; Luginbuehl, Vera

    2014-11-01

    The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

  5. Altered Ca fluxes and contractile state during pH changes in cultured heart cells

    International Nuclear Information System (INIS)

    Kim, D.; Smith, T.W.

    1987-01-01

    The authors studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pH/sub i/) or extracellular (pH 0 ) changes in pH using cultured chick embryo ventricular cells. A change in pH 0 of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45 Ca uptake at 10 s by -29 or +22%, respectively. The pH 0 induced change in Ca uptake was not sensitive to nifedipine but was Na gradient dependent. Changes in pH/sub i/ produced by NH 4 Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45 Ca uptake or efflux. However, larger changes in pH/sub i/ were associated with altered Ca uptake. Changes in pH 0 from 7.5 to 6.0 or to 8.8 were associated with initial changes in 45 Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH 4 Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH 4 Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationships between pH/sub i/ and contractile state. These results indicated that pH 0 -induced changes in contractile state in cultured heart cells are closely correlated with altered transarcolemmal Ca movements and presumably are due to these Ca flux changes

  6. Genetic and epigenetic alterations of the reduced folate carrier in untreated diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Kastrup, I.B.; Worm, J.; Ralfkiaer, E.

    2008-01-01

    The reduced folate carrier (RFC) is a transmembrane protein that mediates cellular uptake of reduced folates and antifolate drugs, including methotrexate (MTX). Acquired alterations of the RFC gene have been associated with resistance to MTX in cancer cell lines and primary osteosarcomas. Here, w...... with adverse outcome. In DLBCL, genetic and epigenetic alterations of RFC were detected at diagnosis in the absence of a selective MTX pressure, suggesting that these alterations may possibly contribute to the development of lymphoma Udgivelsesdato: 2008/1...

  7. Age-related alteration in the composition of immunocompetent blood cells in atomic bomb survivors

    International Nuclear Information System (INIS)

    Kusunoki, Yoichiro; Akiyama, Mitoshi; Kyoizumi, Seishi; Bloom, E.T.; Makinodan, Takashi; California Univ., Los Angeles

    1988-01-01

    1328 survivors of Hiroshima were studied for alterations in the number of blood lymphocytes belonging to T-cell subpopulations, CD19 antigen-positive B cells and Leu 7 and CD16 antigen-positive lymphocytes. With increasing age, significant decreasing trends in the numbers of some lymphocytes in T-cell subpopulations and of B-cells were seen. The number of blood lymphocytes positive for CD5 antigen was significantly lower in those exposed to radiation (> 1Gy) in the older age group (more than 30 years at the time of bombing) and a similar tendency for decreases in the numbers of CD4, CD8, and CD19 antigen-positive cells was observed, but differences were not significant. The results suggest aging of the T-cell related immune system is accelerated in the irradiated people of advanced age, explained by the age-related decrease in thymic function in those subjects. The number of Leu 7 or CD19 antigen-positive cells was found to be increased significantly in the older age group compared to the younger, although there was little dose dependence. (U.K.)

  8. Pseudomonas aeruginosa lipopolysaccharide induces CF-like alteration of protein secretion by human tracheal gland cells.

    Science.gov (United States)

    Kammouni, W; Figarella, C; Baeza, N; Marchand, S; Merten, M D

    1997-12-18

    Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection by Pseudomonas aeruginosa in the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment with Pseudomonas aeruginosa LPS resulted in a significant dose-dependent increase in the basal production of SLPI (+ 250 +/- 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated by Pseudomonas aeruginosa LPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

  9. Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults.

    Science.gov (United States)

    Calderón-Garcidueñas, Lilian; Solt, Anna C; Henríquez-Roldán, Carlos; Torres-Jardón, Ricardo; Nuse, Bryan; Herritt, Lou; Villarreal-Calderón, Rafael; Osnaya, Norma; Stone, Ida; García, Raquel; Brooks, Diane M; González-Maciel, Angelica; Reynoso-Robles, Rafael; Delgado-Chávez, Ricardo; Reed, William

    2008-02-01

    Air pollution is a serious environmental problem. We investigated whether residency in cities with high air pollution is associated with neuroinflammation/neurodegeneration in healthy children and young adults who died suddenly. We measured mRNA cyclooxygenase-2, interleukin-1beta, and CD14 in target brain regions from low (n = 12) or highly exposed residents (n = 35) aged 25.1 +/- 1.5 years. Upregulation of cyclooxygenase-2, interleukin-1beta, and CD14 in olfactory bulb, frontal cortex, substantia nigrae and vagus nerves; disruption of the blood-brain barrier; endothelial activation, oxidative stress, and inflammatory cell trafficking were seen in highly exposed subjects. Amyloid beta42 (Abeta42) immunoreactivity was observed in 58.8% of apolipoprotein E (APOE) 3/3 < 25 y, and 100% of the APOE 4 subjects, whereas alpha-synuclein was seen in 23.5% of < 25 y subjects. Particulate material (PM) was seen in olfactory bulb neurons, and PM < 100 nm were observed in intraluminal erythrocytes from lung, frontal, and trigeminal ganglia capillaries. Exposure to air pollution causes neuroinflammation, an altered brain innate immune response, and accumulation of Abeta42 and alpha-synuclein starting in childhood. Exposure to air pollution should be considered a risk factor for Alzheimer's and Parkinson's diseases, and carriers of the APOE 4 allele could have a higher risk of developing Alzheimer's disease if they reside in a polluted environment.

  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. Ultrastructural alterations in ciliary cells exposed to ionizing radiation. A scanning and transmission electron microscopic study

    Energy Technology Data Exchange (ETDEWEB)

    Baldetorp, L; Mecklenburg, C v; Haakansson, C H [Lund Univ. (Sweden). Hospital; Lund Univ. (Sweden). Dept. of Zoology)

    1977-01-01

    Early effects of ionizing radiation were investigated in an experimental in vitro system using the ciliary cells of the tracheal mucous membrane of the rabbit, irradiated at 30/sup 0/C and at more than 90% humidity. The changes in physiological activities of the ciliary cells caused by irradation were continously registered during the irradation. The specimens were examined immediately after irradiation electron microscopically. The morphological changes in irradiated material after 10-70 Gy are compared with normal material. After 40-70 Gy, scanning electron microscopy revealed the formation of vesicles on cilia, and club-like protrusions and adhesion of their tips. After 30-70 Gy, a swelling of mitochondrial membranes and cristae was apparent transmission electron microscopically. The membrane alterations caused by irradiation are assumed to disturb the permeability and flow of ATP from the mitochondria, which in turn leads to the recorded changes in the activity of the ciliated cells.

  12. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    Science.gov (United States)

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

    Science.gov (United States)

    Fu, J; Tay, S S W; Ling, E A; Dheen, S T

    2006-05-01

    Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the

  14. A 3D-printed microbial cell culture platform with in situ PEGDA hydrogel barriers for differential substrate delivery.

    Science.gov (United States)

    Kadilak, Andrea L; Rehaag, Jessica C; Harrington, Cameron A; Shor, Leslie M

    2017-09-01

    Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200  μ m. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.

  15. Herbicide effects on freshwater benthic diatoms: Induction of nucleus alterations and silica cell wall abnormalities

    Energy Technology Data Exchange (ETDEWEB)

    Debenest, T. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Silvestre, J. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France); Coste, M.; Delmas, F. [Cemagref, 50 avenue de Verdun, 33612 Cestas Cedex (France); Pinelli, E. [Ecolab UMR 5245 (INP ENSAT, CNRS, UPS), Equipe ECOGEN, Avenue de l' Agrobiopole - BP 32607 Auzeville Tolosane, 31326 Castanet Tolosan Cedex (France)], E-mail: pinelli@ensat.fr

    2008-06-02

    Benthic diatoms are well known bio-indicators of river pollution by nutrients (nitrogen and phosphorus). Biological indexes, based on diatom sensitivity for non-toxic pollution, have been developed to assess the water quality. Nevertheless, they are not reliable tools to detect pollution by pesticides. Many authors have suggested that toxic agents, like pesticides, induce abnormalities of the diatom cell wall (frustule). High abnormal frustule abundances have been reported in natural diatom communities sampled in streams contaminated by pesticides. However, no direct link was found between the abundances of abnormal frustules in these communities and the pesticide concentrations in stream water. In the present study, a freshwater benthic diatom community, isolated from natural biofilm and cultured under controlled conditions, was treated with a known genotoxic herbicide, maleic hydrazide (MH). Cells were exposed to three concentrations of MH (5 x 10{sup -6}, 10{sup -6}, 10{sup -7} M) for 6 h followed by a 24 h-recovery time. After MH treatments, nucleus alterations were observed: abnormal nucleus location, micronucleus, multinuclear cell or disruption of the nuclear membrane. A dose-dependent increase of nuclear alterations was observed. The difference between the control (9.65 nuclear alterations per 1000 cells observed (9.65 per mille), S.D. = 4.23) and the highest concentrations (29.40 per mille, S.D. = 8.49 for 10{sup -6} M and 35.96 per mille , S.D. = 3.71 for 5 x 10{sup -6} M) was statistically significant (Tukey test, P < 0.05). Diatoms also exhibited frustules with deformed morphology and abnormal ornamentation. Significantly increased abundances of abnormal frustules were observed for the highest concentrations (10{sup -6} and 5 x 10{sup -6} M; Tukey test, P < 0.05). These two parameters tended to increase together (Pearson correlation = 0.702, P < 0.05). The results suggest that the induction of abnormal frustules could be associated with the genotoxic

  16. Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells

    Directory of Open Access Journals (Sweden)

    Eugenia Mata-Greenwood

    2017-05-01

    Full Text Available Background : Hypoxia inducible factor 1 alpha (HIF1A is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia.Results : We examined the effect of chronic hypoxia (3 days; 0.5% oxygen on human brain micro endothelial cells (HBMEC viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia.Conclusions : We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.

  17. Potential Strategies to Address the Major Clinical Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review.

    Science.gov (United States)

    Nguyen, Patricia K; Neofytou, Evgenios; Rhee, June-Wha; Wu, Joseph C

    2016-11-01

    Although progress continues to be made in the field of stem cell regenerative medicine for the treatment of cardiovascular disease, significant barriers to clinical implementation still exist. To summarize the current barriers to the clinical implementation of stem cell therapy in patients with cardiovascular disease and to discuss potential strategies to overcome them. Information for this review was obtained through a search of PubMed and the Cochrane database for English-language studies published between January 1, 2000, and July 25, 2016. Ten randomized clinical trials and 8 systematic reviews were included. One of the major clinical barriers facing the routine implementation of stem cell therapy in patients with cardiovascular disease is the limited and inconsistent benefit observed thus far. Reasons for this finding are unclear but may be owing to poor cell retention and survival, as suggested by numerous preclinical studies and a small number of human studies incorporating imaging to determine cell fate. Additional studies in humans using imaging to determine cell fate are needed to understand how these factors contribute to the limited efficacy of stem cell therapy. Treatment strategies to address poor cell retention and survival are under investigation and include the following: coadministration of immunosuppressive and prosurvival agents, delivery of cardioprotective factors packaged in exosomes rather than the cells themselves, and use of tissue-engineering strategies to provide structural support for cells. If larger grafts are achieved using these strategies, it will be imperative to carefully monitor for the potential risks of tumorigenicity, immunogenicity, and arrhythmogenicity. Despite important achievements to date, stem cell therapy is not yet ready for routine clinical implementation. Significant research is still needed to address the clinical barriers outlined herein before the next wave of large clinical trials is under way.

  18. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

    Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... pathways across the barrier in ischemic and postischemic brain endothelium is important for developing new medical therapies capable to exploit the barrier changes occurring during/after ischemia to permeate in the brain and treat this devastating disease. Materials and Methods - Primary cultures...... the wall of brain capillaries. The restrictive nature of the BBB is due to the tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during...

  19. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Science.gov (United States)

    Fernández, Esteban R; Olivera, Gabriela C; Quebrada Palacio, Luz P; González, Mariela N; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L; Ledesma Patiño, Oscar S; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  20. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Esteban R Fernández

    Full Text Available Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  1. Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

    Directory of Open Access Journals (Sweden)

    Konstantin V Salojin

    Full Text Available Mammalian sterile 20-like kinase 1 (Mst1 is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

  2. Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

    Science.gov (United States)

    Pathak, Amit

    2018-04-12

    Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

  3. Proteomic Alterations in Response to Hypoxia Inducible Factor 2α in Normoxic Neuroblastoma Cells.

    Science.gov (United States)

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Persano, Luca; Vitale, Monica; Sassi, Mauro; Bresolin, Silvia; Serafin, Valentina; Zambrano, Nicola; Scaloni, Andrea; Basso, Giuseppe; Iolascon, Achille; Capasso, Mario

    2016-10-07

    Hypoxia inducible factor (HIF)-2α protein expression in solid tumors promotes stem-like phenotype in cancer stem cells and increases tumorigenic potential in nonstem cancer cells. Recently, we have shown that HIF-1/2α gene expression is correlated to neuroblastoma (NB) poor survival and to undifferentiated tumor state; HIF-2α protein was demonstrated to enhance aggressive features of the disease. In this study, we used proteomic experiments on NB cells to investigate HIF-2α downstream-regulated proteins or pathways with the aim of providing novel therapeutic targets or bad prognosis markers. We verified that pathways mostly altered by HIF-2α perturbation are involved in tumor progression. In particular, HIF-2α induces alteration of central metabolism and splicing control pathways. Simultaneously, WNT, RAS/MAPK, and PI3K/AKT activity or expression are affected and may impact the sensitivity and the intensity of HIF-2α-regulated pathways. Furthermore, genes coding the identified HIF-2α-related markers built a signature able to stratify NB patients with unfavorable outcome. Taken together, our findings underline the relevance of dissecting the downstream effects of a poor survival marker in developing targeted therapy and improving patient stratification. Future prospective studies are needed to translate the use of these data into the clinical practice.

  4. Altered Distribution of Peripheral Blood Maturation-Associated B-Cell Subsets in Chronic Alcoholism.

    Science.gov (United States)

    Almeida, Julia; Polvorosa, Maria Angeles; Gonzalez-Quintela, Arturo; Madruga, Ignacio; Marcos, Miguel; Pérez-Nieto, Maria Angeles; Hernandez-Cerceño, Maria Luisa; Orfao, Alberto; Laso, Francisco Javier

    2015-08-01

    Although decreased counts of peripheral blood (PB) B cells-associated with an apparently contradictory polyclonal hypergammaglobulinemia-have been reported in chronic alcoholism, no information exists about the specific subsets of circulating B cells altered and their relationship with antibody production. Here, we analyzed for the first time the distribution of multiple maturation-associated subpopulations of PB B cells in alcoholism and its potential relationship with the onset of liver disease. PB samples from 35 male patients-20 had alcoholic hepatitis (AH) and 15 chronic alcoholism without liver disease (AWLD)-were studied, in parallel to 19 male healthy donors (controls). The distribution of PB B-cell subsets (immature/regulatory, naïve, CD27(-) and CD27(+) memory B lymphocytes, and circulating plasmablasts of distinct immunoglobulin-Ig-isotypes) was analyzed by flow cytometry. Patients with AH showed significantly decreased numbers of total PB B lymphocytes (vs. controls and AWLD), at the expense of immature, memory, and, to a lesser extent, also naïve B cells. AWLD showed reduced numbers of immature and naïve B cells (vs. controls), but higher PB counts of plasmablasts (vs. the other 2 groups). Although PB memory B cells were reduced among the patients, the percentage of surface (s)IgA(+) cells (particularly CD27(-) /sIgA(+) cells) was increased in AH, whereas both sIgG(+) and sIgA(+) memory B cells were significantly overrepresented in AWLD versus healthy donors. Regarding circulating plasmablasts, patients with AH only showed significantly reduced counts of sIgG(+) cells versus controls. In contrast, the proportion of both sIgA(+) and sIgG(+) plasmablasts-from all plasmablasts-was reduced in AH and increased in AWLD (vs. the other 2 groups). AH and AWLD patients display a significantly reduced PB B-cell count, at the expense of decreased numbers of recently produced immature/regulatory B cells and naïve B cells, together with an increase in Ig

  5. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor

    Science.gov (United States)

    The phenylpropanoid biosynthesis pathway that generates lignin subunits represents a significant target to alter the abundance and composition of lignin. The major regulators of phenylpropanoid metabolism are myb transcription factors, which have been shown to modulate secondary cell wall compositi...

  6. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  7. Role of Nrf2 in preventing oxidative stress induced chloride current alteration in human lung cells.

    Science.gov (United States)

    Canella, Rita; Benedusi, Mascia; Martini, Marta; Cervellati, Franco; Cavicchio, Carlotta; Valacchi, Giuseppe

    2018-08-01

    The lung tissue is one of the main targets of oxidative stress due to external sources and respiratory activity. In our previous work, we have demonstrated in that O 3 exposure alters the Cl - current-voltage relationship, with the appearance of a large outward rectifier component mainly sustained by outward rectifier chloride channels (ORCCs) in human lung epithelial cells (A549 line). In the present study, we have performed patch clamp experiments, in order to identify which one of the O 3 byproducts (4hydroxynonenal (HNE) and/or H 2 O 2 ) was responsible for chloride current change. While 4HNE exposition (up to 25 μM for 30' before electrophysiological analysis) did not reproduce O 3 effect, H 2 O 2 produced by glucose oxidase 10 mU for 24 hr before electrophysiological analysis mimicked O 3 response. This result was confirmed treating the cell with catalase (CAT) before O 3 exposure (1,000 U/ml for 2 hr): CAT was able to rescue Cl - current alteration. Since CAT is regulated by Nrf2 transcription factor, we pre-treated the cells with the Nrf2 activators, resveratrol and tBHQ. Immunochemical and immunocytochemical results showed Nrf2 activation with both substances that lead to prevent OS effect on Cl - current. These data bring new insights into the mechanisms involved in OS-induced lung tissue damage, pointing out the role of H 2 O 2 in chloride current alteration and the ability of Nfr2 activation in preventing this effect. © 2017 Wiley Periodicals, Inc.

  8. Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

    Science.gov (United States)

    Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

    2015-04-01

    Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  9. *NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

    Science.gov (United States)

    Blasig, I E; Giese, H; Schroeter, M L; Sporbert, A; Utepbergenov, D I; Buchwalow, I B; Neubert, K; Schönfelder, G; Freyer, D; Schimke, I; Siems, W E; Paul, M; Haseloff, R F; Blasig, R

    2001-09-01

    To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature. Copyright 2001 Academic Press.

  10. Time-sequential observation of spindle and phragmoplast orientation in BY-2 cells with altered cortical actin microfilament patterning.

    Science.gov (United States)

    Kojo, Kei H; Yasuhara, Hiroki; Hasezawa, Seiichiro

    2014-01-01

    Precise division plane determination is essential for plant development. At metaphase, a dense actin microfilament meshwork appears on both sides of the cell center, forming a characteristic cortical actin microfilament twin peak pattern in BY-2 cells. We previously reported a strong correlation between altered cortical actin microfilament patterning and an oblique mitotic spindle orientation, implying that these actin microfilament twin peaks play a role in the regulation of mitotic spindle orientation. In the present study, time-sequential observation was used to reveal the progression from oblique phragmoplast to oblique cell plate orientation in cells with altered cortical actin microfilament patterning. In contrast to cells with normal actin microfilament twin peaks, oblique phragmoplast reorientation was rarely observed in cells with altered cortical actin microfilament patterning. These results support the important roles of cortical actin microfilament patterning in division plane orientation.

  11. Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells

    OpenAIRE

    Kim, Brandon J.; Bee, Olivia B.; McDonagh, Maura A.; Stebbins, Matthew J.; Palecek, Sean P.; Doran, Kelly S.; Shusta, Eric V.

    2017-01-01

    ABSTRACT Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with a...

  12. Barrier effect of AlN film in flexible Cu(In,Ga)Se{sub 2} solar cells on stainless steel foil and solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Li, Boyan; Li, Jianjun [Institute of Photo-electronic Thin Film Devices and Technology, Key Laboratory of Photo-electronic Thin Film Devices and Technology of Tianjin, Nankai University, Tianjin 300071 (China); Wu, Li [The MOE Key Laboratory of Weak-Light Nonlinear Photonics, School of Physics, Nankai University, Tianjin 300071 (China); Liu, Wei; Sun, Yun [Institute of Photo-electronic Thin Film Devices and Technology, Key Laboratory of Photo-electronic Thin Film Devices and Technology of Tianjin, Nankai University, Tianjin 300071 (China); Zhang, Yi, E-mail: yizhang@nankai.edu.cn [Institute of Photo-electronic Thin Film Devices and Technology, Key Laboratory of Photo-electronic Thin Film Devices and Technology of Tianjin, Nankai University, Tianjin 300071 (China)

    2015-04-05

    Highlights: • The adhension between AlN film and Mo are verygood. • AlN film can be effectively used as the barrier of flexible CIGS solar cell on SS substrate. • AlN film is suitable as the insulation barrier of flexible CIGS solar cell on SS substrate. - Abstract: The AlN film deposited by DC magnetron sputtering on stainless steel (SS) foils was used as the barrier in flexible Cu(In,Ga)Se{sub 2} (CIGS) solar cells on stainless steel foil and characterized comprehensively by X-ray diffraction (XRD), scanning electron microscopy (SEM), I–V, and QE measurements study. The study of AlN as insulation barrier in the flexible CIGS solar cell showed that the adhesion strength between the SS foil and the deposited AlN film was very strong even after annealing at high temperature at 530 °C. More importantly, a high resistance of over 10 MΩ was remained with the film with thickness of around 200 nm after annealing. This indicates that the AlN film is suitable as an effective insulation barrier in flexible CIGS solar cells based on SS foil. In addition, the XRD and SEM results showed that the AlN film did not influence the crystal structure of the Mo film which was deposited upon the AlN layer and used as the electrical contact in CIGS solar cells. It was found that the AlN film contributed to an improved crystallinity of the Mo contact layer compared to the bare SS foil. The combined results of secondary ion mass spectrometry, I–V and EQE measurements of the corresponding flexible CIGS solar cells confirmed that 1 μm-thick AlN film could be used as an efficient barrier layer in CIGS solar cells on SS foil.

  13. Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation.

    Science.gov (United States)

    Ost, Kyla S; Esher, Shannon K; Leopold Wager, Chrissy M; Walker, Louise; Wagener, Jeanette; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

    2017-01-31

    Compared to other fungal pathogens, Cryptococcus neoformans is particularly adept at avoiding detection by innate immune cells. To explore fungal cellular features involved in immune avoidance, we characterized cell surface changes of the C. neoformans rim101Δ mutant, a strain that fails to organize and shield immunogenic epitopes from host detection. These cell surface changes are associated with an exaggerated, detrimental inflammatory response in mouse models of infection. We determined that the disorganized strain rim101Δ cell wall increases macrophage detection in a contact-dependent manner. Using biochemical and microscopy methods, we demonstrated that the rim101Δ strain shows a modest increase in the levels of both cell wall chitin and chitosan but that it shows a more dramatic increase in chito-oligomer exposure, as measured by wheat germ agglutinin staining. We also created a series of mutants with various levels of cell wall wheat germ agglutinin staining, and we demonstrated that the staining intensity correlates with the degree of macrophage activation in response to each strain. To explore the host receptors responsible for recognizing the rim101Δ mutant, we determined that both the MyD88 and CARD9 innate immune signaling proteins are involved. Finally, we characterized the immune response to the rim101Δ mutant in vivo, documenting a dramatic and sustained increase in Th1 and Th17 cytokine responses. These results suggest that the Rim101 transcription factor actively regulates the C. neoformans cell wall to prevent the exposure of immune stimulatory molecules within the host. These studies further explored the ways in which immune cells detect C. neoformans and other fungal pathogens by mechanisms that include sensing N-acetylglucosamine-containing structures, such as chitin and chitosan. Infectious microorganisms have developed many ways to avoid recognition by the host immune system. For example, pathogenic fungi alter their cell surfaces to

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

    Directory of Open Access Journals (Sweden)

    Matthew D Martin

    2015-10-01

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

  15. Lipoteichoic acid from Staphylococcus aureus induces lung endothelial cell barrier dysfunction: role of reactive oxygen and nitrogen species.

    Directory of Open Access Journals (Sweden)

    Amy Barton Pai

    Full Text Available Tunneled central venous catheters (TCVCs are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA, a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2. The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS. The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam((3CSK((4 induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS activation (as measured by the p-eNOS(ser1177:p-eNOS(thr495 ratio. The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.

  16. Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

    2017-05-01

    Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

  17. Excreted/secreted Trichuris suis products reduce barrier function and suppress inflammatory cytokine production of intestinal epithelial cells

    DEFF Research Database (Denmark)

    Hiemstra, I. H.; Klaver, E. J.; Vrijland, K.

    2014-01-01

    The administration of helminths is considered a promising strategy for the treatment of autoimmune diseases due to their immunomodulatory properties. Currently, the application of the helminth Trichuris suis as a treatment for Crohn's disease is being studied in large multi-center clinical trials....... The intestinal epithelium forms an efficient barrier between the intestinal lumen containing the microbial flora and helminths, and dendritic cells (DCs) present in the lamina propria that determine the TH response. Here, we investigated how excreted/secreted (E/S) products of T. suis affect the barrier function...... of intestinal epithelial cells (IECs) in order to reach the DCs and modulate the immune response. We show that T. suis E/S products reduce the barrier function and the expression of the tight junction proteins EMP-1 and claudin-4 in IEC CMT93/69 monolayers in a glycan-dependent manner. This resulted...

  18. Altered B cell homeostasis and Toll-like receptor 9-driven response in patients affected by autoimmune polyglandular syndrome Type 1: Altered B cell phenotype and dysregulation of the B cell function in APECED patients.

    Science.gov (United States)

    Perri, Valentina; Gianchecchi, Elena; Scarpa, Riccardo; Valenzise, Mariella; Rosado, Maria Manuela; Giorda, Ezio; Crinò, Antonino; Cappa, Marco; Barollo, Susi; Garelli, Silvia; Betterle, Corrado; Fierabracci, Alessandra

    2017-02-01

    APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation. Total B and switched memory (SM) B cells were reduced while IgM memory were increased in patients. In those having more than 15 years from the first clinical manifestation the defect included also mature and transitional B cells; total memory B cells were increased, while SM were unaffected. In patients with shorter disease duration, total B cells were unaltered while SM and IgM memory behaved as in the total group. A defective B cell proliferation was detected after 4day-stimulation. In conclusion APECED patients show, in addition to a significant alteration of the B cell phenotype, a dysregulation of the B cell function involving peripheral innate immune mechanisms particularly those with longer disease duration. Copyright © 2016 Elsevier GmbH. All rights reserved.

  19. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

    Science.gov (United States)

    Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

    2016-01-06

    Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients.

    Science.gov (United States)

    Sugita, Kazunari; Steer, Catherine A; Martinez-Gonzalez, Itziar; Altunbulakli, Can; Morita, Hideaki; Castro-Giner, Francesc; Kubo, Terufumi; Wawrzyniak, Paulina; Rückert, Beate; Sudo, Katsuko; Nakae, Susumu; Matsumoto, Kenji; O'Mahony, Liam; Akdis, Mübeccel; Takei, Fumio; Akdis, Cezmi A

    2018-01-01

    Bronchial epithelial barrier leakiness and type 2 innate lymphoid cells (ILC2s) have been separately linked to asthma pathogenesis; however, the influence of ILC2s on the bronchial epithelial barrier has not been investigated previously. We investigated the role of ILC2s in the regulation of bronchial epithelial tight junctions (TJs) and barrier function both in bronchial epithelial cells of asthmatic patients and healthy subjects and general innate lymphoid cell- and ILC2-deficient mice. Cocultures of human ILC2s and bronchial epithelial cells were used to determine transepithelial electrical resistance, paracellular flux, and TJ mRNA and protein expressions. The effect of ILC2s on TJs was examined by using a murine model of IL-33-induced airway inflammation in wild-type, recombination-activating gene 2 (Rag2) -/- , Rag2 -/- Il2rg -/- , and Rora sg/sg mice undergoing bone marrow transplantation to analyze the in vivo relevance of barrier disruption by ILC2s. ILC2s significantly impaired the epithelial barrier, as demonstrated by reduced transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran permeability in air-liquid interface cultures of human bronchial epithelial cells. This was in parallel to decreased mRNAs and disrupted protein expression of TJ proteins and was restored by neutralization of IL-13. Intranasal administration of recombinant IL-33 to wild-type and Rag2 -/- mice lacking T and B cells triggered TJ disruption, whereas Rag2 -/- Il2rg -/- and Rora sg/sg mice undergoing bone marrow transplantation that lack ILC2s did not show any barrier leakiness. Direct nasal administration of IL-13 was sufficient to induce deficiency in the TJ barrier in the bronchial epithelium of mice in vivo. These data highlight an essential mechanism in asthma pathogenesis by demonstrating that ILC2s are responsible for bronchial epithelial TJ barrier leakiness through IL-13. Copyright © 2017 American Academy of Allergy, Asthma & Immunology

  1. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    Directory of Open Access Journals (Sweden)

    Thiel Cora S

    2012-01-01

    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  2. Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

    Science.gov (United States)

    Chou, Susan S; Clegg, Michael S; Momma, Tony Y; Niles, Brad J; Duffy, Jodie Y; Daston, George P; Keen, Carl L

    2004-10-01

    Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

  3. Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance.

    Science.gov (United States)

    Kalluri, Udaya C; Yin, Hengfu; Yang, Xiaohan; Davison, Brian H

    2014-12-01

    Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma.

    Science.gov (United States)

    Landen, Jaren W; Lang, Roland; McMahon, Steve J; Rusan, Nasser M; Yvon, Anne-Marie; Adams, Ashley W; Sorcinelli, Mia D; Campbell, Ross; Bonaccorsi, Paola; Ansel, John C; Archer, David R; Wadsworth, Patricia; Armstrong, Cheryl A; Joshi, Harish C

    2002-07-15

    Cellular microtubules, polymers of tubulin, alternate relentlessly between phases of growth and shortening. We now show that noscapine, a tubulin-binding agent, increases the time that cellular microtubules spend idle in a paused state. As a result, most mammalian cell types observed arrest in mitosis in the presence of noscapine. We demonstrate that noscapine-treated murine melanoma B16LS9 cells do not arrest in mitosis but rather become polyploid followed by cell death, whereas primary melanocytes reversibly arrest in mitosis and resume a normal cell cycle after noscapine removal. Furthermore, in a syngeneic murine model of established s.c. melanoma, noscapine treatment resulted in an 85% inhibition of tumor volume on day 17 when delivered by gavage compared with untreated animals (P cells through alterations in microtubule dynamics, with no detected toxicity to the host. Consequently, noscapine could be a valuable chemotherapeutic agent, alone or in combination, for the treatment of advanced melanoma.

  5. X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

    Science.gov (United States)

    Hu, Yueyuan; Lau, Patrick; Baumstark-Khan, Christa; Hellweg, Christine E.; Reitz, Günther

    2012-05-01

    To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.

  6. Alteration of T cell function in healthy persons with a history of thymic x irradiation

    International Nuclear Information System (INIS)

    Rieger, C.H.L.; Kraft, S.C.; Rothberg, R.M.

    1975-01-01

    The possible late effects of x irradiation to the infantile thymus were investigated by studying immune functions in 12 healthy persons with a history of thymic x irradiation and healthy control subjects. No differences were found in serum immunoglobulin values, humoral antibody levels, lymphocyte counts, and lymphocyte reactivity to phytohemagglutinin, vaccinia virus, purified protein derivative (PPD), and allogeneic cells. The irradiation group exhibited cellular hyperresponsiveness to streptokinase-streptodornase (SK-SD). In contrast, mean skin and in vitro lymphocyte responses to Candida albicans were depressed in the patients with thymic irradiation. A dissociation of these two Candida responses was found in only 1 of 14 healthy control subjects but in 7 of 12 irradiated individuals. While thymic irradiation did not result in impaired immunologic defenses leading to clinical disease, it caused alterations in T cell responses similar to those reported in patients with chronic mucocutaneous candidiasis

  7. Alteration of T cell function in healthy persons with a history of thymic x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Rieger, C.H.L.; Kraft, S.C.; Rothberg, R.M.

    1975-10-01

    The possible late effects of x irradiation to the infantile thymus were investigated by studying immune functions in 12 healthy persons with a history of thymic x irradiation and healthy control subjects. No differences were found in serum immunoglobulin values, humoral antibody levels, lymphocyte counts, and lymphocyte reactivity to phytohemagglutinin, vaccinia virus, purified protein derivative (PPD), and allogeneic cells. The irradiation group exhibited cellular hyperresponsiveness to streptokinase-streptodornase (SK-SD). In contrast, mean skin and in vitro lymphocyte responses to Candida albicans were depressed in the patients with thymic irradiation. A dissociation of these two Candida responses was found in only 1 of 14 healthy control subjects but in 7 of 12 irradiated individuals. While thymic irradiation did not result in impaired immunologic defenses leading to clinical disease, it caused alterations in T cell responses similar to those reported in patients with chronic mucocutaneous candidiasis.

  8. Zika Virus Infects, Activates, and Crosses Brain Microvascular Endothelial Cells, without Barrier Disruption

    Science.gov (United States)

    Papa, Michelle P.; Meuren, Lana M.; Coelho, Sharton V. A.; Lucas, Carolina G. de Oliveira; Mustafá, Yasmin M.; Lemos Matassoli, Flavio; Silveira, Paola P.; Frost, Paula S.; Pezzuto, Paula; Ribeiro, Milene R.; Tanuri, Amilcar; Nogueira, Mauricio L.; Campanati, Loraine; Bozza, Marcelo T.; Paula Neto, Heitor A.; Pimentel-Coelho, Pedro M.; Figueiredo, Claudia P.; de Aguiar, Renato S.; de Arruda, Luciana B.

    2017-01-01

    Zika virus (ZIKV) has been associated to central nervous system (CNS) harm, and virus was detected in the brain and cerebrospinal fluids of microcephaly and meningoencephalitis cases. However, the mechanism by which the virus reaches the CNS is unclear. Here, we addressed the effects of ZIKV replication in human brain microvascular endothelial cells (HBMECs), as an in vitro model of blood brain barrier (BBB), and evaluated virus extravasation and BBB integrity in an in vivo mouse experimental model. HBMECs were productively infected by African and Brazilian ZIKV strains (ZIKVMR766 and ZIKVPE243), which induce increased production of type I and type III IFN, inflammatory cytokines and chemokines. Infection with ZIKVMR766 promoted earlier cellular death, in comparison to ZIKVPE243, but infection with either strain did not result in enhanced endothelial permeability. Despite the maintenance of endothelial integrity, infectious virus particles crossed the monolayer by endocytosis/exocytosis-dependent replication pathway or by transcytosis. Remarkably, both viruses' strains infected IFNAR deficient mice, with high viral load being detected in the brains, without BBB disruption, which was only detected at later time points after infection. These data suggest that ZIKV infects and activates endothelial cells, and might reach the CNS through basolateral release, transcytosis or transinfection processes. These findings further improve the current knowledge regarding ZIKV dissemination pathways. PMID:29312238

  9. Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

    Directory of Open Access Journals (Sweden)

    Marcel A Schijf

    Full Text Available Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+CD25(+Foxp3(+ regulatory T-cells (Tregs in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+ (Th1 activated CD69(+CD4(+ T cells (p<0.001 and reduced percentage of Gata-3(+ (Th2 activated CD69(+CD4(+T cells (p<0.001 was detected in the mesenteric lymph nodes (MLN of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+Foxp3(+ could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+ /T-bet(+ (Th1-Tregs was significantly reduced (p<0.05 in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.

  10. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

    International Nuclear Information System (INIS)

    Rousseau, Matthieu; Gaugler, Marie-Hélène; Rodallec, Audrey; Bonnaud, Stéphanie; Paris, François; Corre, Isabelle

    2011-01-01

    Highlights: ► We explore the role of RhoA in endothelial cell response to ionizing radiation. ► RhoA is rapidly activated by single high-dose of radiation. ► Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. ► Radiation-induced apoptosis does not require the RhoA/ROCK pathway. ► Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.

  11. Danazol alters mitochondria metabolism of fibrocystic breast Mcf10A cells.

    Science.gov (United States)

    Irgebay, Zhazira; Yeszhan, Banu; Sen, Bhaswati; Tuleukhanov, Sultan; Brooks, Ari D; Sensenig, Richard; Orynbayeva, Zulfiya

    2017-10-01

    Fibrocystic Breast Disease (FBD) or Fibrocystic change (FC) affects about 60% of women at some time during their life. Although usually benign, it is often associated with pain and tenderness (mastalgia). The synthetic steroid danazol has been shown to be effective in reducing the pain associated with FBD, but the cellular and molecular mechanisms for its action have not been elucidated. We investigated the hypothesis that danazol acts by affecting energy metabolism. Effects of danazol on Mcf10A cells homeostasis, including mechanisms of oxidative phosphorylation, cytosolic calcium signaling and oxidative stress, were assessed by high-resolution respirometry and flow cytometry. In addition to fast physiological responses the associated genomic modulations were evaluated by Affimetrix microarray analysis. The alterations of mitochondria membrane potential and respiratory activity, downregulation of energy metabolism transcripts result in suppression of energy homeostasis and arrest of Mcf10A cells growth. The data obtained in this study impacts the recognition of direct control of mitochondria by cellular mechanisms associated with altered energy metabolism genes governing the breast tissue susceptibility and response to medication by danazol. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

    Science.gov (United States)

    Nagy, Zsolt; Acs, Bence; Butz, Henriett; Feldman, Karolina; Marta, Alexa; Szabo, Peter M; Baghy, Kornelia; Pazmany, Tamas; Racz, Karoly; Liko, Istvan; Patocs, Attila

    2016-01-01

    The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Early Effects of Altered Gravity Environments on Plant Cell Growth and Cell Proliferation: Characterization of Morphofunctional Nucleolar Types in an Arabidopsis Cell Culture System

    Energy Technology Data Exchange (ETDEWEB)

    Manzano, Ana I.; Herranz, Raúl; Manzano, Aránzazu [Centro de Investigaciones Biológicas (CSIC), Madrid (Spain); Loon, Jack J. W. A. van [Department of Oral and Maxillofacial Surgery/Oral Pathology, Dutch Experiment Support Center, VU University Medical Center, Amsterdam (Netherlands); Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam (Netherlands); ESA-ESTEC, TEC-MMG, Noordwijk (Netherlands); Medina, F. Javier, E-mail: fjmedina@cib.csic.es [Centro de Investigaciones Biológicas (CSIC), Madrid (Spain)

    2016-02-05

    Changes in the cell growth rate of an in vitro cellular system in Arabidopsis thaliana induced by short exposure to an altered gravity environment have been estimated by a novel approach. The method consisted of defining three structural nucleolar types which are easy and reliable indicators of the ribosome biogenesis activity and, consequently, of protein biosynthesis, a parameter strictly correlated to cell growth in this cellular system. The relative abundance of each nucleolar type was statistically assessed in different conditions of gravity. Samples exposed to simulated microgravity for 200 min showed a significant decrease in nucleolar activity compared to 1g controls, whereas samples exposed to hypergravity (2g) for the same period showed nucleolar activity slightly increased. These effects could be considered as an early cellular response to the environmental alteration, given the short duration of the treatment. The functional significance of the structural data was validated by a combination of several different well-known parameters, using microscopical, flow cytometry, qPCR, and proteomic approaches, which showed that the decreased cell growth rate was decoupled from an increased cell proliferation rate under simulated microgravity, and the opposite trend was observed under hypergravity. Actually, not all parameters tested showed the same quantitative changes, indicating that the response to the environmental alteration is time-dependent. These results are in agreement with previous observations in root meristematic cells and they show the ability of plant cells to produce a response to gravity changes, independently of their integration into plant organs.

  14. Efficient, air-stable colloidal quantum dot solar cells encapsulated using atomic layer deposition of a nanolaminate barrier

    KAUST Repository

    Ip, Alexander H.; Labelle, André J.; Sargent, Edward H.

    2013-01-01

    Atomic layer deposition was used to encapsulate colloidal quantum dot solar cells. A nanolaminate layer consisting of alternating alumina and zirconia films provided a robust gas permeation barrier which prevented device performance degradation over a period of multiple weeks. Unencapsulated cells stored in ambient and nitrogen environments demonstrated significant performance losses over the same period. The encapsulated cell also exhibited stable performance under constant simulated solar illumination without filtration of harsh ultraviolet photons. This monolithically integrated thin film encapsulation method is promising for roll-to-roll processed high efficiency nanocrystal solar cells. © 2013 AIP Publishing LLC.

  15. Efficient, air-stable colloidal quantum dot solar cells encapsulated using atomic layer deposition of a nanolaminate barrier

    KAUST Repository

    Ip, Alexander H.

    2013-12-23

    Atomic layer deposition was used to encapsulate colloidal quantum dot solar cells. A nanolaminate layer consisting of alternating alumina and zirconia films provided a robust gas permeation barrier which prevented device performance degradation over a period of multiple weeks. Unencapsulated cells stored in ambient and nitrogen environments demonstrated significant performance losses over the same period. The encapsulated cell also exhibited stable performance under constant simulated solar illumination without filtration of harsh ultraviolet photons. This monolithically integrated thin film encapsulation method is promising for roll-to-roll processed high efficiency nanocrystal solar cells. © 2013 AIP Publishing LLC.

  16. Murine cytomegalovirus infection of neural stem cells alters neurogenesis in the developing brain.

    Directory of Open Access Journals (Sweden)

    Manohar B Mutnal

    2011-01-01

    Full Text Available Congenital cytomegalovirus (CMV brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV and the pattern of injury to the developing brain.We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons.

  17. Tributyltin alters secretion of interleukin 1 beta from human immune cells.

    Science.gov (United States)

    Brown, Shyretha; Whalen, Margaret

    2015-08-01

    Tributyltin (TBT) has been used as a biocide in industrial applications such as wood preservation, antifouling paint and antifungal agents. Owing to its many uses, it contaminates the environment and has been found in human blood samples. Interleukin-1 beta (IL-1β) is a pro-inflammatory cytokine that promotes cell growth, tissue repair and immune response regulation. Produced predominately by both monocytes and macrophages, IL-1β appears to increase the invasiveness of certain tumors. This study shows that TBT modifies the secretion of IL-1β from increasingly reconstituted preparations of human immune cells. IL-1β secretion was examined after 24-, 48-h or 6-day exposures to TBT in highly enriched human natural killer (NK) cells, monocyte-depleted peripheral blood mononuclear cells (MD-PBMCs), PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes (PBMCs+granulocytes). TBT altered IL-1β secretion from all of the cell preparations. The 200 nM concentration of TBT normally blocked the secretion of IL-1β, whereas lower concentrations (usually 5-50 nM) elevated secretion of IL-1β. Examination of the signaling pathway(s) responsible for the elevated secretion of IL-1β was carried out in MD-PBMCs. Pathways examined were IL-1β processing (Caspase-1), mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB). Results indicated that MAPK pathways (p44/42 and p38) appear to be the targets of TBT that lead to increased IL-1β secretion from immune cells. These results from human immune cells show IL-1β dysregulation by TBT is occurring ex vivo. Thus, the potential for in vivo effects on pro-inflammatory cytokine levels may possibly be a consequence of TBT exposures. Copyright © 2014 John Wiley & Sons, Ltd.

  18. Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Sagarika; Mukherji, Suparna [Indian Institute of Technology Bombay, Mumbai (India). Centre for Environmental Science and Engineering (CESE)

    2012-04-15

    Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50-52 ) was in the same range for both strains while zeta potential at neutral pH was -38 and -31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75 and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

  19. Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

    Science.gov (United States)

    van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

    2014-09-01

    The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Importins α and β signaling mediates endothelial cell inflammation and barrier disruption.

    Science.gov (United States)

    Leonard, Antony; Rahman, Arshad; Fazal, Fabeha

    2018-04-01

    Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and β importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser 536 , showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin β1, but not β2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or β1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or β1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and β1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Diffusion chamber system for testing of collagen-based cell migration barriers for separation of ligament enthesis zones in tissue-engineered ACL constructs.

    Science.gov (United States)

    Hahner, J; Hoyer, M; Hillig, S; Schulze-Tanzil, G; Meyer, M; Schröpfer, M; Lohan, A; Garbe, L-A; Heinrich, G; Breier, A

    2015-01-01

    A temporary barrier separating scaffold zones seeded with different cell types prevents faster growing cells from overgrowing co-cultured cells within the same construct. This barrier should allow sufficient nutrient diffusion through the scaffold. The aim of this study was to test the effect of two variants of collagen-based barriers on macromolecule diffusion, viability, and the spreading efficiency of primary ligament cells on embroidered scaffolds. Two collagen barriers, a thread consisting of a twisted film tape and a sponge, were integrated into embroidered poly(lactic-co-caprolactone) and polypropylene scaffolds, which had the dimension of lapine anterior cruciate ligaments (ACL). A diffusion chamber system was designed and established to monitor nutrient diffusion using fluorescein isothiocyanate-labeled dextran of different molecular weights (20, 40, 150, 500 kDa). Vitality of primary lapine ACL cells was tested at days 7 and 14 after seeding using fluorescein diacetate and ethidium bromide staining. Cell spreading on the scaffold surface was measured using histomorphometry. Nuclei staining of the cross-sectioned scaffolds revealed the penetration of ligament cells through both barrier types. The diffusion chamber was suitable to characterize the diffusivity of dextran molecules through embroidered scaffolds with or without integrated collagen barriers. The diffusion coefficients were generally significantly lower in scaffolds with barriers compared to those without barriers. No significant differences between diffusion coefficients of both barrier types were detected. Both barriers were cyto-compatible and prevented most of the ACL cells from crossing the barrier, whereby the collagen thread was easier to handle and allowed a higher rate of cell spreading.

  2. Poly (A+ transcriptome assessment of ERBB2-induced alterations in breast cell lines.

    Directory of Open Access Journals (Sweden)

    Dirce Maria Carraro

    Full Text Available We report the first quantitative and qualitative analysis of the poly (A⁺ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

  3. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  4. Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

    Science.gov (United States)

    Liu, Y; Cheng, H; Zhou, Y; Zhu, Y; Bian, R; Chen, Y; Li, C; Ma, Q; Zheng, Q; Zhang, Y; Jin, H; Wang, X; Chen, Q; Zhu, D

    2013-01-01

    Myostatin, a member of the transforming growth factor-β superfamily, regulates the glucose metabolism of muscle cells, while dysregulated myostatin activity is associated with a number of metabolic disorders, including muscle cachexia, obesity and type II diabetes. We observed that myostatin induced significant mitochondrial metabolic alterations and prolonged exposure of myostatin induced mitochondria-dependent apoptosis in cancer cells addicted to glycolysis. To address the underlying mechanism, we found that the protein levels of Hexokinase II (HKII) and voltage-dependent anion channel 1 (VDAC1), two key regulators of glucose metabolisms as well as metabolic stress-induced apoptosis, were negatively correlated. In particular, VDAC1 was dramatically upregulated in cells that are sensitive to myostatin treatment whereas HKII was downregulated and dissociated from mitochondria. Myostatin promoted the translocation of Bax from cytosol to mitochondria, and knockdown of VDAC1 inhibited myostatin-induced Bax translocation and apoptosis. These apoptotic changes can be partially rescued by repletion of ATP, or by ectopic expression of HKII, suggesting that perturbation of mitochondrial metabolism is causally linked with subsequent apoptosis. Our findings reveal novel function of myostatin in regulating mitochondrial metabolism and apoptosis in cancer cells. PMID:23412387

  5. Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species.

    Science.gov (United States)

    Morikawa, Yoshifumi; Shibata, Akinobu; Okumura, Naoko; Ikari, Akira; Sasajima, Yasuhide; Suenami, Koichi; Sato, Kiyohito; Takekoshi, Yuji; El-Kabbani, Ossama; Matsunaga, Toshiyuki

    2017-01-01

    Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10μM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.

    Directory of Open Access Journals (Sweden)

    Nagendra N Mishra

    Full Text Available The lipopeptide antibiotic, daptomycin (DAP interacts with the bacterial cell membrane (CM. Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712 and E. faecium (S447 vs. R446 recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG, cardiolipin, lysyl-phosphatidylglycerol (L-PG and glycerolphospho-diglycodiacylglycerol (GP-DGDAG. In addition, E. faecalis CMs (but not E. faecium also contained: i phosphatidic acid; and ii two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447. Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM

  7. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    International Nuclear Information System (INIS)

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-01-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes

  8. Modified mesenchymal stem cells using miRNA transduction alter lung injury in a bleomycin model.

    Science.gov (United States)

    Huleihel, Luai; Sellares, Jacobo; Cardenes, Nayra; Álvarez, Diana; Faner, Rosa; Sakamoto, Koji; Yu, Guoying; Kapetanaki, Maria G; Kaminski, Naftali; Rojas, Mauricio

    2017-07-01

    Although different preclinical models have demonstrated a favorable role for bone marrow-derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (antifibrotic) or miR-154 (profibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors, we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation, and the mice were euthanized at day 14 Bleomycin-injured animals that were treated with let-7d cells were found to recover quicker from the initial weight loss compared with the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45-positive cells in the lung suggests that the differential effects observed in both miRNA mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis. Copyright © 2017 the American Physiological Society.

  9. Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.

    Science.gov (United States)

    Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin

    2014-05-01

    Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.

  10. Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation.

    Science.gov (United States)

    Hegazy, Ahmed N; West, Nathaniel R; Stubbington, Michael J T; Wendt, Emily; Suijker, Kim I M; Datsi, Angeliki; This, Sebastien; Danne, Camille; Campion, Suzanne; Duncan, Sylvia H; Owens, Benjamin M J; Uhlig, Holm H; McMichael, Andrew; Bergthaler, Andreas; Teichmann, Sarah A; Keshav, Satish; Powrie, Fiona

    2017-11-01

    Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4 + T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4 + T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4 + T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction. Circulating and gut-resident CD4 + T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4 + T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These

  11. Altered Natural Killer Cell Subsets in Seropositive Arthralgia and Early Rheumatoid Arthritis Are Associated with Autoantibody Status

    NARCIS (Netherlands)

    Chalan, Paulina; Bijzet, Johan; Kroesen, Bart-Jan; Boots, Annemieke M. H.; Brouwer, Elisabeth

    Objective. The role of natural killer (NK) cells in the immunopathogenesis of rheumatoid arthritis (RA) is unclear. Therefore, numerical and functional alterations of CD56(dim) and CD56(bright) NK cells in the early stages of RA development were studied. Methods. Whole blood samples from newly

  12. Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

    Directory of Open Access Journals (Sweden)

    Victoria L. Campodónico

    2018-03-01

    Full Text Available Background:Mycobacterium tuberculosis (Mtb rpoB mutations are associated with global metabolic remodeling. However, the net effects of rpoB mutations on Mtb physiology, metabolism and function are not completely understood. Based on previous work, we hypothesized that changes in the expression of cell wall molecules in Mtb mutant RpoB 526D lead to changes in cell wall permeability and to altered resistance to environmental stresses and drugs.Methods: The phenotypes of a fully drug-susceptible clinical strain of Mtb and its paired rifampin-monoresistant, RpoB H526D mutant progeny strain were compared.Results: The rpoB mutant showed altered colony morphology, bacillary length and cell wall thickness, which were associated with increased cell wall permeability and susceptibility to the cell wall detergent sodium dodecyl sulfate (SDS after exposure to nutrient starvation. Relative to the isogenic rifampin-susceptible strain, the RpoB H526D mutant showed altered bacterial cellular metabolic activity and an eightfold increase in susceptibility to the cell-wall acting drug vancomycin.Conclusion: Our data suggest that RpoB mutation H526D is associated with altered cell wall physiology and resistance to cell wall-related stress. These findings are expected to contribute to an improved understanding of the pathogenesis of drug-resistant M. tuberculosis infections.

  13. Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro-dielectric barrier discharge plasma.

    Science.gov (United States)

    Choi, Hyeongwon; Choi, Eun Ha; Kim, Kyung Sook

    2017-10-01

    Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro-dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro-DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro-DBD plasma for various exposure times. The elasticity of a single cell was determined by force-distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma-treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro-DBD plasma were related to cell mechanical changes. © 2017 Wiley Periodicals, Inc.

  14. Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment?

    Science.gov (United States)

    Sayed, Blayne A; Christy, Alison L; Walker, Margaret E; Brown, Melissa A

    2010-06-15

    Mast cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis, a rodent model of the human demyelinating disease multiple sclerosis. Yet their site and mode of action is unknown. In both diseases, myelin-specific T cells are initially activated in peripheral lymphoid organs. However, for disease to occur, these cells must enter the immunologically privileged CNS through a breach in the relatively impermeable blood-brain barrier. In this study, we demonstrate that a dense population of resident mast cells in the meninges, structures surrounding the brain and spinal cord, regulate basal CNS barrier function, facilitating initial T cell CNS entry. Through the expression of TNF, mast cells recruit an early wave of neutrophils to the CNS. We propose that neutrophils in turn promote the blood-brain barrier breach and together with T cells lead to further inflammatory cell influx and myelin damage. These findings provide specific targets for intervention in multiple sclerosis as well as other immune-mediated CNS diseases.

  15. Na effect on flexible Cu(In,Ga)Se{sub 2} photovoltaic cell depending on diffusion barriers (SiOx, i-ZnO) on stainless steel

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Woo-Jung; Cho, Dae-Hyung; Wi, Jae-Hyung; Han, Won Seok [Electronics and Telecommunications Research Institute, Daejeon 305-700 (Korea, Republic of); Kim, Jeha [Insitute of Photovoltaics, Cheongju University, Cheongju 360-764 (Korea, Republic of); Chung, Yong-Duck, E-mail: ydchung@etri.re.kr [Electronics and Telecommunications Research Institute, Daejeon 305-700 (Korea, Republic of); Department of Advanced Device Engineering, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of)

    2014-10-15

    Cu(In,Ga)Se{sub 2} (CIGS) based-photovoltaic (PV) cells with different diffusion barriers of SiOx and i-ZnO were fabricated on stainless steel (STS) substrate and their electrical characteristics were investigated by measuring J–V curves under illuminated and dark conditions. The physical properties of the CIGS film depending on type of diffusion barrier were also analyzed using X-ray diffraction and secondary ion mass spectroscopy. The efficiency of the CIGS-PV cell with i-ZnO barrier was approximately 2% higher than that with the SiOx barrier. Through the analysis of dark J–V curves, we discovered that distinctive defects were formed in the band gap of CIGS based on which diffusion barrier contacted the STS. The diffraction pattern showed a slightly different tendency of the peak intensity ratio of (220/204)/(112) in the PV cell with the i-ZnO barrier, which was slightly higher than that in the PV cell with SiOx barrier. In elemental depth profile, a deficient Ga profile was observed near the surface of the CIGS film with the SiOx barrier, and an abundant Na profile within the CIGS film with the i-ZnO barrier was detected. This is attributed to a difference in thermal conduction through the diffusion barriers during CIGS film growth, originating from the larger thermal conductivity of ZnO compared with SiOx. - Highlights: • We fabricated CIGS-PV cells with diffusion barriers of SiOx and i-ZnO on STS. • The efficiency of CIGS-PV cell with i-ZnO was ∼2% higher than that with SiOx. • Distinctive defects were formed into CIGS absorber depending on diffusion barrier.

  16. 3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells.

    Science.gov (United States)

    Chiasserini, Davide; Davidescu, Magdalena; Orvietani, Pier Luigi; Susta, Federica; Macchioni, Lara; Petricciuolo, Maya; Castigli, Emilia; Roberti, Rita; Binaglia, Luciano; Corazzi, Lanfranco

    2017-01-30

    Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We

  17. Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

    Science.gov (United States)

    Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

    2017-01-01

    Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

  18. Mitochondrial Morphofunctional Alterations in Smooth Muscle Cells of Aorta in Rats

    Science.gov (United States)

    Tarán, Mariana; Llorens, Candelaria; Balceda, Ariel; Scribano, María de La Paz; Pons, Patricia; Moya, Mónica

    2014-01-01

    In an experimental model of atherogenesis induced by hyperfibrinogenemia (HF), the pharmacological response of vitamin E was studied in order to assess its antioxidant effect on the mitochondrial morphofunctional alterations in aortic smooth muscle cells. Three groups of male rats were used: (Ctr) control, (AI) atherogenesis induced for 120 days, and (AIE) atherogenesis induced for 120 days and treated with vitamin E. HF was induced by adrenalin injection (0.1 mg/day/rat) for 120 days. AIE group was treated with the administration of 3.42 mg/day/rat of vitamin E for 105 days after the first induction. Mitochondria morphology was analyzed by electronic microscopy (EM) and mitochondrial complexes (MC) by spectrophotometry. In group AI the total and mean number of mitochondria reduced significantly, the intermembranous matrix increased, and swelling was observed with respect to Ctr and AIE (P < 0.01). These damages were related to a significant decrease in the activity of citrate synthase and complexes I, II, III, and IV in group AI in comparison to Ctr (P < 0.001). Similar behavior was presented by group AI compared to AIE (P < 0.001). These results show that vitamin E produces a significative regression of inflammatory and oxidative stress process and it resolved the morphofunctional mitochondrial alterations in this experimental model of atherogenic disease. PMID:24653842

  19. Selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice

    International Nuclear Information System (INIS)

    Nueesch, Juerg P.F.; Lachmann, Sylvie; Rommelaere, Jean

    2005-01-01

    During a productive infection, the prototype strain of parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations to the fibroblast host cell A9, resulting in cell lysis and progeny virus release. In order to understand the mechanisms underlying these changes, we characterized the fate of various cytoskeletal filaments and investigated the nuclear/cytoplasmic compartmentalization of infected cells. While most pronounced effects could be seen on micro- and intermediate filaments, manifest in dramatic rearrangements and degradation of filamentous (F-)actin and vimentin structures, only little impact could be seen on microtubules or the nuclear envelope during the entire monitored time of infection. To further analyze the disruption of the cytoskeletal structures, we investigated the viral impact on selective regulatory pathways. Thereby, we found a correlation between microtubule stability and MVM-induced phosphorylation of α/β tubulin. In contrast, disassembly of actin filaments late in infection could be traced back to the disregulation of two F-actin associated proteins gelsolin and Wiscott-Aldrich Syndrome Protein (WASP). Thereby, an increase in the amount of gelsolin, an F-actin severing protein was observed during infection, accounting for the disruption of stress fibers upon infection. Concomitantly, the actin polymerization activity also diminished due to a loss of WASP, the activator protein of the actin polymerization machinery the Arp2/3 complex. No effects could be seen in amount and distribution of other F-actin regulatory factors such as cortactin, cofilin, and profilin. In summary, the selective attack of MVM towards distinct host cell cytoskeletal structures argues for a regulatory feature during infection, rather than a collapse of the host cell as a mere side effect of virus production

  20. Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species

    Energy Technology Data Exchange (ETDEWEB)

    Morikawa, Yoshifumi [Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu 500-8501 (Japan); Shibata, Akinobu; Okumura, Naoko; Ikari, Akira [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Sasajima, Yasuhide; Suenami, Koichi; Sato, Kiyohito; Takekoshi, Yuji [Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu 500-8501 (Japan); El-Kabbani, Ossama [Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan)

    2017-01-01

    Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with > 10 μM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites. - Highlights: • Treatment with sibutramine, an anorexiant, induces endothelial cell apoptosis. • The apoptotic mechanism includes induction of ROS and NO depletion. • There is an inverse relationship between sibutramine cytotoxicity and its metabolism.

  1. A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology

    Directory of Open Access Journals (Sweden)

    Brian R. Mullen

    2016-06-01

    Full Text Available Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors—reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon—gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology.

  2. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Kaushali Thakore-Shah

    Full Text Available REST (RE1 silencing transcription factor, also known as NRSF (neuron-restrictive silencer factor, is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells.

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

    Science.gov (United States)

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

    2011-01-01

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

  4. The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility.

    Science.gov (United States)

    Escalante, Nichole K; Lemire, Paul; Cruz Tleugabulova, Mayra; Prescott, David; Mortha, Arthur; Streutker, Catherine J; Girardin, Stephen E; Philpott, Dana J; Mallevaey, Thierry

    2016-12-12

    The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1 -/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research. © 2016 Escalante et al.

  5. Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction

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    NM Coelho

    2010-06-01

    Full Text Available Considering the structural role of type IV collagen (Col IV in the assembly of the basement membrane (BM and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass and hydrophobic trichloro(octadecylsilane (ODS surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50μg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine – nearly single molecular size – network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC attach less efficiently to the aggregated Col IV (on ODS, as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both α1 and α2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  6. Olfactory aversive conditioning alters olfactory bulb mitral/tufted cell glomerular odor responses

    Directory of Open Access Journals (Sweden)

    Max L Fletcher

    2012-03-01

    Full Text Available The anatomical organization of receptor neuron input into the olfactory bulb (OB allows odor information to be transformed into an odorant-specific spatial map of mitral/tufted cell glomerular activity at the upper level of the olfactory bulb. In other sensory systems, neuronal representations of stimuli can be reorganized or enhanced following learning. While the mammalian OB has been shown to undergo experience-dependent plasticity at the glomerular level, it is still unclear if similar representational change occurs within mitral/tufted cell glomerular odor representations following learning. To address this, odorant-evoked glomerular activity patterns were imaged in mice expressing a GFP-based calcium indicator (GCaMP2 in OB mitral/tufted cells. Glomerular odor responses were imaged before and after olfactory associative conditioning to aversive foot shock. Following conditioning, we found no overall reorganization of the glomerular representation. Training, however, did significantly alter the amplitudes of individual glomeruli within the representation in mice in which the odor was presented together with foot shock. Further, the specific pairing of foot shock with odor presentations lead to increased responses primarily in initially weakly activated glomeruli. Overall, these results suggest that associative conditioning can enhance the initial representation of odors within the olfactory bulb by enhancing responses to the learned odor in some glomeruli.

  7. Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate

    Directory of Open Access Journals (Sweden)

    Priya Srikanth

    2015-09-01

    Full Text Available Genetic and clinical association studies have identified disrupted in schizophrenia 1 (DISC1 as a candidate risk gene for major mental illness. DISC1 is interrupted by a balanced chr(1;11 translocation in a Scottish family in which the translocation predisposes to psychiatric disorders. We investigate the consequences of DISC1 interruption in human neural cells using TALENs or CRISPR-Cas9 to target the DISC1 locus. We show that disruption of DISC1 near the site of the translocation results in decreased DISC1 protein levels because of nonsense-mediated decay of long splice variants. This results in an increased level of canonical Wnt signaling in neural progenitor cells and altered expression of fate markers such as Foxg1 and Tbr2. These gene expression changes are rescued by antagonizing Wnt signaling in a critical developmental window, supporting the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types generated during cortical development.

  8. Impaired function of the blood-testis barrier during aging is preceded by a decline in cell adhesion proteins and GTPases.

    Directory of Open Access Journals (Sweden)

    Catriona Paul

    Full Text Available With increasing age comes many changes in the testis, including germ cell loss. Cell junctions in the testis tether both seminiferous epithelial and germ cells together and assist in the formation of the blood-testis barrier (BTB, which limits transport of biomolecules, ions and electrolytes from the basal to the adluminal compartment and protects post-meiotic germ cells. We hypothesize that as male rats age the proteins involved in forming the junctions decrease and that this alters the ability of the BTB to protect the germ cells. Pachytene spermatocytes were isolated from Brown Norway rat testes at 4 (young and 18 (aged months of age using STA-PUT velocity sedimentation technique. RNA was extracted and gene expression was assessed using Affymetrix rat 230 2.0 whole rat genome microarrays. Microarray data were confirmed by q-RT-PCR and protein expression by Western blotting. Of the genes that were significantly decreased by at least 1.5 fold, 70 were involved in cell adhesion; of these, at least 20 are known to be specifically involved in junction dynamics within the seminiferous epithelium. The mRNA and protein levels of Jam2, Ocln, cdh2 (N-cadherin, ctnna (α-catenin, and cldn11 (involved in adherens junctions, among others, were decreased by approximately 50% in aged spermatocytes. In addition, the GTPases Rac1 and cdc42, involved in the recruitment of cadherins to the adherens junctions, were similarly decreased. It is therefore not surprising that with lower expression of these proteins that the BTB becomes diminished with age. We saw, using a FITC tracer, a gradual collapse of the BTB between 18 and 24 months. This provides the opportunity for harmful substances and immune cells to cross the BTB and cause the disruption of spermatogenesis that is observed with increasing age.

  9. Reduction of microhemorrhages in the spinal cord of symptomatic ALS mice after intravenous human bone marrow stem cell transplantation accompanies repair of the blood-spinal cord barrier.

    Science.gov (United States)

    Eve, David J; Steiner, George; Mahendrasah, Ajay; Sanberg, Paul R; Kurien, Crupa; Thomson, Avery; Borlongan, Cesar V; Garbuzova-Davis, Svitlana

    2018-02-13

    Blood-spinal cord barrier (BSCB) alterations, including capillary rupture, have been demonstrated in animal models of amyotrophic lateral sclerosis (ALS) and ALS patients. To date, treatment to restore BSCB in ALS is underexplored. Here, we evaluated whether intravenous transplantation of human bone marrow CD34 + (hBM34 + ) cells into symptomatic ALS mice leads to restoration of capillary integrity in the spinal cord as determined by detection of microhemorrhages. Three different doses of hBM34 + cells (5 × 10 4 , 5 × 10 5 or 1 × 10 6 ) or media were intravenously injected into symptomatic G93A SOD1 mice at 13 weeks of age. Microhemorrhages were determined in the cervical and lumbar spinal cords of mice at 4 weeks post-treatment, as revealed by Perls' Prussian blue staining for ferric iron. Numerous microhemorrhages were observed in the gray and white matter of the spinal cords in media-treated mice, with a greater number of capillary ruptures within the ventral horn of both segments. In cell-treated mice, microhemorrhage numbers in the cervical and lumbar spinal cords were inversely related to administered cell doses. In particular, the pervasive microvascular ruptures determined in the spinal cords in late symptomatic ALS mice were significantly decreased by the highest cell dose, suggestive of BSCB repair by grafted hBM34 + cells. The study results provide translational outcomes supporting transplantation of hBM34 + cells at an optimal dose as a potential therapeutic strategy for BSCB repair in ALS patients.

  10. Altered gravity causes the changes in the proteins NoA100 in plant cell nucleoli

    Science.gov (United States)

    Sobol, Margarita A.; Gonzalez-Camacho, Fernando; Kordyum, Elizabeth L.; Medina, Francisco Javier

    2005-08-01

    A nucleolar protein homologous to the mammalian nucleolin and to the onion nucleolin-like protein NopA100 was detected in nuclear soluble protein fraction from Lepidium sativum root meristematic cells, using the specific silver staining method and the cross-reaction with the anti-NopA100 antibody. In 2D Western blots of soluble nuclear fraction, NopA100 was revealed as a smear extending through a certain range of pI. In extracts obtained from seedlings grown under clinorotation, the extension of the pI range was shorter than in the stationary control indicating a lower phosphorylation of the protein. This suggests that altered gravity causes a decrease in the rate of nucleolar activity.

  11. [Significance of the alteration of Th17 cells in patients with chronic lymphocytic thyroiditis].

    Science.gov (United States)

    Song, Jin-Zhan; Wu, Han-Ni; Qian, Wei

    2009-10-01

    To investigate the alteration and its significance of T help 17 cells (Th17) in patients with chronic lymphocytic thyroiditis (CLT). Patients were divided into 3 groups: CLT patients with euthyroidism (n=15), CLT patients with hypothyroidism (n=30) and healthy control group (n=20). The ratio of Th17 lymphocytes subpopulations in the peripheral blood were evaluated by technique of flow cytometry. Production of thyroid autoantibody (TPO-Ab, TG-Ab) were measured by electro-chemiluminescence immunoassay (ECLIA). Compared with the healthy control group, in CLT group: The frequencies of Th17 in peripheral blood were found to be significantly higher in patients with CLT than healthy control group (PCLT patients than healthy control group (PCLT which may suggest a potential role for Th17 in the progression and happen of CLT.

  12. Adaptation of innate lymphoid cells to nutrient deprivation promotes type 2 barrier immunity

    Science.gov (United States)

    Survival of the host relies on the establishment of site-specific barrier defense tailored to constrain pressures imposed by commensal and parasitic exposures. The host is confronted with the additional challenge of maintaining barrier immunity in fluctuating states of dietary availability, yet how ...

  13. Recombination barrier layers in solid-state quantum dot-sensitized solar cells

    KAUST Repository

    Roelofs, Katherine E.; Brennan, Thomas P.; Dominguez, Juan C.; Bent, Stacey F.

    2012-01-01

    in situ by successive ion layer adsorption and reaction (SILAR). Aluminum oxide recombination barrier layers were deposited by atomic layer deposition (ALD) at the TiO2/hole-conductor interface. For low numbers of ALD cycles, the Al2O3 barrier layer

  14. Methylglyoxal synthase regulates cell elongation via alterations of cellular methylglyoxal and spermidine content in Bacillus subtilis.

    Science.gov (United States)

    Shin, Sang-Min; Song, Sung-Hyun; Lee, Jin-Woo; Kwak, Min-Kyu; Kang, Sa-Ouk

    2017-10-01

    Methylglyoxal regulates cell division and differentiation through its interaction with polyamines. Loss of their biosynthesizing enzyme causes physiological impairment and cell elongation in eukaryotes. However, the reciprocal effects of methylglyoxal and polyamine production and its regulatory metabolic switches on morphological changes in prokaryotes have not been addressed. Here, Bacillus subtilis methylglyoxal synthase (mgsA) and polyamine biosynthesizing genes encoding arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), were disrupted or overexpressed. Treatment of 0.2mM methylglyoxal and 1mM spermidine led to the elongation and shortening of B. subtilis wild-type cells to 12.38±3.21μm (P<0.05) and 3.24±0.73μm (P<0.01), respectively, compared to untreated cells (5.72±0.68μm). mgsA-deficient (mgsA - ) and -overexpressing (mgsA OE ) mutants also demonstrated cell shortening and elongation, similar to speB- and speE-deficient (speB - and speE - ) and -overexpressing (speB OE and speE OE ) mutants. Importantly, both mgsA-depleted speB OE and speE OE mutants (speB OE /mgsA - and speE OE /mgsA - ) were drastically shortened to 24.5% and 23.8% of parental speB OE and speE OE mutants, respectively. These phenotypes were associated with reciprocal alterations of mgsA and polyamine transcripts governed by the contents of methylglyoxal and spermidine, which are involved in enzymatic or genetic metabolite-control mechanisms. Additionally, biophysically detected methylglyoxal-spermidine Schiff bases did not affect morphogenesis. Taken together, the findings indicate that methylglyoxal triggers cell elongation. Furthermore, cells with methylglyoxal accumulation commonly exhibit an elongated rod-shaped morphology through upregulation of mgsA, polyamine genes, and the global regulator spx, as well as repression of the cell division and shape regulator, FtsZ. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Perioperative dynamic alterations in peripheral regulatory T and B cells in patients with hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Chen Tianxiang

    2012-01-01

    Full Text Available Abstract Background Intratumoral and circulating regulatory T cells (Tregs have been shown to be critical in the pathogenesis of hepatocellular carcinoma (HCC. However there is limited knowledge on the alterations of regulatory B cells (Bregs. We here investigated perioperative dynamic alterations of peripheral circulating Tregs and Bregs in HCC patients to reveal the relationship between regulatory lymphocytes and its clinical implications. Methods 36 patients with HCC, 6 with chronic hepatitis B infection and 10 healthy donors were enrolled for this study. Frequencies of peripheral Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD19 and IL-10 before, and after radical surgery. Then, clinical informatics of HCC patients was achieved through Digital Evaluation Score System (DESS for the assessment of disease severity. Finally, we analysed correlations between digitalized clinical features and kinetics of circulating regulatory lymphocytes. Results Level of circulating CD4+CD25+CD127- Tregs in HCC patients was significantly lower than that in healthy donors and patients with chronic hepatitis B infection before surgery, but was increased after surgery. Preoperative level of CD19+ IL-10+ Bregs in HCC patients was also significantly lower than the other groups. However it dramatically was elevated right after surgery and remained elevated compared to controls (about 7 days after surgery, P = 0.04. Frequency of circulating Tregs was correlated with circulating leukocytes, ferritin, and clinical features suggesting tumor aggressiveness including portal vein thrombosis, hepatic vein involvement and advanced clinical stages. Frequency of circulating Bregs was associated with Hepatitis B e Antigen (HBeAg and Hepatitis B virus (HBV DNA copy number. In addition, DESS was significantly and positively correlated with other staging systems. Conclusion Frequencies of peripheral Tregs and Bregs in HCC patients

  16. Infection with the oncogenic human papillomavirus type 59 alters protein components of the cornified cell envelope

    International Nuclear Information System (INIS)

    Lehr, Elizabeth; Brown, Darron R.

    2003-01-01

    Infection of the genital tract with human papillomaviruses (HPVs) leads to proliferative and dysplastic epithelial lesions. The mechanisms used by the virus to escape the infected keratinocyte are not well understood. Infection of keratinocytes with HPV does not cause lysis, the mechanism used by many viruses to release newly formed virions. For HPV 11, a type associated with a low risk of neoplastic disease, the cornified cell envelope (CCE) of infected keratinocytes is thin and fragile, and transcription of loricrin, the major CCE protein, is reduced. The effects of high-risk HPV infection on components of the CCE have not been previously reported. HPV 59, an oncogenic genital type related to HPV types 18 and 45 was identified in a condylomata acuminata lesion. An extract of this lesion was used to infect human foreskin fragments, which were grown in athymic mice as xenografts. Continued propagation using extracts of xenografts permitted growth of additional HPV 59-infected xenografts. CCEs purified from HPV 59-infected xenografts displayed subtle morphologic abnormalities compared to those derived from uninfected xenografts. HPV 59-infected xenografts revealed dysplastic-appearing cells with mitotic figures. Detection of loricrin, involucrin, and cytokeratin 10 was reduced in HPV 59-infected epithelium, while small proline-rich protein 3 (SPR3) was increased. Reduction in loricrin was most apparent in regions of epithelium containing abundant HPV 59 DNA. Compared to uninfected epithelium, loricrin transcription was decreased in HPV 59-infected epithelium. We conclude that HPV 59 shares with HPV 11 the ability to alter CCE components and to specifically reduce transcription of the loricrin gene. Because loricrin is the major CCE protein, a reduction in this component could alter the physical properties of the CCE, thus facilitating virion release

  17. Altered Expression of Wnt Signaling Pathway Components in Osteogenesis of Mesenchymal Stem Cells in Osteoarthritis Patients.

    Science.gov (United States)

    Tornero-Esteban, Pilar; Peralta-Sastre, Ascensión; Herranz, Eva; Rodríguez-Rodríguez, Luis; Mucientes, Arkaitz; Abásolo, Lydia; Marco, Fernando; Fernández-Gutiérrez, Benjamín; Lamas, José Ramón

    2015-01-01

    Osteoarthritis (OA) is characterized by altered homeostasis of joint cartilage and bone, whose functional properties rely on chondrocytes and osteoblasts, belonging to mesenchymal stem cells (MSCs). WNT signaling acts as a hub integrating and crosstalking with other signaling pathways leading to the regulation of MSC functions. The aim of this study was to evaluate the existence of a differential signaling between Healthy and OA-MSCs during osteogenesis. MSCs of seven OA patients and six healthy controls were isolated, characterised and expanded. During in vitro osteogenesis, cells were recovered at days 1, 10 and 21. RNA and protein content was obtained. Expression of WNT pathway genes was evaluated using RT-qPCR. Functional studies were also performed to study the MSC osteogenic commitment and functional and post-traslational status of β-catenin and several receptor tyrosine kinases. Several genes were downregulated in OA-MSCs during osteogenesis in vitro. These included soluble Wnts, inhibitors, receptors, co-receptors, several kinases and transcription factors. Basal levels of β-catenin were higher in OA-MSCs, but calcium deposition and expression of osteogenic genes was similar between Healthy and OA-MSCs. Interestingly an increased phosphorylation of p44/42 MAPK (ERK1/2) signaling node was present in OA-MSCs. Our results point to the existence in OA-MSCs of alterations in expression of Wnt pathway components during in vitro osteogenesis that are partially compensated by post-translational mechanisms modulating the function of other pathways. We also point the relevance of other signaling pathways in OA pathophysiology suggesting their role in the maintenance of joint homeostasis through modulation of MSC osteogenic potential.

  18. Liposomal curcumin alters chemosensitivity of breast cancer cells to Adriamycin via regulating microRNA expression.

    Science.gov (United States)

    Zhou, Siying; Li, Jian; Xu, Hanzi; Zhang, Sijie; Chen, Xiu; Chen, Wei; Yang, Sujin; Zhong, Shanliang; Zhao, Jianhua; Tang, Jinhai

    2017-07-30

    Emerging evidence suggests that curcumin can overcome drug resistance to classical chemotherapies, but poor bioavailability and low absorption have limited its clinical use and the mechanisms remain unclear. Also, Adriamycin (Adr) is one of the most active cytotoxic agents in breast cancer; however, the high resistant rate of Adr leads to a poor prognosis. We utilized encapsulation in liposomes as a strategy to improve the bioavailability of curcumin and demonstrated that liposomal curcumin altered chemosensitivity of Adr-resistant MCF-7 human breast cancer (MCF-7/Adr) by MTT assay. The miRNA and mRNA expression profiles of MCF-7/S, MCF-7/Adr and curcumin-treated MCF-7/Adr cells were analyzed by microarray and further confirmed by real-time PCR. We focused on differentially expressed miR-29b-1-5p to explore the involvement of miR-29b-1-5p in the resistance of Adr. Candidate genes of dysregulated miRNAs were identified by prediction algorithms based on gene expression profiles. Networks of KEGG pathways were organized by the selected dysregulated miRNAs. Moreover, protein-protein interaction (PPI) was utilized to map protein interaction networks of curcumin regulated proteins. We first demonstrated liposomal curcumin could rescue part of Adriamycin resistance in breast cancer and further identified 67 differentially expressed microRNAs among MCF-7/S, MCF-7/Adr and curcumin-treated MCF-7/Adr. The results showed that lower expressed miR-29b-1-5p decreased the IC50 of MCF-7/Adr cells and higher expressed miR-29b-1-5p, weaken the effects of liposomal curcumin to Adr-resistance. Besides, we found that 20 target genes (mRNAs) of each dysregulated miRNA were not only predicted by prediction algorithms, but also differentially expressed in the microarray. The results showed that MAPK, mTOR, PI3K-Akt, AMPK, TNF, Ras signaling pathways and several target genes such as PPARG, RRM2, SRSF1and EPAS1, may associate with drug resistance of breast cancer cells to Adr. We determined

  19. A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

    Directory of Open Access Journals (Sweden)

    Birte Möhlendick

    Full Text Available Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH of single cells. The protocol is based on an established adapter-linker PCR (WGAM and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost- effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

  20. Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging.

    Science.gov (United States)

    Sahay, Peeyush; Shukla, Pradeep K; Ghimire, Hemendra M; Almabadi, Huda M; Tripathi, Vibha; Mohanty, Samarendra K; Rao, Radhakrishna; Pradhan, Prabhakar

    2017-03-01

    Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10-12 week-old mice fed a Lieber-DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.

  1. Alterations of Intercellular Junctions in Peritoneal Mesothelial Cells from Patients Undergoing Dialysis: Effect of Retinoic Acid

    Science.gov (United States)

    Retana, Carmen; Sanchez, Elsa; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas, Jesus; Cruz, Carmen; Vital, Socorro; Reyes, Jose L.

    2015-01-01

    ♦ Background: Dialysis patients are classified according to their peritoneal permeability as low transporter (LT, low solute permeability) or high transporter (HT, high solute permeability). Tight junction (TJ) proteins are critical to maintain ions, molecules and water paracellular transport through peritoneum. Exposure to peritoneal dialysis solutions causes damage to TJ in human peritoneal mesothelial cells (HPMCs). We analyzed the quantity, distribution and function of TJ proteins: claudin-1, -2 and -8, ZO-1 and occludin, in HPMC cultures from LT and HT patients. Since all-trans retinoic acid (ATRA) might modify the expression of TJ proteins, we studied its effect on HPMCs. ♦ Methods: Control HPMCs were isolated from human omentum, while HT or LT cells were obtained from dialysis effluents. Cells were cultured in presence of ATRA 0, 50 or 100 nM. Transepithelial electrical resistance (TER) measurement, immunostaining and Western blot analyses were performed. ♦ Results: HT exhibited lower TER than control and LT monolayers. Immunofluorescence for TJ was weak and discontinuous along the cell contour, in LT and HT. Furthermore, claudin-1, occludin and ZO-1 expressions were decreased. In all groups, claudin-2 was localized at nuclei. We observed that ATRA improved TJ distribution and increased TJ expression in HT. This retinoid did not modify claudin-2 and -8 expressions. All-trans retinoic acid decreased TER in HT, but had no effect in LT. ♦ Conclusions: Tight junctions were altered in HPMCs from dialyzed patients. The HT monolayer has lower TER than LT, which might be associated with the peritoneal permeability in these patients. ATRA might be a therapeutic alternative to maintain mesothelial integrity, since it improved TJ localization and expression. PMID:24584604

  2. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

    Science.gov (United States)

    Sun, Xiaofei; Park, Craig B; Deng, Wenbo; Potter, S Steven; Dey, Sudhansu K

    2016-04-01

    Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. © FASEB.

  3. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    International Nuclear Information System (INIS)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay

    2015-01-01

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

  4. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  5. Efficiency enhancement of solid-state PbS quantum dot-sensitized solar cells with Al2O3 barrier layer

    KAUST Repository

    Brennan, Thomas P.; Trejo, Orlando; Roelofs, Katherine E.; Xu, John; Prinz, Fritz B.; Bent, Stacey F.

    2013-01-01

    Atomic layer deposition (ALD) was used to grow both PbS quantum dots and Al2O3 barrier layers in a solid-state quantum dot-sensitized solar cell (QDSSC). Barrier layers grown prior to quantum dots resulted in a near-doubling of device efficiency (0.30% to 0.57%) whereas barrier layers grown after quantum dots did not improve efficiency, indicating the importance of quantum dots in recombination processes. © 2013 The Royal Society of Chemistry.

  6. Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations.

    Science.gov (United States)

    Kompisch, Kai Michael; Lange, Claudia; Steinemann, Doris; Skawran, Britta; Schlegelberger, Brigitte; Müller, Reinhard; Schumacher, Udo

    2010-11-01

    Adipose-derived stem cells (ASCs) are reported to display multilineage differentiation potential, including neuroectodermal pathways. The aim of the present study was to critically re-evaluate the potential neurogenic (trans-)differentiation capacity of ASCs using a neurogenic induction protocol based on the combination of isobutylmethylxanthine (IBMX), indomethacin and insulin. ASCs isolated from lipo-aspirate samples of five healthy female donors were characterized and potential neurogenic (trans-)differentiation was assessed by means of immunohistochemistry and gene expression analyses. Cell proliferation and cell cycle alterations were studied, and the expression of CREB/ATF transcription factors was analyzed. ASCs expressed CD59, CD90 and CD105, and were tested negative for CD34 and CD45. Under neurogenic induction, ASCs adopted a characteristic morphology comparable to neur(on)al progenitors and expressed musashi1, β-III-tubulin and nestin. Gene expression analyses revealed an increased expression of β-III-tubulin, GFAP, vimentin and BDNF, as well as SOX4 in induced ASCs. Cell proliferation was significantly reduced under neurogenic induction; cell cycle analyses showed a G2-cell cycle arrest accompanied by differential expression of key regulators of cell cycle progression. Differential expression of CREB/ATF transcription factors could be observed on neurogenic induction, pointing to a decisive role of the cAMP-CREB/ATF system. Our findings may point to a potential neurogenic (trans-)differentiation of ASCs into early neur(on)al progenitors, but do not present definite evidence for it. Especially, the adoption of a neural progenitor cell-like morphology must not automatically be misinterpreted as a specific characteristic of a respective (trans-)differentiation process, as this may as well be caused by alterations of cell cycle progression.

  7. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jose F Abisambra

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

  8. Asthma pregnancy alters postnatal development of chromaffin cells in the rat adrenal medulla.

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    Xiu-Ming Wu

    Full Text Available Adrenal neuroendocrine plays an important role in asthma. The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring remain unknown.This study aims to explore the influence of maternal asthma during pregnancy on the development and function of adrenal medulla in offspring from postnatal day 3 (P3 to postnatal day 60 (P60. Asthmatic pregnant rats (AP, nerve growth factor (NGF-treated pregnant rats (NP and NGF antibody-treated pregnant rats (ANP were sensitized and challenged with ovalbumin (OVA; NP and ANP were treated with NGF and NGF antibody respectively. Offspring rats from the maternal group were divided into four groups: offspring from control pregnant rats (OCP, offspring from AP (OAP, offspring from NP (ONP, and offspring from ANP (OANP. The expressions of phenylethanolamine N-methyltransferase (PNMT protein in adrenal medulla were analyzed. The concentrations of epinephrine (EPI, corticosterone and NGF in serum were measured. Adrenal medulla chromaffin cells (AMCC were prone to differentiate into sympathetic nerve cells in OAP and ONP. Both EPI and PNMT were decreased in OAP from P3 to P14, and then reached normal level gradually from P30 to P60, which were lower from birth to adulthood in ONP. Corticosterone concentration increased significantly in OAP and ONP.Asthma pregnancy may promote AMCC to differentiate into sympathetic neurons in offspring rats and inhibit the synthesis of EPI, resulting in dysfunction of bronchial relaxation.

  9. Ablation of p120-Catenin Altering the Activity of Small GTPase in Human Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nan LIU

    2009-05-01

    Full Text Available Background and objective p120-catenin (p120ctn, a member of the Armadillo gene family, has emerged as an important modulator of small GTPase activities. Therefore, it plays novel roles in tumor malignant phenotype, such as invasion and metastasis, whose mechanism are not well clarified yet. The aim of this study is to explore the roles of p120ctn on the regulation of small GTP family members in lung cancer and the effects to lung cancer invasions andmetastasis. Methods After p120ctn was knocked down by siRNA, in vivo and in vitro analysis was applied to investigate the role and possible mechanism of p120ctn in lung cancer, such as Western Blot, pull-down analysis, and nude mice models. Results p120ctn depletion inactivated RhoA, with the the activity of Cdc42 and Rac1 increased, the invasiveness of lung cancer cells was promoted both in vitro and in vivo . Conclusion p120ctn gene knockdown enhances the metastasis of lung cancer cells, probably by altering expression of small GTPase, such as inactivation of RhoA and activation of Cdc42/Rac1.

  10. Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum

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    Ashish Srivastava

    2017-07-01

    Full Text Available A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L. plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV and Ageratum leaf curl betasatellite (ALCB, respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch’s postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.

  11. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    International Nuclear Information System (INIS)

    Sahijdak, W.M.; Yang, Chin-Rang; Zuckerman, J.S.; Meyers, M.; Boothman, D.A.

    1994-01-01

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs

  12. Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery

    International Nuclear Information System (INIS)

    Brown, Nicole; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2006-01-01

    Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells. In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8 + T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8 + T cells. The density of expression of HY-TCR on CD8 + cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment. Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery

  13. Errantum: Treatment of human astrocytoma U87 cells with silicon dioxide nanoparticles lowers their survival and alters their expression of mitochondrial and cell signaling proteins

    Directory of Open Access Journals (Sweden)

    Lai JCK

    2010-12-01

    Full Text Available Lai JCK, Ananthakrishnan G, Jandhyam S, et al. Treatment of human astrocytoma U87 cells with silicon dioxide nanoparticles lowers their survival and alters their expression of mitochondrial and cell signaling proteins. Int J Nanomedicine. 2010;5:715–723.The wrong image was used in Figure 5 on page 719.

  14. Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism.

    Science.gov (United States)

    Halama, Anna; Guerrouahen, Bella S; Pasquier, Jennifer; Satheesh, Noothan J; Suhre, Karsten; Rafii, Arash

    2017-01-04

    The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment - a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4 + EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer - endothelial cells interaction. We demonstrated that "Warburg effect" is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment.

  15. In a Canine Pneumonia Model of Exchange-Transfusion, Altering the Age but Not the Volume of Older Red Blood Cells Markedly Alters Outcome

    Science.gov (United States)

    Cortés-Puch, Irene; Remy, Kenneth E.; Solomon, Steven B.; Sun, Junfeng; Wang, Dong; Al-Hamad, Mariam; Kelly, Seth M.; Sinchar, Derek; Bellavia, Landon; Kanias, Tamir; Popovsky, Mark A.; Kim-Shapiro, Daniel B.; Klein, Harvey G.; Natanson, Charles

    2015-01-01

    Background Massive exchange-transfusion of 42-day-old red blood cells (RBCs) in a canine model of S. aureus pneumonia resulted in in vivo hemolysis with increases in cell-free hemoglobin (CFH), transferrin bound iron (TBI), non-transferrin bound iron (NTBI), and mortality. We have previously shown that washing 42-day-old RBCs before transfusion significantly decreased NTBI levels and mortality, but washing 7-day-old RBCs increased mortality and CFH levels. We now report the results of altering volume, washing, and age of RBCs. Study Design and Methods Two-year-old purpose-bred infected beagles were transfused with increasing volumes (5-10, 20-40, or 60-80 mL/kg) of either 42- or 7-day-old RBCs (n=36) or 80 mL/kg of either unwashed or washed RBCs with increasing storage age (14, 21, 28, or 35 days) (n=40). Results All volumes transfused (5-80 mL/kg) of 42-day-old RBCs, resulted in alike (i.e., not significantly different) increases in TBI during transfusion as well as in CFH, lung injury, and mortality rates after transfusion. Transfusion of 80 mL/kg of RBCs stored for 14, 21, 28 and 35 days resulted in increased CFH and NTBI in between levels found at 7 and 42 days of storage. However, washing RBCs of intermediate ages (14-35 days) does not alter NTBI and CFH levels or mortality rates. Conclusions Preclinical data suggest that any volume of 42-day-old blood potentially increases risks during established infection. In contrast, even massive volumes of 7-day-old blood result in minimal CFH and NTBI levels and risks. In contrast to the extremes of storage, washing blood stored for intermediate ages does not alter risks of transfusion or NTBI and CFH clearance. PMID:26469998

  16. Neuroblastoma cells undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression.

    Science.gov (United States)

    Rifatbegovic, Fikret; Frech, Christian; Abbasi, M Reza; Taschner-Mandl, Sabine; Weiss, Tamara; Schmidt, Wolfgang M; Schmidt, Iris; Ladenstein, Ruth; Ambros, Inge M; Ambros, Peter F

    2018-01-15

    Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive. The present RNA-Seq study of stage 4/M primary tumors, enriched BM-derived diagnostic and relapse DTCs, as well as the corresponding BM-derived mononuclear cells (MNCs) from 53 patients revealed 322 differentially expressed genes in DTCs as compared to the tumors (q 2). Particularly, the levels of transcripts encoded by mitochondrial DNA were elevated in DTCs, whereas, for example, genes involved in angiogenesis were downregulated. Furthermore, 224 genes were highly expressed in DTCs and only slightly, if at all, in MNCs (q  6). Interestingly, we found the transcriptome of relapse DTCs largely resembling those of diagnostic DTCs with only 113 differentially expressed genes under relaxed cut-offs (q 0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4 and GLTSCR2. This first RNA-Seq analysis of neuroblastoma DTCs revealed their unique expression profile in comparison to the tumors and MNCs, and less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected downregulation of genes encoded by chromosome 19. As these alterations might be associated with treatment failure and disease relapse, further functional studies on DTCs should be considered. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  17. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  18. Alterations in circulating T-cell lymphocyte populations in children with obstructive sleep apnea.

    Science.gov (United States)

    Tan, Hui-Leng; Gozal, David; Wang, Yang; Bandla, Hari P R; Bhattacharjee, Rakesh; Kulkarni, Richa; Kheirandish-Gozal, Leila

    2013-06-01

    Changes in lymphocyte phenotype and functionality have been described in adult patients with obstructive sleep apnea (OSA). We hypothesized that OSA is associated with T lymphocyte alterations in children, particularly in T regulatory lymphocytes (T regs), and aimed to characterize circulating T lymphocyte subsets in children with OSA. Cross-sectional. Kosair Children's Hospital (Louisville, KY, USA) and Comer Children's Hospital (Chicago, IL, USA). Consecutively recruited children being evaluated for habitual snoring. N/A. Overnight polysomnography (PSG) was performed and a fasting blood sample was obtained from the patients. Flow cytometry was performed on peripheral blood mononuclear cells stained for CD3, CD4, CD8, CD25, FOXP3, interleukin-4 (IL-4), interferon-γ (IFN-γ), and IL-17. Patients were divided into three groups based on their PSG: controls (apnea-hypopnea indices [AHI] hTST), moderate-severe OSA (AHI ≥ 5/h TST). The percentage of CD4+ and T reg lymphocytes differed across groups. Children with moderate-severe OSA had significantly reduced T reg than control children (median [interquartile range] 4.8 [3.8-5.7% CD4+] versus 7.8 [7.0-9.2% CD4+]; P < 0.001). There were also significant differences in the percentage of T helper 1 (Th1) lymphocytes and in Th1:Th2 ratios between groups. Children with moderate-severe OSA had increased Th1 cells (P = 0.001) and Th1:Th2 ratios (P = 0.0026) compared with children with mild OSA and control children. Associations between AHI and T reg (P = 0.0003; r = -0.46), CD4+ lymphocytes (P = 0.0047; r = -0.37), and Th1:Th2 ratios (P = 0.0009; r = 0.43) emerged. In addition, the percentage of T reg was inversely correlated with Th1:Th2 ratios (P = 0.029; r = -0.29). Pediatric OSA is associated with reduced T reg population and altered Th1:Th2 balance toward Th1 predominance, suggesting a shift to a proinflammatory state. The changes in lymphocytic phenotypes associated with OSA may contribute to the variance in systemic

  19. The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis

    International Nuclear Information System (INIS)

    Perides, George; Zhuge, Yuzheng; Lin, Tina; Stins, Monique F; Bronson, Roderick T; Wu, Julian K

    2006-01-01

    Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model. Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system. Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg -/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg -/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner. Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain

  20. Intestinal barrier integrity and inflammatory bowel disease

    DEFF Research Database (Denmark)

    Holmberg, Fredrik Eric Olof; Pedersen, Jannie; Jørgensen, Peter

    2018-01-01

    Disruption of normal barrier function is a fundamental factor in the pathogenesis of inflammatory bowel disease, which includes increased epithelial cell death, modified mucus configuration, altered expression and distribution of tight junction-proteins, along with a decreased expression of antim......Disruption of normal barrier function is a fundamental factor in the pathogenesis of inflammatory bowel disease, which includes increased epithelial cell death, modified mucus configuration, altered expression and distribution of tight junction-proteins, along with a decreased expression...... of antimicrobial peptides. Inflammatory bowel disease is associated with life-long morbidity for affected patients, and both the incidence and prevalence is increasing globally, resulting in substantial economic strain for society. Mucosal healing and re-establishment of barrier integrity is associated......, novel treatment strategies to accomplish mucosal healing and to re-establish normal barrier integrity in inflammatory bowel disease are warranted, and luminal stem cell-based approaches might have an intriguing potential. Transplantation of in vitro expanded intestinal epithelial stem cells derived...

  1. Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review.

    Science.gov (United States)

    Kim, Bum Jun; Kim, Jung Han; Kim, Hyeong Su; Zang, Dae Young

    2017-02-21

    The von Hippel-Lindau (VHL) gene is often inactivated in sporadic renal cell carcinoma (RCC) by mutation or promoter hypermethylation. The prognostic or predictive value of VHL gene alteration is not well established. We conducted this meta-analysis to evaluate the association between the VHL alteration and clinical outcomes in patients with RCC. We searched PUBMED, MEDLINE and EMBASE for articles including following terms in their titles, abstracts, or keywords: 'kidney or renal', 'carcinoma or cancer or neoplasm or malignancy', 'von Hippel-Lindau or VHL', 'alteration or mutation or methylation', and 'prognostic or predictive'. There were six studies fulfilling inclusion criteria and a total of 633 patients with clear cell RCC were included in the study: 244 patients who received anti-vascular endothelial growth factor (VEGF) therapy in the predictive value analysis and 419 in the prognostic value analysis. Out of 663 patients, 410 (61.8%) had VHL alteration. The meta-analysis showed no association between the VHL gene alteration and overall response rate (relative risk = 1.47 [95% CI, 0.81-2.67], P = 0.20) or progression free survival (hazard ratio = 1.02 [95% CI, 0.72-1.44], P = 0.91) in patients with RCC who received VEGF-targeted therapy. There was also no correlation between the VHL alteration and overall survival (HR = 0.80 [95% CI, 0.56-1.14], P = 0.21). In conclusion, this meta-analysis indicates that VHL gene alteration has no prognostic or predictive value in patients with clear cell RCC.

  2. Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Weng Yuan-Yuan

    2008-10-01

    Full Text Available Abstract Background CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs. Tumor cells adhesion to extracellular matrix (ECM proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA against CD147 (si-CD147 on hepatocellular carcinoma cells' (SMMC-7721 architecture and functions. Methods Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK, vinculiln and mitogen-activated protein kinase (MAPK expression in SMMC-7721 cells. Results Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression. Conclusion CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

  3. Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

    Science.gov (United States)

    Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

    2012-01-01

    Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

  4. Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

    KAUST Repository

    Diaz-Rua, Ruben

    2016-11-23

    Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases.

  5. MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Wiklund, Erik Digman; Gao, Shan; Hulf, Toby

    2011-01-01

    MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC....

  6. Genetic and epigenetic alterations of the reduced folate carrier in untreated diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Kastrup, Ingelise Bjerring; Worm, Jesper; Ralfkiaer, Elisabeth

    2007-01-01

    The reduced folate carrier (RFC) is a transmembrane protein that mediates cellular uptake of reduced folates and antifolate drugs, including methotrexate (MTX). Acquired alterations of the RFC gene have been associated with resistance to MTX in cancer cell lines and primary osteosarcomas. Here, we...

  7. Treatment of poly(ethylene terephthalate) foils by atmospheric pressure air dielectric barrier discharge and its influence on cell growth

    Science.gov (United States)

    Kuzminova, Anna; Vandrovcová, Marta; Shelemin, Artem; Kylián, Ondřej; Choukourov, Andrei; Hanuš, Jan; Bačáková, Lucie; Slavínská, Danka; Biederman, Hynek

    2015-12-01

    In this contribution an effect of dielectric barrier discharge (DBD) sustained in air at atmospheric pressure on surface properties of poly(ethylene terephthalate) (PET) foils is studied. It is found that exposure of PET to DBD plasma leads to rapid changes of surface chemical composition, wettability, surface morphology as well as mechanical properties of PET surface. In addition, based on biological tests that were performed using two cell types (Saos-2 human osteoblast-like cells and HUVEC human umbilical vein endothelial cells), it may be concluded that DBD plasma treatment positively influences cell growth on PET. This effect was found to be connected predominantly with increased surface energy and oxygen content of the surface of treated PET foils.

  8. Zika Virus Infects Human Sertoli Cells and Modulates the Integrity of the In Vitro Blood-Testis Barrier Model.

    Science.gov (United States)

    Siemann, David N; Strange, Daniel P; Maharaj, Payal N; Shi, Pei-Yong; Verma, Saguna

    2017-11-15

    Confirmed reports of Zika virus (ZIKV) in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune-privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell (SC) barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV compared to the closely related dengue virus and induced the expression of alpha interferon (IFN-α), key cytokines, and cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and intracellular adhesion molecule 1 [ICAM-1]). Furthermore, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with a higher efficiency than in the blood-brain barrier model. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decreases in SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin, and JAM-A; however, exposure of SCs to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of the ZO-1 protein, which correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoon development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and to test the ability of anti-ZIKV drugs to cross the barrier. IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, which has not been reported for other flaviviruses. To be able to sexually transmit for months after the clearance of

  9. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

    Science.gov (United States)

    Wagner, Sylvia; Zensi, Anja; Wien, Sascha L; Tschickardt, Sabrina E; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U; Kreuter, Jörg; von Briesen, Hagen

    2012-01-01

    The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

  10. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

    Directory of Open Access Journals (Sweden)

    Sylvia Wagner

    Full Text Available BACKGROUND: The blood-brain barrier (BBB represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. CONCLUSIONS/SIGNIFICANCE: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

  11. Hyperfractionation as an altered fractionation regimen in primary radiotherapy for squamous cell carcinoma of the larynx

    International Nuclear Information System (INIS)

    Krstevska, V.; Smichkoska, S.

    2006-01-01

    The aim of the study was to investigate the efficacy of hyperfractionation as altered fractionation treatment schedule in comparison with conventional fractionation in primary definitive radiotherapy for laryngeal squamous cell carcinoma. From March 1999 to December 2000, a group of 28 patients with previously untreated squamous cell carcinoma of the larynx were irradiated with conventional fractionation to to total doses of 66 to 70 Gy in 33 to 35 fraction/6.5 to 7 weeks, 2 Gy/fraction/day, 5 days/week. From January 2001 to June 2004, the other 27 patients with the same diagnosis, were treated prospectively with hyperfractionation receiving radiotherapy delivered at 1.2 Gy/fraction, twice daily, 5 days/week to 74.4 to 79.2 Gy/62 to fractions/6.2 to 7 weeks. Complete response rates after two mounts of radiotherapy completion were 78.6% (22 of 28) and 66.7% (18 of 27) in the conventional fractionation and hyperfractionation group, respectively (Fisher exact test; P=0.246). The two year loco-regional control rates were 61 .0%±18.1 (95% CI) in the conventional fractionation group and 45.0%±18.8 (95% CI) in the hyperfractionation group (long-rank test; P=0.075). Overall survival rate at two years was 71.0%±16.8 (95% CI) for the conventional group and 43.0%±18.7 (95% CI) for the hyperfractionation group (long- rank test; P=0.071). The absence of statistically significant differences either in loco-regional control or overall survival observed between the two treatment modalities suggested that hyperfractionation regimen was not more efficacious than conventionally fractionated radiotherapy for previously untreated carcinoma of the larynx.

  12. Controlled meal frequency without caloric restriction alters peripheral blood mononuclear cell cytokine production

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    Longo Dan L

    2011-03-01

    Full Text Available Abstract Background Intermittent fasting (IF improves healthy lifespan in animals by a mechanism involving reduced oxidative damage and increased resistance to stress. However, no studies have evaluated the impact of controlled meal frequency on immune responses in human subjects. Objective A study was conducted to establish the effects of controlled diets with different meal frequencies, but similar daily energy intakes, on cytokine production in healthy male and female subjects. Design In a crossover study design with an intervening washout period, healthy normal weight middle-age male and female subjects (n = 15 were maintained for 2 months on controlled on-site one meal per day (OMD or three meals per day (TMD isocaloric diets. Serum samples and peripheral blood mononuclear cells (PBMCs culture supernatants from subjects were analyzed for the presence of inflammatory markers using a multiplex assay. Results There were no significant differences in the inflammatory markers in the serum of subjects on the OMD or TMD diets. There was an increase in the capacity of PBMCs to produce cytokines in subjects during the first month on the OMD or TMD diets. Lower levels of TNF-α, IL-17, MCP-1 and MIP-1β were produced by PBMCs from subjects on the OMD versus TMD diet. Conclusions PBMCs of subjects on controlled diets exhibit hypersensitivities to cellular stimulation suggesting that stress associated with altered eating behavior might affect cytokine production by immune cells upon stimulation. Moreover, stimulated PBMCs derived from healthy individuals on a reduced meal frequency diet respond with a reduced capability to produce cytokines.

  13. The innate immune response of equine bronchial epithelial cells is altered by training.

    Science.gov (United States)

    Frellstedt, Linda; Gosset, Philippe; Kervoaze, Gwenola; Hans, Aymeric; Desmet, Christophe; Pirottin, Dimitri; Bureau, Fabrice; Lekeux, Pierre; Art, Tatiana

    2015-01-17

    Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-β, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-β after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.

  14. Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable.

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    Julián Tejada

    Full Text Available Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.

  15. Blood banking-induced alteration of red blood cell oxygen release ability.

    Science.gov (United States)

    Li, Yaojin; Xiong, Yanlian; Wang, Ruofeng; Tang, Fuzhou; Wang, Xiang

    2016-05-01

    Current blood banking procedures may not fully preserve red blood cell (RBC) function during storage, contributing to the decrease of RBC oxygen release ability. This study was undertaken to evaluate the impact of routine cold storage on RBC oxygen release ability. RBC units were collected from healthy donors and each unit was split into two parts (whole blood and suspended RBC) to exclude possible donor variability. Oxygen dissociation measurements were performed on blood units stored at 4 °C during a 5-week period. 2,3-diphosphoglycerate levels and fluorescent micrographs of erythrocyte band 3 were also analysed. P50 and oxygen release capacity decreased rapidly during the first 3 weeks, and then did not change significantly. In contrast, the kinetic properties (PO2-t curve and T*50) of oxygen release changed slowly during the first 3 weeks of storage, but then decreased significantly in the last 2 weeks. 2,3-diphosphoglycerate decreased quickly during the first 3 weeks of storage to almost undetectable levels. Band 3 aggregated significantly during the last 2 weeks of storage. RBC oxygen release ability appears to be sensitive to routine cold storage. The thermodynamic characteristics of RBC oxygen release ability changed mainly in the first 3 weeks of storage, due to the decrease of 2,3-diphosphoglycerate, whereas the kinetic characteristics of RBC oxygen release ability decreased significantly at the end of storage, probably affected by alterations of band 3.

  16. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

    Directory of Open Access Journals (Sweden)

    Jeremy S Leventhal

    Full Text Available Sepsis related acute kidney injury (AKI is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS, a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO. Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3 and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

  17. Toward guided tissue and bone regeneration: morphology, attachment, proliferation, and migration of cells cultured on collagen barrier membranes. A systematic review.

    NARCIS (Netherlands)

    Behring, J.; Junker, R.; Walboomers, X.F.; Chessnut, B.; Jansen, J.A.

    2008-01-01

    Collagen barrier membranes are frequently used in both guided tissue regeneration (GTR) and guided bone regeneration (GBR). Collagen used for these devices is available from different species and is often processed to alter the properties of the final product. This is necessary because unprocessed

  18. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  19. Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

    Science.gov (United States)

    Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

    2013-05-01

    Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

  20. The astrocyte/meningeal cell interface is a barrier to neurite outgrowth which can be overcome by manipulation of inhibitory molecules or axonal signalling pathways

    NARCIS (Netherlands)

    Shearer, Morven C; Niclou, Simone P; Brown, David; Asher, Richard A; Holtmaat, Anthony J D G; Levine, Joel M; Verhaagen, J.; Fawcett, James W

    2003-01-01

    Invading meningeal cells form a barrier to axon regeneration after damage to the spinal cord and other parts of the CNS, axons stopping at the interface between meningeal cells and astrocytes. Axon behavior was examined using an in vitro model of astrocyte/meningeal cell interfaces, created by

  1. Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas

    International Nuclear Information System (INIS)

    Gonzaga, Isabela Martins; Andreollo, Nelson Adami; Simão, Tatiana Almeida de; Pinto, Luis Felipe Ribeiro; Soares-Lima, Sheila Coelho; Santos, Paulo Thiago Souza de; Blanco, Tania Cristina Moita; Reis, Bruno Souza Bianchi de; Quintella, Danielle Carvalho; Oliveira, Ivanir Martins de; Faria, Paulo Antonio Silvestre de; Kruel, Cleber Dario Pinto

    2012-01-01

    Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluated EGFR mutation profile as well as the presence of mutations in hotspots of KRAS and BRAF in ESCC patients. We performed RT-qPCR, immunohistochemistry and Fluorescent in situ hybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis. Our results showed an increased EGFR mRNA expression in tumors compared to surrounding tissue (p <0.05), with 11% of the cases presenting at least a four-fold difference between tumor and paired adjacent mucosa. EGFR protein overexpression was present only in 4% of the cases. The median expression of HER2 mRNA was not different between tumors and adjacent mucosa. Still, 7% of the tumors presented at least a 25-fold higher expression of this gene when compared to its paired counterpart. Immunohistochemical analysis revealed that 21% of the tumors were positive for HER2 (scores 2+ and 3+), although only 3+ tumors presented amplification of this gene. Mutation analysis for EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (V600E) showed no mutations in any of the hotspots of these genes in almost 100 patients analyzed. EGFR presented synonymous polymorphisms at codon 836 (C>T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations of EGFR, KRAS and BRAF as well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this

  2. Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

    Science.gov (United States)

    Arai, Eri; Gotoh, Masahiro; Tian, Ying; Sakamoto, Hiromi; Ono, Masaya; Matsuda, Akio; Takahashi, Yoriko; Miyata, Sayaka; Totsuka, Hirohiko; Chiku, Suenori; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Matsumoto, Kenji; Yamada, Tesshi; Yoshida, Teruhiko; Kanai, Yae

    2015-12-01

    CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10(-6))" and "Spindle assembly and chromosome separation (p = 9.260 × 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.

  3. Chronic tinnitus and unipolar brush cell alterations in the cerebellum and dorsal cochlear nucleus.

    Science.gov (United States)

    Brozoski, Thomas; Brozoski, Daniel; Wisner, Kurt; Bauer, Carol

    2017-07-01

    Animal model research has shown that the central features of tinnitus, the perception of sound without an acoustic correlate, include elevated spontaneous and stimulus-driven activity, enhanced burst-mode firing, decreased variance of inter-spike intervals, and distortion of tonotopic frequency representation. Less well documented are cell-specific correlates of tinnitus. Unipolar brush cell (UBC) alterations in animals with psychophysical evidence of tinnitus has recently been reported. UBCs are glutamatergic interneurons that appear to function as local-circuit signal amplifiers. UBCs are abundant in the dorsal cochlear nucleus (DCN) and very abundant in the flocculus (FL) and paraflocculus (PFL) of the cerebellum. In the present research, two indicators of UBC structure and function were examined: Doublecortin (DCX) and epidermal growth factor receptor substrate 8 (Eps8). DCX is a protein that binds to microtubules where it can modify their assembly and growth. Eps8 is a cell-surface tyrosine kinase receptor mediating the response to epidermal growth factor; it appears to have a role in actin polymerization as well as cytoskeletal protein interactions. Both functions could contribute to synaptic remodeling. In the present research UBC Eps8 and DCX immunoreactivity (IR) were determined in 4 groups of rats distinguished by their exposure to high-level sound and psychophysical performance: Unexposed, exposed to high-level sound with behavioral evidence of tinnitus, and two exposed groups without behavioral evidence of tinnitus. Compared to unexposed controls, exposed animals with tinnitus had Eps8 IR elevated in their PFL; other structures were not affected, nor was DCX IR affected. This was interpreted as UBC upregulation in animals with tinnitus. Exposure that failed to produce tinnitus did not increase either Eps8 or DCX IR. Rather Eps8 IR was decreased in the FL and DCN of one subgroup (Least-Tinnitus), while DCX IR decreased in the FL of the other subgroup (No

  4. Efficacy of In2S3 interfacial recombination barrier layer in PbS quantum-dot-sensitized solar cells

    International Nuclear Information System (INIS)

    Basit, Muhammad Abdul; Abbas, Muhammad Awais; Bang, Jin Ho; Park, Tae Joo

    2015-01-01

    In 2 S 3 interfacial recombination barrier layer (IBL) via successive ionic layer adsorption and reaction (SILAR) was successfully employed between PbS quantum dots and mesoporous TiO 2 in quantum-dot-sensitized solar cells (QDSSCs). In 2 S 3 IBL significantly increased the resistance against back electron transfer from TiO 2 , resulting an increment in the photocurrent density (J SC ) for the cell with single SILAR cycle of In 2 S 3 IBL. Further increase in the number of SILAR cycles of In 2 S 3 IBL deteriorated the J SC , whereas open-circuit voltage sustained the increasing trend. Therefore, an optimal photo-conversion efficiency of ∼2.2% was obtained for the cell with 2 SILAR cycles of In 2 S 3 IBL, which strategically reached a value of ∼2.70% after annealing (increased by 40% compared to the control cell without IBL). In 2 S 3 IBL not only improved the recombination resistance and electron life time of the cells, but it also enhanced the photostability of the cells. - Highlights: • In 2 S 3 interfacial recombination barrier layer was deposited on TiO 2 photoanode via SILAR process. • Resistance against back electron transfer from TiO 2 (recombination) increased notably. • Fabricated PbS-QDSSCs were characterized using IPCE, OCVD and EIS techniques. • In 2 S 3 IBL improved chemical capacitance, electron life time and photostability of modified cells. • 2In 2 S 3 IBL showed optimal performance, yielding 40% improvement in PCE after heat treatment.

  5. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  6. Can a Proper T-Cell Development Occur in an Altered Thymic Epithelium? Lessons From EphB-Deficient Thymi

    Directory of Open Access Journals (Sweden)

    Juan José Muñoz

    2018-04-01

    Full Text Available For a long time, the effects of distinct Eph tyrosine kinase receptors and their ligands, ephrins on the structure, immunophenotype, and development of thymus and their main cell components, thymocytes (T and thymic epithelial cells (TECs, have been studied. In recent years, the thymic phenotype of mutant mice deficient in several Ephs and ephrins B has been determined. Remarkably, thymic stroma in these animals exhibits important defects that appear early in ontogeny but little alterations in the proportions of distinct lymphoid cell populations. In the present manuscript, we summarize and extend these results discussing possible mechanisms governing phenotypical and functional thymocyte maturation in an absence of the critical T–TEC interactions, concluding that some signaling mediated by key molecules, such as MHCII, CD80, β5t, Aire, etc. could be sufficient to enable a proper maturation of thymocytes, independently of morphological alterations affecting thymic epithelium.

  7. Maternal Inflammation Contributes to Brain Overgrowth and Autism-Associated Behaviors through Altered Redox Signaling in Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Janel E. Le Belle

    2014-11-01

    Full Text Available A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function. Pregnant mice treated with low-dose lipopolysaccharide at embryonic day 9 had offspring with brain overgrowth, with a more pronounced effect in PTEN heterozygotes. Exposure to maternal inflammation also enhanced NADPH oxidase (NOX-PI3K pathway signaling, stimulated the hyperproliferation of neural stem and progenitor cells, increased forebrain microglia, and produced abnormal autism-associated behaviors in affected pups. Our evidence supports the idea that a prenatal neuroinflammatory dysregulation in neural stem cell redox signaling can act in concert with underlying genetic susceptibilities to affect cellular responses to environmentally altered cellular levels of reactive oxygen species.

  8. Dielectric and diffusion barrier multilayer for Cu(In,Ga)Se{sub 2} solar cells integration on stainless steel sheet

    Energy Technology Data Exchange (ETDEWEB)

    Amouzou, Dodji, E-mail: dodji.amouzou@fundp.ac.be [Research Centre in Physics of Matter and Radiation (PMR), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur (Belgium); Guaino, Philippe; Fourdrinier, Lionel; Richir, Jean-Baptiste; Maseri, Fabrizio [CRM-Group, Boulevard de Colonster, B 57, 4000 Liège (Belgium); Sporken, Robert [Research Centre in Physics of Matter and Radiation (PMR), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur (Belgium)

    2013-09-02

    For the fabrication of monolithically integrated flexible Cu(In, Ga)Se{sub 2}, CIGS modules on stainless steel, individual photovoltaic cells must be insulated from metal substrates by a barrier layer that can sustain high thermal treatments. In this work, a combination of sol–gel (organosilane-sol) and sputtered SiAlxOy forming thin diffusion barrier layers (TDBL) was prepared on stainless steel substrates. The deposition of organosilane-sol dielectric layers on the commercial stainless steel (maximal roughness, Rz = 500 nm and Root Mean Square roughness, RMS = 56 nm) induces a planarization of the surface (RMS = 16.4 nm, Rz = 176 nm). The DC leakage current through the dielectric layers was measured for the metal-insulator-metal (MIM) junctions that act as capacitors. This method allowed us to assess the quality of our TDBL insulating layer and its lateral uniformity. Indeed, evaluating a ratio of the number of valid MIM capacitors to the number of tested MIM capacitors, a yield of ∼ 95% and 50% has been reached respectively with non-annealed and annealed samples based on sol–gel double layers. A yield of 100% was achieved for sol–gel double layers reinforced with a sputtered SiAlxOy coating and a third sol–gel monolayer. Since this yield is obtained on several samples, it can be extrapolated to any substrate size. Furthermore, according to Glow Discharge Optical Emission Spectroscopy and Time of Flight Secondary Ion Mass Spectroscopy measurements, these barrier layers exhibit excellent barrier properties against the diffusion of undesired atoms which could otherwise spoil the electronic and optical properties of CIGS photovoltaic cells. - Highlights: • We functionalize steel for monolithically integrated Cu(In,Ga)Se{sub 2} solar cells • Thin dielectric and diffusion barrier layers (TDDBL) prepared on steel • Reliability and breakdown voltage of dielectric layers have been studied. • Investigation of thermal treatment effect on dielectric

  9. Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

    Science.gov (United States)

    Curis, Céline; Percher, Florent; Jeannin, Patricia; Montange, Thomas; Chevalier, Sébastien A; Seilhean, Danielle; Cartier, Luis; Couraud, Pierre-Olivier; Gout, Olivier; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel; Afonso, Philippe V

    2016-08-15

    Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear

  10. Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

    Science.gov (United States)

    Lagaye, S.; Derrien, M.; Menu, E.; Coïto, C.; Tresoldi, E.; Mauclère, P.; Scarlatti, G.; Chaouat, G.; Barré-Sinoussi, F.; Bomsel, M.

    2001-01-01

    Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies. PMID:11312350

  11. Molecular biologic study about the non-small cell lung carcinoma (2) : p53 gene alteration in non-small cell lung carcinoma

    International Nuclear Information System (INIS)

    Park, Jong Ho; Zo, Jae Ill; Paik, Hee Jong; Kim, Mi Hee

    1996-12-01

    The main purpose of this research was to identify of the p53 and 3p gene alteration in non-small cell lung cancer patients residing in Korea. Furthermore, we analyzed the relationship between the p53 and 3p gene alterations and the clinicopathologic results of lung cancer patients. And we have investigated the role of PCR-LOH in analyzing tumor samples for LOH of defined chromosomal loci. We have used the 40 samples obtained from the lung cancer patients who were diagnosed and operated curatively at Korea Cancer Center Hospital. We have isolated the high molecular weight. DNA from the tumors and normal tissues. And we have amplified the DNA with PCR method and used the microsatellite assay method to detect the altered p53 and 3p gene. The conclusions were as follow: 1) The 3p gene alteration was observed in 9/39 (23.1%) and p53 gene alteration was observed in 15/40 (37.5%) of resected non-small cell lung cancer. 2) There was no correlations between the 3p or p53 gene alterations and prognosis of patients, but further study is necessary. 3) PCR-LOH is a very useful tool for analyzing small amount of tumor samples for loss of heterozygosity of defined chromosomal loci. (author). 10 refs

  12. Influence of the oxygen electrode and inter-diffusion barrier on the degradation of solid oxide electrolysis cells

    DEFF Research Database (Denmark)

    Hjalmarsson, Per; Sun, Xiufu; Liu, Yi-Lin

    2013-01-01

    -diffusion barrier sandwiched between the YSZ electrolyte and an LSCF:CGO oxygen electrode. Impedance Spectroscopy was used during the tests to diagnose the change in electrochemical response of the different components of the SOECs. The results showed a significantly lower degradation rate for the cell with an LSCF......Two Solid Oxide Electrolysis Cells (SOECs) with different oxygen electrodes have been tested in galvanostatic tests carried out at −1.5 Acm−2 and 800 °C converting 60% of a 50:50% mixture of H2O and CO2 (co-electrolysis). One of the cells had an LSM:YSZ oxygen electrode. The other had an CGO inter...

  13. [Changes in expression of Slingshot protein in hypoxic human intestinal epithelial cell and its relation with barrier function of the cells].

    Science.gov (United States)

    Zhang, Jian; Wang, Pei; He, Wen; Wang, Fengjun

    2016-04-01

    To study the effect of hypoxia on Slingshot protein expression in human intestinal epithelial cell and its relation with changes in barrier function of the cells. The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. One portion of the monolayer-cell specimens were divided into six parts according to the random number table, and they were respectively exposed to hypoxia for 0 (without hypoxia), 1, 2, 6, 12, and 24 h. Transepithelial electrical resistance (TER) was determined with an ohmmeter. Another portion of the monolayer-cell specimens were exposed to hypoxia as above. Western blotting was used to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, claudin-1, Slingshot-1, Slingshot-2, and Slingshot-3. The remaining portion of the monolayer-cell specimens were also exposed to hypoxia as above. The content of fibrous actin (F-actin) and globular actin (G-actin) was determined by fluorescence method. The sample number of above-mentioned 3 experiments was respectively 10, 10, and 18 at each time point. Data were processed with one-way analysis of variance and Dunnett test. (1) Compared with that of cells exposed to hypoxia for 0 h, TER of cells exposed to hypoxia for 1 to 24 h was significantly reduced (P values below 0.01). (2) Compared with those of cells exposed to hypoxia for 0 h (all were 1.00), the protein expressions of ZO-1, occludin, and claudin-1 of cells exposed to hypoxia for 1 to 24 h were generally lower, especially those of cells exposed to hypoxia for 12 h or 24 h (respectively 0.69 ± 0.20, 0.47 ± 0.15, and 0.47 ± 0.22, Pprotein expressions of Slingshot-1 and Slingshot-3 of cells exposed to hypoxia for 1 to 24 h were not obviously changed (P values above 0.05). The protein expression of Slingshot-2 of cells was decreased at first and then gradually increased from hypoxia hour 1 to 24. The protein expression of Slingshot-2 of cells exposed to hypoxia for 24 h (1.54 ± 0.57) was significantly

  14. Secretion of Interferon gamma (IFNγ) from Human Immune Cells is Altered by Exposure to Tributyltin (TBT) and Dibutyltin (DBT)

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2013-01-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and also alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 μM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from NK cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure. PMID:24357260

  15. Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

    Science.gov (United States)

    Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

    2008-06-01

    Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

  16. Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

    Science.gov (United States)

    Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

    2015-04-23

    With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory