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Sample records for activated primary human

  1. Flame Retardant BDE-47 Effectively Activates Nuclear Receptor CAR in Human Primary Hepatocytes

    PubMed Central

    Sueyoshi, Tatsuya

    2014-01-01

    Polybrominated diphenyl ether BDE-47 (2,2′,4,4′-tetrabromodiphenyl ether) is a thyroid hormone disruptor in mice; hepatic induction of various metabolic enzymes and transporters has been suggested as the mechanism for this disruption. Utilizing Car −/− and Pxr −/− mice as well as human primary hepatocytes, here we have demonstrated that BDE-47 activated both mouse and human nuclear receptor constitutive activated/androstane receptor (CAR). In mouse livers, CAR, not PXR, was responsible for Cyp2b10 mRNA induction by BDE-47. In human primary hepatocytes, BDE-47 was able to induce translocation of YFP-tagged human CAR from the cytoplasm to the nucleus andCYP2B6 and CYP3A4 mRNAs expressions. BDE-47 activated human CAR in a manner akin to the human CAR ligand CITCO (6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) in luciferase-reporter assays using Huh-7 cells. In contrast, mouse CAR was not potently activated by BDE-47 in the same reporter assays. Furthermore, human pregnane X receptor (PXR) was effectively activated by BDE-47 while mouse PXR was weakly activated in luciferase-reporter assays. Our results indicate that BDE-47 induces CYP genes through activation of human CAR in addition to the previously identified pathway through human PXR. PMID:24218150

  2. Lysozyme: primary bactericidin in human plasma serum active against Bacillus subtilis.

    PubMed Central

    Selsted, M E; Martinez, R J

    1978-01-01

    The in vitro bactericidal reaction of human plasma serum against Bacillus subtilis was investigated. Human lysozyme was purified to homogeneity, and antiserum was prepared against the enzyme. The anti-lysozyme immunoglobulin G was used as a specific inhibitor in bactericidal and bacteriolytic reactions. It was found that at low serum concentrations lysozyme was the primary bactericide active against B. subtilis. At appreciably higher serum concentrations, a lysozyme-independent bactericidal activity was also demonstrated. Images PMID:97236

  3. Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

    PubMed Central

    Kortmann, Jens; Brubaker, Sky W.

    2015-01-01

    Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

  4. Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin.

    PubMed

    Kortmann, Jens; Brubaker, Sky W; Monack, Denise M

    2015-08-01

    Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

  5. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression

    PubMed Central

    Balint, Klara; Xiao, Min; Pinnix, Chelsea C.; Soma, Akinobu; Veres, Imre; Juhasz, Istvan; Brown, Eric J.; Capobianco, Anthony J.; Herlyn, Meenhard; Liu, Zhao-Jun

    2005-01-01

    Notch is a highly conserved transmembrane receptor that determines cell fate. Notch signaling denotes cleavage of the Notch intracellular domain, its translocation to the nucleus, and subsequent activation of target gene transcription. Involvement of Notch signaling in several cancers is well known, but its role in melanoma remains poorly characterized. Here we show that the Notch1 pathway is activated in human melanoma. Blocking Notch signaling suppressed whereas constitutive activation of the Notch1 pathway enhanced primary melanoma cell growth both in vitro and in vivo yet had little effect on metastatic melanoma cells. Activation of Notch1 signaling enabled primary melanoma cells to gain metastatic capability. Furthermore, the oncogenic effect of Notch1 on primary melanoma cells was mediated by β-catenin, which was upregulated following Notch1 activation. Inhibiting β-catenin expression reversed Notch1-enhanced tumor growth and metastasis. Our data therefore suggest a β-catenin–dependent, stage-specific role for Notch1 signaling in promoting the progression of primary melanoma. PMID:16239965

  6. Complexity of the primary genetic response to mitogenic activation of human T cells

    SciTech Connect

    Zipfel, P.F.; Siebenlist, U. ); Irving, S.G.; Kelly, K. )

    1989-03-01

    The authors describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cylcosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.

  7. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells

    PubMed Central

    Reinhard, Nathalie R.; van Helden, Suzanne F.; Anthony, Eloise C.; Yin, Taofei; Wu, Yi I.; Goedhart, Joachim; Gadella, Theodorus W. J.; Hordijk, Peter L.

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

  8. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

    PubMed

    Reinhard, Nathalie R; van Helden, Suzanne F; Anthony, Eloise C; Yin, Taofei; Wu, Yi I; Goedhart, Joachim; Gadella, Theodorus W J; Hordijk, Peter L

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

  9. Gene modification of primary tumor cells for active immunotherapy of human breast and ovarian cancer.

    PubMed

    Philip, R; Clary, B; Brunette, E; Kilinski, L; Murugesh, D; Sorich, M; Yau, J; Lebkowski, J; Lyerly, H K; Philip, M

    1996-01-01

    We have previously shown that cationic liposomes facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of breast and ovarian cancers, we have constructed an expression plasmid pMP6IL2 and investigated the use of liposome-mediated gene delivery into primary, uncultured human breast and ovarian tumor cells to produce interleukin 2 (IL-2)-secreting tumor cells. We have demonstrated significant levels of IL-2 expression in tumor cell lines and primary breast and ovarian tumor cells using this AAV-based expression plasmid complexed to cationic liposomes. Transfections with the non-AAV plasmid containing the identical expression cassette as the AAV plasmid induced IL-2 expression in the tumor cell line but failed to produce IL-2 in primary tumor cells. Significant levels of IL-2 were induced with the AAV plasmid regardless of liposome compositions used for transfection. The transfected breast cell line and primary tumor cells were able to express the transgene product for up to 28 days after lethal radiation. The transfection efficiency was comparable for both the tumor cell line and primary tumor cells and ranged from 20 to 50% for both cell types as assessed by intracellular IL-2 staining. Although the primary tumor cell preparations consist of mixed population of cells, at least 40% of the tumor cells expressed the transgene as assessed by immunostaining for IL-2. The ability to efficiently express transgenes in freshly isolated, nondividing tumor cells may potentiate active immunotherapy strategies for gene-based cancer treatment.

  10. 1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation

    PubMed Central

    Meredith, Anna; Boroomand, Seti; Carthy, Jon; Luo, Zongshu; McManus, Bruce

    2015-01-01

    Aims Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples. Methods and Results Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation. Conclusions Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis. PMID:26061181

  11. Effects of primary metabolites of organophosphate flame retardants on transcriptional activity via human nuclear receptors.

    PubMed

    Kojima, Hiroyuki; Takeuchi, Shinji; Van den Eede, Nele; Covaci, Adrian

    2016-03-14

    Organophosphate flame retardants (OPFRs) have been used in a wide variety of applications and detected in several environmental matrices, including indoor air and dust. Continuous human exposure to these chemicals is of growing concern. In this study, the agonistic and/or antagonistic activities of 12 primary OPFR-metabolites against ten human nuclear receptors were examined using cell-based transcriptional assays, and compared to those of their parent compounds. As a result, 3-hydroxylphenyl diphenyl phosphate and 4-hydroxylphenyl diphenyl phosphate showed more potent estrogen receptor α (ERα) and ERβ agonistic activity than did their parent, triphenyl phosphate (TPHP). In addition, these hydroxylated TPHP-metabolites also showed ERβ antagonistic activity at higher concentrations and exhibited pregnane X receptor (PXR) agonistic activity as well as androgen receptor (AR) and glucocorticoid receptor (GR) antagonistic activities at similar levels to those of TPHP. Bis(2-butoxyethyl) 3'-hydroxy-2-butoxyethyl phosphate and 2-hydroxyethyl bis(2-butoxyethyl) phosphate act as PXR agonists at similar levels to their parent, tris(2-butoxyethyl) phosphate. On the other hand, seven diester OPFR-metabolites and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate did not show any receptor activity. Taken together, these results suggest that hydroxylated TPHP-metabolites show increased estrogenicity compared to the parent compound, whereas the diester OPFR-metabolites may have limited nuclear receptor activity compared to their parent triester OPFRs.

  12. Electrically Activated Primary Human Fibroblasts Improve In Vitro and In Vivo Skin Regeneration.

    PubMed

    Rouabhia, Mahmoud; Park, Hyun Jin; Zhang, Ze

    2016-08-01

    Electrical stimulation (ES) changes cellular behaviors and thus constitutes a potential strategy to promote wound healing. However, well-controlled in vitro findings have yet to be translated to in vivo trials. This study was to demonstrate the feasibility and advantages of transplanting electrically activated cells (E-Cells) to help wound healing. Primary human skin fibroblasts were activated through well defined ES and cultured with keratinocytes to generate engineered human skin (EHS), which were transplanted to nu/nu mice. The electrically activated EHS grafts were analyzed at 20 and 30 days post-grafting, showing faster wound closure, thick epidermis, vasculature, and functional basement membrane containing laminin and type IV collagen that were totally produced by the implanted human cells. Because a variety of cells can be electrically activated, E-Cells may become a new cell source and the transplantation of E-Cells may represent a new strategy in wound healing and tissue engineering. J. Cell. Physiol. 231: 1814-1821, 2016. © 2015 Wiley Periodicals, Inc. PMID:26661681

  13. Electrically Activated Primary Human Fibroblasts Improve In Vitro and In Vivo Skin Regeneration.

    PubMed

    Rouabhia, Mahmoud; Park, Hyun Jin; Zhang, Ze

    2016-08-01

    Electrical stimulation (ES) changes cellular behaviors and thus constitutes a potential strategy to promote wound healing. However, well-controlled in vitro findings have yet to be translated to in vivo trials. This study was to demonstrate the feasibility and advantages of transplanting electrically activated cells (E-Cells) to help wound healing. Primary human skin fibroblasts were activated through well defined ES and cultured with keratinocytes to generate engineered human skin (EHS), which were transplanted to nu/nu mice. The electrically activated EHS grafts were analyzed at 20 and 30 days post-grafting, showing faster wound closure, thick epidermis, vasculature, and functional basement membrane containing laminin and type IV collagen that were totally produced by the implanted human cells. Because a variety of cells can be electrically activated, E-Cells may become a new cell source and the transplantation of E-Cells may represent a new strategy in wound healing and tissue engineering. J. Cell. Physiol. 231: 1814-1821, 2016. © 2015 Wiley Periodicals, Inc.

  14. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  15. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  16. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  17. Activity, inhibition, and induction of cytochrome P450 2J2 in adult human primary cardiomyocytes.

    PubMed

    Evangelista, Eric A; Kaspera, Rüdiger; Mokadam, Nahush A; Jones, J P; Totah, Rheem A

    2013-12-01

    Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar K(m) value of 1.5 μM. The V(max) of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity. PMID:24021950

  18. Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells

    PubMed Central

    Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

    2015-01-01

    Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our

  19. Reduced DPP4 activity improves insulin signaling in primary human adipocytes.

    PubMed

    Röhrborn, Diana; Brückner, Julia; Sell, Henrike; Eckel, Jürgen

    2016-03-11

    DPP4 is a ubiquitously expressed cell surface protease which is also released to the circulation as soluble DPP4 (sDPP4). Recently, we identified DPP4 as a novel adipokine oversecreted in obesity and thus potentially linking obesity to the metabolic syndrome. Furthermore, sDPP4 impairs insulin signaling in an autocrine and paracrine fashion in different cell types. However, it is still unknown which functional role DPP4 might play in adipocytes. Therefore, primary human adipocytes were treated with a specific DPP4 siRNA. Adipocyte differentiation was not affected by DPP4 silencing. Interestingly, DPP4 reduction improved insulin responsiveness of adipocytes at the level of insulin receptor, proteinkinase B (Akt) and Akt substrate of 160 kDa. To investigate whether the observed effects could be attributed to the enzymatic activity of DPP4, human adipocytes were treated with the DPP4 inhibitors sitagliptin and saxagliptin. Our data show that insulin-stimulated activation of Akt is augmented by DPP4 inhibitor treatment. Based on our previous observation that sDPP4 induces insulin resistance in adipocytes, and that adipose DPP4 levels are higher in obese insulin-resistant patients, we now suggest that the abundance of DPP4 might be a regulator of adipocyte insulin signaling. PMID:26872429

  20. Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages.

    PubMed

    Snodgrass, Ryan G; Boß, Marcel; Zezina, Ekaterina; Weigert, Andreas; Dehne, Nathalie; Fleming, Ingrid; Brüne, Bernhard; Namgaladze, Dmitry

    2016-01-01

    Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation. PMID:26578520

  1. Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Cacicedo, Jose M; Chen, Tai C; Breton, Lionel; Ruderman, Neil B

    2012-01-01

    We investigated the effects of AMPK on H(2)O(2)-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H(2)O(2) for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21(CIP1) (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H(2)O(2)-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H(2)O(2). As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H(2)O(2) at low concentrations causes premature senescence in human keratinocytes by activating p53-p21(CIP1) signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific. PMID:22514710

  2. Human Spaceflight. Activities for the Primary Student. Aerospace Education Services Project.

    ERIC Educational Resources Information Center

    Hartsfield, John W.; Hartsfield, Kendra J.

    Since its beginning, the space program has caught the attention of young people. This space science activity booklet was designed to provide information and learning activities for students in elementary grades. It contains chapters on: (1) primitive beliefs about flight; (2) early fantasies of flight; (3) the United States human spaceflight…

  3. HIV-1 reduces Aβ-degrading enzymatic activities in primary human mononuclear phagocytes1

    PubMed Central

    Lan, Xiqian; Xu, Jiqing; Kiyota, Tomomi; Peng, Hui; Zheng, Jialin C.; Ikezu, Tsuneya

    2011-01-01

    The advent and wide introduction of antiretroviral therapy (ART) has greatly improved the survival and longevity of HIV-infected patients. Unfortunately, despite ART treatment, these patients are still afflicted with many complications including cognitive dysfunction. There is a growing body of reports indicating accelerated deposition of amyloid plaques, which are composed of amyloid-β peptide (Aβ), in HIV-infected brains. Though how HIV viral infection precipitates Aβ accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Aβ. Mononuclear phagocytes (macrophages and microglia) that are productively infected by HIV in brains play a pivotal role in Aβ degradation through the expression and execution of two endopeptidases: neprilysin (NEP) and insulin-degrading enzyme (IDE). Here we report that NEP has the dominant endopeptidase activity towards Aβ in macrophages. Further, we demonstrate that monomeric Aβ degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accompanied with great reduction of NEP endopeptidase activity, which might be due to the diminished transport of NEP to cell surface and intracellular accumulation at the endoplasmic reticulum and lysosomes. Therefore, these data suggest that malfunction of NEP in infected macrophages may contribute to acceleration of beta amyloidosis in HIV-inflicted brains and modulation of macrophages may be a potential preventative target of Aβ-related cognitive disorders in HIV-affected patients. PMID:21551363

  4. Ozone induces a proinflammatory response in primary human bronchial epithelial cells through mitogen-activated protein kinase activation without nuclear factor-κB activation.

    PubMed

    McCullough, Shaun D; Duncan, Kelly E; Swanton, Samantha M; Dailey, Lisa A; Diaz-Sanchez, David; Devlin, Robert B

    2014-09-01

    Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-mediated expression of inflammatory cytokines with activation of the canonical NF-κB pathway. In this study, we sought to characterize the O3-mediated activation of cellular signaling pathways using primary human bronchial epithelial cells obtained from a panel of donors. We demonstrate that the O3-induced expression of proinflammatory cytokines requires the activation of the epidermal growth factor receptor/MEK/ERK and MKK4/p38 mitogen-activated signaling pathways but does not appear to involve activation of canonical NF-κB signaling. In addition to providing a novel mechanistic model for the O3-mediated induction of proinflammatory cytokines, these findings highlight the importance of using primary cells over cell lines in mechanistic studies.

  5. Adhesion, cytoskeletal architecture and activation status of primary human macrophages on a diamond-like carbon coated surface.

    PubMed

    Linder, Stefan; Pinkowski, Wolfhard; Aepfelbacher, Martin

    2002-02-01

    Diamond-like carbon is a promising surface coating for biomedicinal implants like coronary stents or hip joints. Before widespread clinical use of this material, its biocompatibility has to be thoroughly assessed. Cells likely to encounter a diamond-like coated implant in the human body are cells of the monocytic lineage. Their interaction with the diamond-like carbon coated surface will probably critically influence the fate of the implant, as monocytes orchestrate inflammatory reactions and also affect osseointegration of implants. We therefore investigated adhesion, cytoarchitecture and activation status of primary human monocytes and their differentiated derivatives, macrophages, on diamond-like coated glass coverslips using immunofluorescence technique. We show that adhesion of primary monocytes to a diamond-like-coated coverslip is slightly, but not significantly, enhanced in comparison to uncoated coverslips, while the actin and microtubule cytoskeletons of mature macrophages show a normal development. The activation status of macrophages, as judged by polarization of the cell body, was not affected by growth on a diamond-like carbon surface. We conclude that diamond-like carbon shows good indications for biocompatibility to blood monocytes in vitro. It is therefore unlikely that contact with a diamond-like carbon coated surface in the human body will elicit inflammatory signals by these cells.

  6. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

    PubMed

    Välimäki, Elina; Miettinen, Juho J; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A

    2013-03-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  7. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells

    SciTech Connect

    Lu Haitian Crawford, Robert B. Kaplan, Barbara L.F. Kaminski, Norbert E.

    2011-09-15

    Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells. - Highlights: > In this study primary human and mouse B cell toxicity to TCDD was compared. > TCDD altered the expression of Blimp-1 and Pax5 in mouse but not human B cells. > TCDD markedly suppressed human B cell activation as characterized by CD80, CD86 and CD69 expression. > TCDD inhibited ERK, p38, and Akt phosphorylation in human B cells.

  8. Evaluation of Perfluoroalkyl Acid Activity Using Primary Mouse and Human Hepatocytes.

    EPA Science Inventory

    While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) in the environment. Using a transient transfection assay developed in COS-1 cells, our group h...

  9. EVALUATION OF PERFLUOROALKYL ACID ACTIVITY USING PRIMARY MOUSE AND HUMAN HEPATOCYTES

    EPA Science Inventory

    While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is know about the biological activity of other environmental perfluoroalkyl acids (pFAAs). Using a transient transfection assay developed in COS-l cells, our group has previ...

  10. Primary Cilium-Autophagy-Nrf2 (PAN) Axis Activation Commits Human Embryonic Stem Cells to a Neuroectoderm Fate.

    PubMed

    Jang, Jiwon; Wang, Yidi; Lalli, Matthew A; Guzman, Elmer; Godshalk, Sirie E; Zhou, Hongjun; Kosik, Kenneth S

    2016-04-01

    Under defined differentiation conditions, human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate, the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening, a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification, and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2, increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus, we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. PMID:27020754

  11. Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock-in (Hupki) mouse embryo fibroblasts.

    PubMed

    Kucab, Jill E; Phillips, David H; Arlt, Volker M

    2012-04-01

    Approximately 50% of human tumors have a mutation in TP53. The pattern and spectra of TP53 mutations often differ between cancer types, perhaps due to different etiological factors. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay is useful for studying mutagenesis in the human TP53 gene by environmental carcinogens. Prior to initiating an immortalization assay, carcinogen treatment conditions must be optimized, which can require a large number of cells. As primary HUF cultures senesce within 2 weeks, restricting their use, we investigated whether immortalized HUFs retaining wild-type TP53 can be surrogates for primary HUFs in initial treatment optimization. DNA damage by eight compounds found in diesel exhaust, benzo[a]pyrene, 3-nitrobenzanthrone, 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, 6-nitrochrysene, and 3-nitrofluorene, was assessed by (32) P-postlabeling and the alkaline comet assay in primary HUFs and in an immortal HUF cell line J201. For most compounds, higher levels of DNA adducts accumulated in J201 cells than in primary HUFs. This difference was not reflected in the comet assay or by cell viability changes. Experiments in three additional immortal HUF cell lines (AAI49, U56, and E2-143) confirmed strong differences in DNA adduct levels compared with primary HUFs. However, these did not correlate with the protein expression of Nqo1 or Nat1/2, or with gene expression of Cyp1a1 or Cyp1b1. Our results show that using immortal HUFs as surrogates for primary HUFs in genotoxicity screening has limitations and that DNA adduct formation is the best measure of genotoxicity of the nitro-polycyclic aromatic hydrocarbons tested in HUFs. PMID:22351035

  12. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  13. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human

    PubMed Central

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  14. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma.

  15. CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells

    PubMed Central

    Steiner, Laurie A.; Schulz, Vincent; Makismova, Yelena; Lezon-Geyda, Kimberly; Gallagher, Patrick G.

    2016-01-01

    Background CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Results Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. Conclusion These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis. PMID:27219007

  16. Distinct activation of primary human BDCA1(+) dendritic cells upon interaction with stressed or infected β cells.

    PubMed

    Schulte, B M; Kers-Rebel, E D; Bottino, R; Piganelli, J D; Galama, J M D; Engelse, M A; de Koning, E J P; Adema, G J

    2016-06-01

    Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic β cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf β cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-α/β responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in β cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect β cells and required RNA within virally infected cells. DCs encountering enterovirus-infected β cells, but not those incubated with mock-infected or stressed β cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-γ in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed β cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected β cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk.

  17. Low Thymic Activity and Dendritic Cell Numbers Are Associated with the Immune Response to Primary Viral Infection in Elderly Humans.

    PubMed

    Schulz, Axel Ronald; Mälzer, Julia Nora; Domingo, Cristina; Jürchott, Karsten; Grützkau, Andreas; Babel, Nina; Nienen, Mikalai; Jelinek, Tomas; Niedrig, Matthias; Thiel, Andreas

    2015-11-15

    Immunological competence declines progressively with age, resulting in increased susceptibility of the elderly to infection and impaired responses to vaccines. Underlying mechanisms remain largely obscure as they have been related to complex, individual systemic immune properties that are challenging to investigate. In this study, we explored age-related changes in human immunity during a primary virus infection experimentally induced by immunization with live-attenuated yellow fever (YF) vaccine. Applying detailed serology, advanced FACS analysis, and systems biology, we discovered that aged subjects developed fewer neutralizing Abs, mounted diminished YF-specific CD8(+) T cell responses, and showed quantitatively and qualitatively altered YF-specific CD4(+) T cell immunity. Among numerous immune signatures, low in vivo numbers of naive CD4(+) recent thymic emigrants and peripheral dendritic cells correlated well with reduced acute responsiveness and altered long-term persistence of human cellular immunity to YF vaccination. Hence, we reveal in this article that essential elements of immune responses such as recent thymic emigrants and dendritic cells strongly relate to productive immunity in the elderly, providing a conceivable explanation for diminished responsiveness to vaccination with neoantigens and infection with de novo pathogens in the aged population. PMID:26459351

  18. Camphor Induces Proliferative and Anti-senescence Activities in Human Primary Dermal Fibroblasts and Inhibits UV-Induced Wrinkle Formation in Mouse Skin.

    PubMed

    Tran, Thao Anh; Ho, Manh Tin; Song, Yeon Woo; Cho, Moonjae; Cho, Somi Kim

    2015-12-01

    Camphor ((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one), a bicyclic monoterpene, is one of the major constituents of essential oils from various herbs such as rosemary, lavender, and sage. In this study, we investigated the beneficial effects of camphor as a botanical ingredient in cosmetics. Camphor induced the proliferation of human primary dermal fibroblasts in a dose-dependent manner via the PI3K/AKT and ERK signaling pathways. Camphor attenuated the elevation of senescence associated with β-galactosidase (SA-β-gal) activity. Elastase activity decreased, while the total amount of collagen increased, in a dose- and time-dependent manner in human primary dermal fibroblasts treated with camphor. Camphor induced the expression of collagen IA, collagen IIIA, collagen IVA, and elastin in human primary dermal fibroblasts. In addition, posttreatment with 26 and 52 mM camphor for 2 weeks led to a significant reduction in the expression of MMP1 but increases in the expression of collagen IA, IIIA, and elastin in mouse skin exposed to UV for 4 weeks. These posttreatments also reduced the depths of the epidermis and subcutaneous fat layer in UV-exposed mouse skin. Taken together, these findings suggest camphor to be a potent wound healing and antiwrinkle agent with considerable potential for use in cosmeceuticals. PMID:26458283

  19. LACBWR primary shield activation analysis

    SciTech Connect

    Nelson, L.L.; Lahti, G.P.; Johnson, W.J.

    1996-11-01

    Nuclear power plants in the US are required to estimate the costs of decommissioning to ensure that adequate funds are accumulated during the useful life of the plant. A major component of the decommissioning cost is the disposal of radioactive material, including material near the reactor created by neutron activation. An accurate assessment of the residual radioactivity in the reactor`s primary shield is necessary to determine this portion of the decommissioning demolition and disposal cost. This paper describes the efforts used to determine the activation levels remaining in the primary shield of the LaCrosse boiling water reactor (LACBWR), owned and operated by Dairyland Power Cooperative.

  20. Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

    PubMed Central

    VanCott, T C; Mascola, J R; Kaminski, R W; Kalyanaraman, V; Hallberg, P L; Burnett, P R; Ulrich, J T; Rechtman, D J; Birx, D L

    1997-01-01

    Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates. PMID:9151820

  1. Human Immunodeficiency Virus (HIV) Primary Infection

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Human Immunodeficiency Virus (HIV) Primary Infection Information for adults A A ... weeks following exposure to HIV (the human immunodeficiency virus). Chronic infection with this virus can cause AIDS ( ...

  2. Classification of Sasang constitutional body types using immunostimulatory activities of constitution-specific herbal extracts in human primary immune cells.

    PubMed

    Choi, Jae-Ho; Chung, Mi Ja; Oh, Deog-Hwan

    2012-09-01

    Sasang is a Korean traditional constitutional medicine in which individuals are classified into four constitutional types: Taeyangin, Soeumin, Taeumin, and Soyangin. However, the classification of each constitution is empirical and not scientific, and so it is difficult to consistently classify patients into these four constitutional types. Acanthopanacis cortex (AC; Taeyangin), Rehmanniae radix (RR; Soyangin), Ephedra herba (EH; Taeumin), and White ginseng (WG; Soeumin) are herbal medicines that are used to treat and prevent diseases associated with these four constitutions. Therefore, AC, RR, EH, and WG extracts (ACE, RRE, EHE, and WGE) were used as reference extracts to determine constitutional differences in the immunostimulatory activities of primary immune cells isolated from the blood of 15 volunteers. Cellular proliferation, nitric oxide (NO), tumor necrosis factor-α (TNF-α) protein production, TNF-α and interleukin-6, and inducible NO synthase mRNA expression were measured as the immunostimulatory parameters. The increase in cell proliferation of V2, V3, V8, V11, and V12 was highest in EHE-treated lymphocytes among the ACE-, RRE-, EHE- or WGE-treated lymphocytes, and increase in cellular proliferation of V4, V7, V13, V14, and V15 in RRE-treated lymphocytes was significantly better than the other three medicines. The cell proliferation of V1, V5, V6, V9, and V10 responded best to WGE among the four extracts. Results of cell proliferation and other immunostimulatory parameters showed similar trends in regard to individual differences. These extracts may be useful as reference extracts in the development of a rapid and dependable individual lymphocyte- or macrophage-based assay that aids in the Sasang constitutional classification. PMID:22870991

  3. PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

    PubMed

    Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

    2016-04-01

    Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases.

  4. Expression of Phospholipases A2 in Primary Human Lung Macrophages. Role of Cytosolic Phospholipase A2–α in Arachidonic Acid Release and Platelet Activating Factor Synthesis

    PubMed Central

    Giannattasio, Giorgio; Lai, Ying; Granata, Francescopaolo; Mounier, Carine M.; Nallan, Laxman; Oslund, Rob; Leslie, Christina C.; Marone, Gianni; Lambeau, Gérard; Gelb, Michael H.; Triggiani, Massimo

    2009-01-01

    Summary Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA2) and secreted phospholipases A2 (sPLA2) to the generation of lipid mediators in human macrophages is unclear. We investigated the expression and role of different PLA2s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA2 (iPLA2). Two structurally-divergent inhibitors of group IV cPLA2 completely block arachidonic acid release by macrophages in response to non-physiological (Ca2+ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA2s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA2s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA2 inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA2-alpha, iPLA2 and several sPLA2s. Cytosolic PLA2-alpha is the major enzyme responsible for lipid mediator production in human macrophages. PMID:19130898

  5. Dietary Phenolic Compounds Interfere with the Fate of Hydrogen Peroxide in Human Adipose Tissue but Do Not Directly Inhibit Primary Amine Oxidase Activity

    PubMed Central

    Carpéné, Christian; Hasnaoui, Mounia; Balogh, Balázs; Matyus, Peter; Fernández-Quintela, Alfredo; Rodríguez, Víctor; Mercader, Josep; Portillo, Maria P.

    2016-01-01

    Resveratrol has been reported to inhibit monoamine oxidases (MAO). Many substrates or inhibitors of neuronal MAO interact also with other amine oxidases (AO) in peripheral organs, such as semicarbazide-sensitive AO (SSAO), known as primary amine oxidase, absent in neurones, but abundant in adipocytes. We asked whether phenolic compounds (resveratrol, pterostilbene, quercetin, and caffeic acid) behave as MAO and SSAO inhibitors. AO activity was determined in human adipose tissue. Computational docking and glucose uptake assays were performed in 3D models of human AO proteins and in adipocytes, respectively. Phenolic compounds fully inhibited the fluorescent detection of H2O2 generated during MAO and SSAO activation by tyramine and benzylamine. They also quenched H2O2-induced fluorescence in absence of biological material and were unable to abolish the oxidation of radiolabelled tyramine and benzylamine. Thus, phenolic compounds hampered H2O2 detection but did not block AO activity. Only resveratrol and quercetin partially impaired MAO-dependent [14C]-tyramine oxidation and behaved as MAO inhibitors. Phenolic compounds counteracted the H2O2-dependent benzylamine-stimulated glucose transport. This indicates that various phenolic compounds block downstream effects of H2O2 produced by biogenic or exogenous amine oxidation without directly inhibiting AO. Phenolic compounds remain of interest regarding their capacity to limit oxidative stress rather than inhibiting AO. PMID:26881018

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

    PubMed Central

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

    2016-01-01

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

  7. Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A; Breton, Lionel; Ruderman, Neil B

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by

  8. Whole venom of Loxosceles similis activates caspases-3, -6, -7, and -9 in human primary skin fibroblasts.

    PubMed

    Dantas, Arthur Estanislau; Horta, Carolina Campolina Rebello; Martins, Thais M M; do Carmo, Anderson Oliveira; Mendes, Bárbara Bruna Ribeiro de Oliveira; Goes, Alfredo M; Kalapothakis, Evanguedes; Gomes, Dawidson A

    2014-06-01

    Spiders of the Loxosceles genus represent a risk to human health due to the systemic and necrotic effects of their bites. The main symptoms of these bites vary from dermonecrosis, observed in the majority of cases, to occasional systemic hemolysis and coagulopathy. Although the systemic effects are well characterized, the mechanisms of cell death triggered by the venom of these spiders are poorly characterized. In this study, we investigated the cell death mechanisms induced by the whole venom of the spider Loxosceles similis in human skin fibroblasts. Our results show that the venom initiates an apoptotic process and a caspase cascade involving the initiator caspase-9 and the effector caspases-3, -6, and -7.

  9. CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    PubMed Central

    Tiede, Imke; Fritz, Gerhard; Strand, Susanne; Poppe, Daniela; Dvorsky, Radovan; Strand, Dennis; Lehr, Hans Anton; Wirtz, Stefan; Becker, Christoph; Atreya, Raja; Mudter, Jonas; Hildner, Kai; Bartsch, Brigitte; Holtmann, Martin; Blumberg, Richard; Walczak, Henning; Iven, Heiko; Galle, Peter R.; Ahmadian, Mohammad Reza; Neurath, Markus F.

    2003-01-01

    Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-κB, and bcl-xL was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation. PMID:12697733

  10. Illumination from light-emitting diodes (LEDs) disrupts pathological cytokines expression and activates relevant signal pathways in primary human retinal pigment epithelial cells.

    PubMed

    Shen, Ye; Xie, Chen; Gu, Yangshun; Li, Xiuyi; Tong, Jianping

    2016-04-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in the aged people. The latest systemic review of epidemiological investigations revealed that excessive light exposure increases the risk of AMD. With the drastically increasing use of high-energy light-emitting diodes (LEDs) light in our domestic environment nowadays, it is supposed to pose a potential oxidative threat to ocular health. Retinal pigment epithelium (RPE) is the major ocular source of pathological cytokines, which regulate local inflammation and angiogenesis. We hypothesized that high-energy LED light might disrupt the pathological cytokine expression of retinal pigment epithelium (RPE), contributing to the pathogenesis of AMD. Primary human RPE cells were isolated from eyecups of normal eye donors and seeded into plate wells for growing to confluence. Two widely used multichromatic white light-emitting diodes (LEDs) with correlated color temperatures (CCTs) of 2954 and 7378 K were used in this experiment. The confluent primary RPE cells were under white LEDs light exposure until 24 h. VEGF-A, IL-6, IL-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs), Akt, Janus kinase (JAK)2 and Nuclear factor (NF)-κB signal pathways after LEDs illumination were evaluated by western blotting analysis. The level of reactive oxygen species (ROS) using chloromethyl- 2',7'-dichlorodihydrofluorescein diacetate. Inhibitors of relevant signal pathways and anti-oxidants were added to the primary RPE cells before LEDs illumination to evaluate their biological functions. We found that 7378 K light, but not 2954 K upregulated the VEGF-A, IL-6, IL-8 and downregulated MCP-1 proteins and mRNAs levels in a time-dependent manner. In parallel, initial activation of MAPKs and NF-κB signal pathways were also observed after 7378 K light exposure. Mechanistically, antioxidants for eliminating reactive oxygen

  11. Primary Planets and Elementary Moons: Activities for Primary Students.

    ERIC Educational Resources Information Center

    Winrich, Ralph A.; Samuel, Mary

    This booklet was designed to supplement existing classroom studies on the subject of the solar system at the primary level. Science and mathematics activities for studying moons, planets, and space craft are presented. (PR)

  12. Isolation of Microarray-Quality RNA from Primary Human Cells after Intracellular Immunostaining and Fluorescence-Activated Cell Sorting

    PubMed Central

    Iglesias-Ussel, Maria; Marchionni, Luigi; Romerio, Fabio

    2013-01-01

    Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that – although the RNA isolated from these samples presented some degradation – it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis. PMID:23434645

  13. Young Scientists Explore the Human Body. Book 11 Primary Level.

    ERIC Educational Resources Information Center

    Penn, Linda

    Designed to present interesting facts about science and to heighten the curiosity of primary age students, this book contains activities about the natural world and numerous black and white illustrations. The activities specifically focus on the human body and encourage a positive self-concept. The theme of the first section is air--the breath of…

  14. Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells

    PubMed Central

    2011-01-01

    Background Numerous reports have identified therapeutic roles for plants and their extracts and constituents. The aim of this study was to assess the efficacies of three plant extracts for their potential antioxidant and anti-inflammatory activity in primary human skin fibroblasts. Methods Aqueous extracts and formulations of white tea, witch hazel and rose were subjected to assays to measure anti-collagenase, anti-elastase, trolox equivalent and catalase activities. Skin fibroblast cells were employed to determine the effect of each extract/formulation on IL-8 release induced by the addition of hydrogen peroxide. Microscopic examination along with Neutral Red viability testing was employed to ascertain the effects of hydrogen peroxide directly on cell viability. Results Considerable anti-collagenase, anti-elastase, and antioxidant activities were measured for all extracts apart from the witch hazel distillate which showed no activity in the collagenase assay or in the trolox equivalence assay. All of the extracts and products tested elicited a significant decrease in the amount of IL-8 produced by fibroblast cells compared to the control (p < 0.05). None of the test samples exhibited catalase activity or had a significant effect on the spontaneous secretion of IL-8 in the control cells which was further corroborated with the microscopy results and the Neutral Red viability test. Conclusions These data show that the extracts and products tested have a protective effect on fibroblast cells against hydrogen peroxide induced damage. This approach provides a potential method to evaluate the claims made for plant extracts and the products in which these extracts are found. PMID:21995704

  15. HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

    PubMed Central

    Granato, M.; Zompetta, C.; Vescarelli, E.; Rizzello, C.; Cardi, A.; Valia, S.; Antonelli, G.; Marchese, C.; Torrisi, M. R.; Faggioni, A.; Cirone, M.

    2016-01-01

    Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis. PMID:27476557

  16. Effect of ozone on platelet-activating factor production in phorbol-differentiated HL60 cells, a human bronchial epithelial cell line (BEAS S6), and primary human bronchial epithelial cells.

    PubMed

    Samet, J M; Noah, T L; Devlin, R B; Yankaskas, J R; McKinnon, K; Dailey, L A; Friedman, M

    1992-11-01

    Platelet-activating factor (PAF) is a phospholipid with a wide spectrum of pro-inflammatory properties. In the lung, PAF induces airway hyperresponsiveness, neutrophil sequestration, and increased vascular permeability. The alveolar macrophage and the bronchial epithelium are tissues that are exposed to inhaled ozone (O3). We studied the effect of an in vitro O3 exposure on PAF production in a macrophage-like HL60 human cell line (dHL60), a human bronchial epithelial cell line (BEAS S6), and also in primary human bronchial epithelial cells. PAF was quantified by thin-layer chromatographic separation of lipid extracts from cells radiolabeled with [3H]lysoPAF and by radioimmunoassay. In vitro exposure of dHL60 cells to 0.05 to 1.0 ppm O3 for 15 to 120 min was found to significantly increase PAF levels above air control values at all exposure levels and time points (average increase of 92%). Similarly, BEAS S6 cells grown on collagen-coated filter supports and exposed to 0.05 to 1.0 ppm O3 for 60 min released an average increase in PAF of 626% above control values. Primary human bronchial epithelial cells also demonstrated significant increases in [3H]PAF release (average increase of 289% after exposure to 1.0 ppm O3 for 60 min) compared with paired air controls. These findings suggest that some of the effects of O3 inhalation may be mediated by PAF.

  17. [Primary human demodicosis. A disease sui generis].

    PubMed

    Hsu, C-K; Zink, A; Wei, K-J; Dzika, E; Plewig, G; Chen, W

    2015-03-01

    Human Demodex mites (Demodex folliculorum and Demodex brevis) are unique in that they are an obligate human ectoparasite that can inhabit the pilosebaceous unit lifelong without causing obvious host immune response in most cases. The mode of symbiosis between humans and human Demodex mites is unclear, while the pathogenicity of human Demodex mites in many inflammatory skin diseases is now better understood. Primary human demodicosis is a skin disease sui generis not associated with local or systemic immunosuppression. Diagnosis is often underestimated and differentiation from folliculitis, papulopustular rosacea and perioral dermatitis is not always straightforward. Dependent on the morphology and degree of inflammation, the clinical manifestations can be classified into spinulate, papulopustular, nodulocystic, crustic and fulminant demodicosis. Therapy success can be achieved only with acaricides/arachidicides. The effective doses, optimal regimen and antimicrobial resistance remain to be determined. PMID:25744530

  18. The multisensory function of the human primary visual cortex.

    PubMed

    Murray, Micah M; Thelen, Antonia; Thut, Gregor; Romei, Vincenzo; Martuzzi, Roberto; Matusz, Pawel J

    2016-03-01

    It has been nearly 10 years since Ghazanfar and Schroeder (2006) proposed that the neocortex is essentially multisensory in nature. However, it is only recently that sufficient and hard evidence that supports this proposal has accrued. We review evidence that activity within the human primary visual cortex plays an active role in multisensory processes and directly impacts behavioural outcome. This evidence emerges from a full pallet of human brain imaging and brain mapping methods with which multisensory processes are quantitatively assessed by taking advantage of particular strengths of each technique as well as advances in signal analyses. Several general conclusions about multisensory processes in primary visual cortex of humans are supported relatively solidly. First, haemodynamic methods (fMRI/PET) show that there is both convergence and integration occurring within primary visual cortex. Second, primary visual cortex is involved in multisensory processes during early post-stimulus stages (as revealed by EEG/ERP/ERFs as well as TMS). Third, multisensory effects in primary visual cortex directly impact behaviour and perception, as revealed by correlational (EEG/ERPs/ERFs) as well as more causal measures (TMS/tACS). While the provocative claim of Ghazanfar and Schroeder (2006) that the whole of neocortex is multisensory in function has yet to be demonstrated, this can now be considered established in the case of the human primary visual cortex. PMID:26275965

  19. Essential Oil from Berries of Lebanese Juniperus excelsa M. Bieb Displays Similar Antibacterial Activity to Chlorhexidine but Higher Cytocompatibility with Human Oral Primary Cells.

    PubMed

    Azzimonti, Barbara; Cochis, Andrea; Beyrouthy, Marc El; Iriti, Marcello; Uberti, Francesca; Sorrentino, Rita; Landini, Manuela Miriam; Rimondini, Lia; Varoni, Elena Maria

    2015-05-21

    Chlorhexidine (CHX), one of the most effective drugs administered for periodontal treatment, presents collateral effects including toxicity when used for prolonged periods; here, we have evaluated the bactericidal potency and the cytocompatibility of Juniperus excelsa M. Bieb essential oil (EO) in comparison with 0.05% CHX. The EO was extracted from berries by hydrodistillation and components identified by gas chromatography and mass spectrometry. Bacterial inhibition halo analysis, quantitative cell viability 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenyl amino) carbonyl]-2H-tetrazolium hydroxide assay (XTT), and colony forming unit (CFU) count were evaluated against the two biofilm formers Aggregatibacter actinomycetemcomitans and Streptococcus mutans. Finally, cytocompatibility was assessed with human primary gingival fibroblasts (HGF) and mucosal keratinocytes (HK). The resulting EO was mainly composed of monoterpene hydrocarbons and oxygenated monoterpenes. An inhibition halo test demonstrated that both bacteria were sensitive to the EO; XTT analysis and CFU counts confirmed that 10-fold-diluted EO determined a statistically significant (p < 0.05) reduction in bacteria count and viability towards both biofilm and planktonic forms in a comparable manner to those obtained with CHX. Moreover, EO displayed higher cytocompatibility than CHX (p < 0.05). In conclusion, EO exhibited bactericidal activity similar to CHX, but a superior cytocompatibility, making it a promising antiseptic alternative to CHX.

  20. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    PubMed

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

  1. Human Appropriation of Net Primary Production - Can Earth Keep Up?

    NASA Technical Reports Server (NTRS)

    Imhoff, Marc L.

    2006-01-01

    The amount of Earth's vegetation or net primary production required to support human activities is powerful measure of aggregate human impacts on the biosphere. Biophysical models applied to consumption statistics were used to estimate the annual amount of net primary production in the form of elemental carbon required for food, fibre, and fuel-wood by the global population. The calculations were then compared to satellite-based estimates of Earth's average net primary production to produce a geographically explicit balance sheet of net primary production "supply" and "demand". Humans consume 20% of Earth's net primary production (11.5 petagrams carbon) annually and this percentage varies regionally from 6% (South America) to over 70% (Europe and Asia), and locally from near 0% (central Australia) to over 30,000% (New York City, USA). The uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations are vulnerable to climate change and suggest policy options for slowing future growth of NPP demand.

  2. Primary Bioassay of Human Myeloma Stem Cells

    PubMed Central

    Hamburger, Anne; Salmon, Sydney E.

    1977-01-01

    The ability to clone primary tumors in soft agar has proven useful in the study of the kinetics and biological properties of tumor stem cells. We report the development of an in vitro assay which permits formation of colonies of human monoclonal plasma cells in soft agar. Colony growth has been observed from bone marrow aspirates from 75% of the 70 patients with multiple myeloma or related monoclonal disorders studied. Growth was induced with either 0.02 ml of human type O erythrocytes or 0.25 ml of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. 5-500 colonies appeared after 2-3 wk in culture yielding a plating efficiency of 0.001-0.1%. The number of myeloma colonies was proportional to the number of cells plated between concentrations of 105-106 and back-extrapolated through zero, suggesting that colonies were clones derived from single myeloma stem cells. Morphological, histochemical, and functional criteria showed the colonies to consist of immature plasmablasts and mature plasma cells. 60-80% of cells picked from colonies contained intracytoplasmic monoclonal immunoglobulin. Colony growth was most easily achieved from the bone marrow cells of untreated patients or those in relapse. Only 50% of bone marrow samples from patients in remission were successfully cultured. Tritiated thymidine suicide studies provided evidence that for most myeloma patients, a very high proportion of myeloma colony-forming cells was actively in transit through the cell cycle. Velocity sedimentation at 1 g showed myeloma stem cells sedimented in a broad band with a peak at 13 mm/h. Antibody to granulocyte colony-stimulating factor did not reduce the number or size of the colonies. Increased numbers of myeloma colonies were seen when the marrow was depleted of colony-stimulating factor elaborating adherent cells before plating. This bioassay should prove useful in studying the in vitro biological behavior of certain bone marrow-derived (B

  3. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    SciTech Connect

    Coppel, R.L.; McNeilage, L.J.; Surh, C.D.; Van De Water, J.; Spithill, T.W.; Whittingham, S.; Gershwin, M.E. )

    1988-10-01

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a M{sub r} 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

  4. The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

    PubMed

    Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

    2016-05-01

    Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps.

  5. Health Activities for Primary School Students.

    ERIC Educational Resources Information Center

    Peace Corps, Washington, DC. Information Collection and Exchange Div.

    This manual targets new and second-year Peace Corps volunteers, presenting health lessons and activities for primary school students in Thailand. Each section of the manual outlines basic technical information about the topic, contains several detailed lesson plans, and lists quick activities that can be carried out at schools. Songs and recipes…

  6. The VST active primary mirror support system

    NASA Astrophysics Data System (ADS)

    Schipani, Pietro; Capaccioli, Massimo; D'Orsi, Sergio; Ferragina, Luigi; Marty, Laurent; Molfese, Cesare; Perrotta, Francesco; De Paris, Giacinto; Fierro, Davide; Tomelleri, Raffaele; Rossettini, Pierfrancesco; Perina, Francesco; Recchia, Stefano; Magrin, Demetrio

    2010-07-01

    The 2.6-m primary mirror of the VST telescope is equipped with an active optics system in order to correct low-order aberrations, constantly monitoring the optical quality of the image and controlling the relative position and the shape of the optical elements. Periodically an image analyser calculates the deviation of the image from the best quality. VST is equipped with both a Shack-Hartmann in the probe system and a curvature sensor embedded in the OmegaCAM instrument. The telescope control software decomposes the deviation into single optical contributions and calculates the force correction that each active element has to perform to achieve the optimal quality. The set of correction forces, one for each axial actuator, is computed by the telescope central computer and transmitted to the local control unit of the primary mirror system for execution. The most important element of the VST active optics is the primary mirror, with its active support system located within the primary mirror cell structure. The primary mirror support system is composed by an axial and a lateral independent systems and includes an earthquake safety system. The system is described and the results of the qualification test campaign are discussed.

  7. RANK and RANK ligand expression in primary human osteosarcoma.

    PubMed

    Branstetter, Daniel; Rohrbach, Kathy; Huang, Li-Ya; Soriano, Rosalia; Tometsko, Mark; Blake, Michelle; Jacob, Allison P; Dougall, William C

    2015-09-01

    Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential mediator of osteoclast formation, function and survival. In patients with solid tumor metastasis to the bone, targeting the bone microenvironment by inhibition of RANKL using denosumab, a fully human monoclonal antibody (mAb) specific to RANKL, has been demonstrated to prevent tumor-induced osteolysis and subsequent skeletal complications. Recently, a prominent functional role for the RANKL pathway has emerged in the primary bone tumor giant cell tumor of bone (GCTB). Expression of both RANKL and RANK is extremely high in GCTB tumors and denosumab treatment was associated with tumor regression and reduced tumor-associated bone lysis in GCTB patients. In order to address the potential role of the RANKL pathway in another primary bone tumor, this study assessed human RANKL and RANK expression in human primary osteosarcoma (OS) using specific mAbs, validated and optimized for immunohistochemistry (IHC) or flow cytometry. Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79) of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy

  8. Immortalization of primary human smooth muscle cells.

    PubMed Central

    Perez-Reyes, N; Halbert, C L; Smith, P P; Benditt, E P; McDougall, J K

    1992-01-01

    Primary human aortic and myometrial smooth muscle cells (SMCs) were immortalized using an amphotropic recombinant retroviral construct containing the E6 and E7 open reading frames (ORFs) of human papillomavirus type 16. The SMCs expressing the E6/E7 ORFs have considerably elevated growth rates when compared with nonimmortalized control cells and show no signs of senescence with long-term passage. The first SMC line derived in this study has been maintained in continuous tissue culture for greater than 1 year (greater than 180 population doublings). The immortalized SMCs have decreased cell size and decreased content of muscle-specific alpha-actin filaments as determined by indirect immunofluorescence. Southern blot analysis has demonstrated the stable integration of the E6/E7 ORFs in the retrovirally infected cells, and radioimmunoprecipitation has confirmed the continued expression of the E6 and E7 genes. Cytogenetic studies of the SMC lines have revealed essentially diploid populations except for the myometrial clonal line, which became aneuploid at late passage (greater than 125 doublings). These cell lines were not tumorigenic in nude mice. Images PMID:1311088

  9. Energy Activities for the Primary Classroom. Revised.

    ERIC Educational Resources Information Center

    Tierney, Blue, Comp.

    An energy education program at the primary level should help students to understand the nature and importance of energy, consider different energy sources, learn about energy conservation, prepare for energy related careers, and become energy conscious in other career fields. The activities charts, readings, and experiments provided in this…

  10. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

    PubMed Central

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A.; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  11. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

    PubMed

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

    2016-07-11

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

  12. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

    PubMed

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  13. Exploring primary care activities in ACT teams.

    PubMed

    Vanderlip, Erik R; Williams, Nancy A; Fiedorowicz, Jess G; Katon, Wayne

    2014-05-01

    People with serious mental illness often receive inadequate primary and preventive care services. Federal healthcare reform endorses team-based care that provides high quality primary and preventive care to at risk populations. Assertive community treatment (ACT) teams offer a proven, standardized treatment approach effective in improving mental health outcomes for the seriously mentally ill. Much is known about the effectiveness of ACT teams in improving mental health outcomes, but the degree to which medical care needs are addressed is not established. The purpose of this study was to explore the extent to which ACT teams address the physical health of the population they serve. ACT team leaders were invited to complete an anonymous, web-based survey to explore attitudes and activities involving the primary care needs of their clients. Information was collected regarding the use of health screening tools, physical health assessments, provision of medical care and collaboration with primary care systems. Data was analyzed from 127 team leaders across the country, of which 55 completed the entire survey. Nearly every ACT team leader believed ACT teams have a role in identifying and managing the medical co-morbidities of their clientele. ACT teams report participation in many primary care activities. ACT teams are providing a substantial amount of primary and preventive services to their population. The survey suggests standardization of physical health identification, management or referral processes within ACT teams may result in improved quality of medical care. ACT teams are in a unique position to improve physical health care by virtue of having medically trained staff and frequent, close contact with their clients.

  14. Global patterns in human consumption of net primary production

    NASA Astrophysics Data System (ADS)

    Imhoff, Marc L.; Bounoua, Lahouari; Ricketts, Taylor; Loucks, Colby; Harriss, Robert; Lawrence, William T.

    2004-06-01

    The human population and its consumption profoundly affect the Earth's ecosystems. A particularly compelling measure of humanity's cumulative impact is the fraction of the planet's net primary production that we appropriate for our own use. Net primary production-the net amount of solar energy converted to plant organic matter through photosynthesis-can be measured in units of elemental carbon and represents the primary food energy source for the world's ecosystems. Human appropriation of net primary production, apart from leaving less for other species to use, alters the composition of the atmosphere, levels of biodiversity, energy flows within food webs and the provision of important ecosystem services. Here we present a global map showing the amount of net primary production required by humans and compare it to the total amount generated on the landscape. We then derive a spatial balance sheet of net primary production `supply' and `demand' for the world. We show that human appropriation of net primary production varies spatially from almost zero to many times the local primary production. These analyses reveal the uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations depend on net primary production `imports' and suggest policy options for slowing future growth of human appropriation of net primary production.

  15. Global patterns in human consumption of net primary production.

    PubMed

    Imhoff, Marc L; Bounoua, Lahouari; Ricketts, Taylor; Loucks, Colby; Harriss, Robert; Lawrence, William T

    2004-06-24

    The human population and its consumption profoundly affect the Earth's ecosystems. A particularly compelling measure of humanity's cumulative impact is the fraction of the planet's net primary production that we appropriate for our own use. Net primary production--the net amount of solar energy converted to plant organic matter through photosynthesis--can be measured in units of elemental carbon and represents the primary food energy source for the world's ecosystems. Human appropriation of net primary production, apart from leaving less for other species to use, alters the composition of the atmosphere, levels of biodiversity, energy flows within food webs and the provision of important ecosystem services. Here we present a global map showing the amount of net primary production required by humans and compare it to the total amount generated on the landscape. We then derive a spatial balance sheet of net primary production 'supply' and 'demand' for the world. We show that human appropriation of net primary production varies spatially from almost zero to many times the local primary production. These analyses reveal the uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations depend on net primary production 'imports' and suggest policy options for slowing future growth of human appropriation of net primary production.

  16. Global Patterns in Human Consumption of Net Primary Production

    NASA Technical Reports Server (NTRS)

    Imhoff, Marc L.; Bounoua, Lahouari; Ricketts, Taylor; Loucks, Colby; Harriss, Robert; Lawrence William T.

    2004-01-01

    The human population and its consumption profoundly affect the Earth's ecosystems. A particularly compelling measure of humanity's cumulative impact is the fraction of the planet's net primary production that we appropriate for our Net primary production-the net amount of solar energy converted to plant organic matter through photosynthesis-can be measured in units of elemental carbon and represents the primary food energy source for the world's ecosystems. Human appropriation of net primary production, apart from leaving less for other species to use, alters the composition of the atmosphere, levels of biodiversity, flows within food webs and the provision of important primary production required by humans and compare it to the total amount generated on the landscape. We then derive a spatial ba!mce sheet of net primary production supply and demand for the world. We show that human appropriation of net primary production varies spatially from almost zero to many times the local primary production. These analyses reveal the uneven footprint of human consumption and related environmental impacts, indicate the degree to which human populations depend on net primary production "imports" and suggest policy options for slowing future growth of human appropriation of net primary production.

  17. Activity of 8F4, a T cell receptor-like anti-PR1/HLA-A2 antibody, against primary human AML in vivo

    PubMed Central

    Sergeeva, Anna; He, Hong; Ruisaard, Kathryn; St. John, Lisa; Alatrash, Gheath; Clise-Dwyer, Karen; Li, Dan; Patenia, Rebecca; Hong, Richard; Sukhumalchandra, Pariya; You, M. James; Gagea, Mihai; Ma, Qing; Molldrem, Jeffrey J.

    2016-01-01

    The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high affinity T cell receptor-like murine monoclonal antibody, 8F4, which binds to the PR1/HLA-A2 complex, mediates lysis of AML, and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, co-incubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML. PMID:27055866

  18. The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.

    PubMed

    Oppici, Elisa; Montioli, Riccardo; Dindo, Mirco; Maccari, Laura; Porcari, Valentina; Lorenzetto, Antonio; Chellini, Sara; Voltattorni, Carla Borri; Cellini, Barbara

    2015-10-16

    The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands. PMID:26161999

  19. Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes.

    PubMed

    Sharma, Vyom; Singh, Suman K; Anderson, Diana; Tobin, Desmond J; Dhawan, Alok

    2011-05-01

    Zinc oxide (ZnO) nanoparticles are widely used in cosmetics and sunscreens. Human epidermal keratinocytes may serve as the first portal of entry for these nanoparticles either directly through topically applied cosmetics or indirectly through any breaches in the skin integrity. Therefore, the objective of the present study was to assess the biological interactions of ZnO nanoparticles in primary human epidermal keratinocytes (HEK) as they are the most abundant cell type in the human epidermis. Cellular uptake of nanoparticles was investigated by scanning electron microscopy using back scattered electrons imaging as well as transmission electron microscopy. The electron microscopy revealed the internalization of ZnO nanoparticles in primary HEK after 6 h exposure at 14 microg/ml concentration. ZnO nanoparticles exhibited a time (6-24 h) as well as concentration (8-20 microg/ml) dependent inhibition of mitochondrial activity as evident by the MTT assay. A significant (p < 0.05) induction in DNA damage was observed in cells exposed to ZnO nanoparticles for 6 h at 8 and 14 microg/ml concentrations compared to control as evident in the Comet assay. This is the first study providing information on biological interactions of ZnO nanoparticles with primary human epidermal keratinocytes. Our findings demonstrate that ZnO nanoparticles are internalized by the human epidermal keratinocytes and elicit a cytotoxic and genotoxic response. Therefore, caution should be taken while using consumer products containing nanoparticles as any perturbation in the skin barrier could expose the underlying cells to nanoparticles.

  20. Purification and primary structure determination of human lysosomal dipeptidase.

    PubMed

    Dolenc, Iztok; Mihelic, Marko

    2003-02-01

    The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.

  1. Effect of silver nanoparticles on human primary keratinocytes.

    PubMed

    Szmyd, Radoslaw; Goralczyk, Anna Grazyna; Skalniak, Lukasz; Cierniak, Agnieszka; Lipert, Barbara; Filon, Francesca Larese; Crosera, Matteo; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

  2. Prediction of primary somatosensory neuron activity during active tactile exploration

    PubMed Central

    Campagner, Dario; Evans, Mathew Hywel; Bale, Michael Ross; Erskine, Andrew; Petersen, Rasmus Strange

    2016-01-01

    Primary sensory neurons form the interface between world and brain. Their function is well-understood during passive stimulation but, under natural behaving conditions, sense organs are under active, motor control. In an attempt to predict primary neuron firing under natural conditions of sensorimotor integration, we recorded from primary mechanosensory neurons of awake, head-fixed mice as they explored a pole with their whiskers, and simultaneously measured both whisker motion and forces with high-speed videography. Using Generalised Linear Models, we found that primary neuron responses were poorly predicted by whisker angle, but well-predicted by rotational forces acting on the whisker: both during touch and free-air whisker motion. These results are in apparent contrast to previous studies of passive stimulation, but could be reconciled by differences in the kinematics-force relationship between active and passive conditions. Thus, simple statistical models can predict rich neural activity elicited by natural, exploratory behaviour involving active movement of sense organs. DOI: http://dx.doi.org/10.7554/eLife.10696.001 PMID:26880559

  3. Integrated Modular Teaching of Human Biology for Primary Care Practitioners

    ERIC Educational Resources Information Center

    Glasgow, Michael S.

    1977-01-01

    Describes the use of integrated modular teaching of the human biology component of the Health Associate Program at Johns Hopkins University, where the goal is to develop an understanding of the sciences as applied to primary care. Discussion covers the module sequence, the human biology faculty, goals of the human biology faculty, laboratory…

  4. Regulation of human renin expression in chorion cell primary cultures

    SciTech Connect

    Duncan, K.G.; Haidar, M.A.; Baxter, J.D.; Reudelhuber, T.L. )

    1990-10-01

    The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3{endash} to 6{endash}fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'{endash}flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'{endash}flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

  5. Zika virus productively infects primary human placenta-specific macrophages

    PubMed Central

    Jurado, Kellie Ann; Simoni, Michael K.; Tang, Zhonghua; Uraki, Ryuta; Hwang, Jesse; Householder, Sarah; Wu, Mingjie; Lindenbach, Brett D.; Abrahams, Vikki M.; Guller, Seth

    2016-01-01

    The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus–associated pregnancy complications. PMID:27595140

  6. Zika virus productively infects primary human placenta-specific macrophages

    PubMed Central

    Jurado, Kellie Ann; Simoni, Michael K.; Tang, Zhonghua; Uraki, Ryuta; Hwang, Jesse; Householder, Sarah; Wu, Mingjie; Lindenbach, Brett D.; Abrahams, Vikki M.; Guller, Seth; Fikrig, Erol

    2016-01-01

    The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus–associated pregnancy complications.

  7. Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy.

    PubMed

    Eriksen, Agnete Bratsberg; Torgersen, Maria Lyngaas; Holm, Kristine Lillebø; Abrahamsen, Greger; Spurkland, Anne; Moskaug, Jan Øivind; Simonsen, Anne; Blomhoff, Heidi Kiil

    2015-01-01

    In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

  8. Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

    PubMed

    Schuetz, E G; Hazelton, G A; Hall, J; Watkins, P B; Klaassen, C D; Guzelian, P S

    1986-06-25

    We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

  9. Kaposi's sarcoma-associated herpesvirus induces sustained NF-kappaB activation during de novo infection of primary human dermal microvascular endothelial cells that is essential for viral gene expression.

    PubMed

    Sadagopan, Sathish; Sharma-Walia, Neelam; Veettil, Mohanan Valiya; Raghu, Hari; Sivakumar, Ramu; Bottero, Virginie; Chandran, Bala

    2007-04-01

    In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, growth factors, and angiogenic factors. Since NF-kappaB is a key molecule involved in the regulation of several of these factors, here, we examined NF-kappaB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-kappaB was observed as early as 5 to 15 min postinfection by KSHV, and translocation of p65-NF-kappaB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IkappaB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-kappaB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-kappaB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-kappaB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription factors, including AP-1 family members, as well as several cytokines, growth factors, and angiogenic factors, which were significantly affected by NF-kappaB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-kappaB to regulate viral and

  10. Hedgehog activity controls opening of the primary mouth.

    PubMed

    Tabler, Jacqueline M; Bolger, Trióna G; Wallingford, John; Liu, Karen J

    2014-12-01

    To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes.

  11. Activation of primary sensory neurons by the topical application of capsaicin on the epidermis of a re-innervated organotypic human skin model.

    PubMed

    Lebonvallet, Nicolas; Pennec, Jean-Pierre; Le Gall-Ianotto, Christelle; Chéret, Jérémy; Jeanmaire, Christine; Carré, Jean-Luc; Pauly, Gilles; Misery, Laurent

    2014-01-01

    Using an ex vivo skin-nerve preparation, skin and nerve cells were reconstituted into a single unit and maintained in a nutrient medium bath until required experimentally. Our objective was to use the epidermis as a relay for the induction of an electric current to the neurons following the topical application of capsaicin on the skin epidermis of the skin explant, an agonist of the TRPV1 channel implicated in pruritus and pain. After 10-20 days of coculture to form the re-innervated skin model, we applied a solution of capsaicin directly on the epidermis of the skin explant (4 μm). The resulting current was recorded using a path-clamp technique on the neuronal fibres. Following the topical application of capsaicin, spontaneous activity was triggered, as characterised by repetitive spikes with periods of 125, 225 or 275 ms. This study demonstrates that the skin explant and nerve cells preparation may receive stimuli and be used to screen molecules or to study signal transmission.

  12. Activation of hypoxia-inducible factor 1 in human T-cell leukaemia virus type 1-infected cell lines and primary adult T-cell leukaemia cells

    PubMed Central

    Tomita, Mariko; Semenza, Gregg L.; Michiels, Canine; Matsuda, Takehiro; Uchihara, Jun-Nosuke; Okudaira, Taeko; Tanaka, Yuetsu; Taira, Naoya; Ohshiro, Kazuiku; Mori, Naoki

    2007-01-01

    HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1α in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1α protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1α by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1α protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1α protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics. PMID:17576198

  13. Effect of matrine on primary human hepatocytes in vitro.

    PubMed

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  14. Transcranial focused ultrasound stimulation of human primary visual cortex

    PubMed Central

    Lee, Wonhye; Kim, Hyun-Chul; Jung, Yujin; Chung, Yong An; Song, In-Uk; Lee, Jong-Hwan; Yoo, Seung-Schik

    2016-01-01

    Transcranial focused ultrasound (FUS) is making progress as a new non-invasive mode of regional brain stimulation. Current evidence of FUS-mediated neurostimulation for humans has been limited to the observation of subjective sensory manifestations and electrophysiological responses, thus warranting the identification of stimulated brain regions. Here, we report FUS sonication of the primary visual cortex (V1) in humans, resulting in elicited activation not only from the sonicated brain area, but also from the network of regions involved in visual and higher-order cognitive processes (as revealed by simultaneous acquisition of blood-oxygenation-level-dependent functional magnetic resonance imaging). Accompanying phosphene perception was also reported. The electroencephalo graphic (EEG) responses showed distinct peaks associated with the stimulation. None of the participants showed any adverse effects from the sonication based on neuroimaging and neurological examinations. Retrospective numerical simulation of the acoustic profile showed the presence of individual variability in terms of the location and intensity of the acoustic focus. With exquisite spatial selectivity and capability for depth penetration, FUS may confer a unique utility in providing non-invasive stimulation of region-specific brain circuits for neuroscientific and therapeutic applications. PMID:27658372

  15. Transcranial focused ultrasound stimulation of human primary visual cortex

    NASA Astrophysics Data System (ADS)

    Lee, Wonhye; Kim, Hyun-Chul; Jung, Yujin; Chung, Yong An; Song, In-Uk; Lee, Jong-Hwan; Yoo, Seung-Schik

    2016-09-01

    Transcranial focused ultrasound (FUS) is making progress as a new non-invasive mode of regional brain stimulation. Current evidence of FUS-mediated neurostimulation for humans has been limited to the observation of subjective sensory manifestations and electrophysiological responses, thus warranting the identification of stimulated brain regions. Here, we report FUS sonication of the primary visual cortex (V1) in humans, resulting in elicited activation not only from the sonicated brain area, but also from the network of regions involved in visual and higher-order cognitive processes (as revealed by simultaneous acquisition of blood-oxygenation-level-dependent functional magnetic resonance imaging). Accompanying phosphene perception was also reported. The electroencephalo graphic (EEG) responses showed distinct peaks associated with the stimulation. None of the participants showed any adverse effects from the sonication based on neuroimaging and neurological examinations. Retrospective numerical simulation of the acoustic profile showed the presence of individual variability in terms of the location and intensity of the acoustic focus. With exquisite spatial selectivity and capability for depth penetration, FUS may confer a unique utility in providing non-invasive stimulation of region-specific brain circuits for neuroscientific and therapeutic applications.

  16. Intravenous immunoglobulin treatment preserves and protects primary rat hippocampal neurons and primary human brain cultures against oxidative insults.

    PubMed

    Lahiri, Debomoy K; Ray, Balmiki

    2014-01-01

    Alzheimer's disease (AD) is characterized by deleterious accumulation of amyloid-β (Aβ) peptide into senile plaque, neurofibrillary tangles formed from hyperphosphorylated tau protein, and loss of cholinergic synapses in the cerebral cortex. The deposition of Aβ-loaded plaques results in microglial activation and subsequent production of reactive oxygen species (ROS), including free radicals. Neurons in aging and AD brains are particularly vulnerable to ROS and other toxic stimuli. Therefore, agents that decrease the vulnerability of neurons against ROS may provide therapeutic values for the treatment or prevention of AD. In the present study, our goal was to test whether intravenous immunoglobulin (IVIG) treatment could preserve as well as protect neurons from oxidative damage. We report that treatment with IVIG protects neuronal viability and synaptic proteins in primary rat hippocampal neurons. Further, we demonstrate the tolerability of IVIG treatment in the primary human fetal mixed brain cultures. Indeed, a high dose (20 mg/ml) of IVIG treatment was well-tolerated by primary human brain cultures that exhibit a normal neuronal phenotype. We also observed a potent neuropreservatory effect of IVIG against ROS-mediated oxidative insults in these human fetal brain cultures. These results indicate that IVIG treatment has great potential to preserve and protect primary human neuronal-enriched cultures and to potentially rescue dying neurons from oxidative insults. Therefore, our findings suggest that IVIG treatment may represent an important therapeutic agent for clinical trials designed to prevent and delay the onset of neurodegeneration as well as AD pathology. PMID:25115544

  17. Steroid synthesis by primary human keratinocytes; implications for skin disease

    SciTech Connect

    Hannen, Rosalind F.; Michael, Anthony E.; Jaulim, Adil; Bhogal, Ranjit; Burrin, Jacky M.; Philpott, Michael P.

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  18. Noradrenaline release and the pathophysiology of primary human hypertension

    SciTech Connect

    Esler, M.; Jennings, G.; Lambert, G.

    1989-03-01

    Measurements of the overflow of norepinephrine to plasma from individual organs (using radiotracer methodology) were used to delineate the pattern of sympathetic nervous system activation present in primary human hypertension. Mean total norepinephrine (NE) spillover in hypertensive patients was 418 ng/min, 42% (124 ng/min) higher than in subjects with normal blood pressure (BP)(P less than .05). Norepinephrine spillover among hypertensive patients was a function of age, only being elevated in patients under 40 years of age. Half of the excess in total norepinephrine release in hypertensive patients was accounted for by increased cardiorenal spillover. Mean renal norepinephrine spillover was 120 ng/min, compared with 69 ng/min in healthy subjects (P less than .02). Renal spillover was highest in younger patients. Corresponding cardiac norepinephrine spillover values were 12.6 ng/min and 5.1 ng/min (P less than .01). The balance of the excess total norepinephrine spillover comes from undetermined sites, but not the lungs or hepatomesenteric circulation. These measurements of regional norepinephrine overflow suggest that sympathetic nervous outflow to the kidneys and heart is selectively activated in early hypertension. 21 references.

  19. ESA Human Exploration Activities

    NASA Astrophysics Data System (ADS)

    Hovland, Scott

    The long term goal of the Aurora Exploration Programme is Human exploration of Mars. In preparation for this, exploration of the Moon is a necessary step to provide demonstration of capabilities, mandatory for long duration human spaceflight. With the European Columbus module attached to the ISS, Europe has access to a world class laboratory in space for microgravity research, technology demonstration and preparation for future human exploration missions. The ongoing phase of the exploration programme has been focused on defining the overall European strategy and exploration architecture within the global exploration environment. System studies as well as focused technology developments are in progress (e.g. development of regenerative life support).

  20. Promoting Physical Activity in Primary Care

    PubMed Central

    Singer, Joel; Lindsay, Elizabeth A.; Wilson, Douglas M.C.

    1991-01-01

    The principle barriers preventing health care professionals from promoting physical activity include an incomplete understanding of the evidence linking physical activity and health, difficulty in translating research findings into a feasible and efficacious clinical intervention, resistance to adopting a preventive orientation, and concerns about the risks of physical activity. Low level activities likely provide benefit with little risk. PMID:21229089

  1. Finite element analysis of lightweight active primary mirror

    NASA Astrophysics Data System (ADS)

    Lu, Wei Xin; Guan, Chun Lin; Rao, Chang Hui

    2012-09-01

    With the increasing requirement on spatial resolution to achieve ideal performance in space-based optical imaging system, there is a need to enlarge primary apertures. However, primary mirrors of such systems cannot maintain its optical tolerances across the mirror surface after sending to space, because of gravity change and varying ambient temperature. It necessitates active optics technology of primary mirror surface correction. Since mass-to-orbit is expensive and limited, lightweight primary mirror is needed. The paper investigates a lightweight, active primary mirror. This primary mirror structure includes lightweight face sheet and substrate with surface-parallel actuators embedded in the recess of web support ribs. Finite element models of lightweight, active primary mirror structures with different structural parameters are established and simulated. Using the response function matrixes acquired from finite element analysis, the fitting errors for Zernike polynomials are computed by MATLAB. Correctability comparisons of lightweight, active primary mirror structures with different parameters are carried out. To get best correctability, the mirrors should have small recess depth, high and thin ribs, thick face sheets and long actuators. The structural analysis result will be valuable for the design of lightweight, active primary mirror.

  2. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    PubMed

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  3. Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1--prostaglandin D2 synthase activity in the absence of NADP(H).

    PubMed

    Nagata, Nanae; Kusakari, Yukiko; Fukunishi, Yoshifumi; Inoue, Tsuyoshi; Urade, Yoshihiro

    2011-04-01

    Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F(2α) (PGF(2α)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2α). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP(+). Both PGD(2) and PGF(2α) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP(+) or after heat treatment of these enzymes at 100 °C for 5 min. The K(m), V(max), pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μM, 151 and 57 nmol·min(-1)·(mg protein)(-1), 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2α) synthase activity were 29 and 33 μM, 169 and 240 nmol·min(-1)·(mg protein)(-1), 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2α) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2α) in the absence of NADPH or NADP(+) and in the presence of NADPH, respectively.

  4. Humanization policy in primary health care: a systematic review

    PubMed Central

    Nora, Carlise Rigon Dalla; Junges, José Roque

    2013-01-01

    OBJECTIVE To analyze humanization practices in primary health care in the Brazilian Unified Health System according to the principles of the National Humanization Policy. METHODS A systematic review of the literature was carried out, followed by a meta-synthesis, using the following databases: BDENF (nursing database), BDTD (Brazilian digital library of theses and dissertations), CINAHL (Cumulative Index to nursing and allied health literature), LILACS (Latin American and Caribbean health care sciences literature), MedLine (International health care sciences literature), PAHO (Pan-American Health Care Organization Library) and SciELO (Scientific Electronic Library Online). The following descriptors were used: Humanization; Humanizing Health Care; Reception: Humanized care: Humanization in health care; Bonding; Family Health Care Program; Primary Care; Public Health and Sistema Único de Saúde (the Brazilian public health care system). Research articles, case studies, reports of experiences, dissertations, theses and chapters of books written in Portuguese, English or Spanish, published between 2003 and 2011, were included in the analysis. RESULTS Among the 4,127 publications found on the topic, 40 studies were evaluated and included in the analysis, producing three main categories: the first referring to the infrastructure and organization of the primary care service, made clear the dissatisfaction with the physical structure and equipment of the services and with the flow of attendance, which can facilitate or make difficult the access. The second, referring to the health work process, showed issues about the insufficient number of professionals, fragmentation of the work processes, the professional profile and responsibility. The third category, referring to the relational technologies, indicated the reception, bonding, listening, respect and dialog with the service users. CONCLUSIONS Although many practices were cited as humanizing they do not produce changes

  5. Humanities-Oriented Accents in Teaching Mathematics to Prospective Primary School Teachers

    ERIC Educational Resources Information Center

    Tabov, Jordan; Gortcheva, Iordanka

    2016-01-01

    Our research includes undergraduate students who major in primary school education. Their academic background is prevailingly in the humanities. This poses specific demands on their mathematics instruction at university. To attract them to their mathematics course and raise its effectiveness, we use a series of activities. Writing assignments…

  6. Interaction between arsenic trioxide and human primary cells: emphasis on human cells of myeloid origin.

    PubMed

    Binet, François; Antoine, Francis; Girard, Denis

    2009-03-01

    Arsenic trioxide (As(2)O(3); ATO) is considered to be one of the most potent drugs in cancer chemotherapy and is highly effective in the treatment of acute promyelocytic leukemia (APL). It is well established that treatment of APL patients with ATO is associated with the disappearance of the PML-RARalpha fusion transcript, the characteristic APL gene product of the chromosomal translocation t(15;17). Although its mode of action is still not fully understood, ATO is known to induce cell apoptosis via generation of reactive oxygen species and activation of caspases. Several reports have indicated that ATO acts principally by inducing cell apoptosis not only in APL, but in a variety of non-APL cells including myeloma cells, chronic myeloid leukemia cells and cells of immune origin, including B or T lymphocytes, macrophages and, more recently, neutrophils. There is an increasing amount of data, including some from our laboratory, concerning the interaction between ATO and human primary cells. The focus of this review will be to cover the role of ATO in human immune primary cells with special emphasis on cells of myeloid origin.

  7. Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages.

    PubMed

    Graham, Joseph G; MacDonald, Laura J; Hussain, S Kauser; Sharma, Uma M; Kurten, Richard C; Voth, Daniel E

    2013-06-01

    The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii-host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short-termphosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C

  8. Primary pulmonary hypertension associated with human immunodeficiency virus infection.

    PubMed Central

    Golpe, R.; Fernandez-Infante, B.; Fernandez-Rozas, S.

    1998-01-01

    Several cardiorespiratory diseases can complicate human immunodeficiency virus infection. Primary pulmonary hypertension is a rare clinical disorder which carries a bad prognosis. More than 90 cases of HIV-associated primary pulmonary hypertension have been reported to date. Although its pathogenesis remains unknown, some evidence suggests a possible role for the virus itself in its development. Genetic susceptibility may also be implicated. The clinical and histopathologic features of this entity do not differ from those of classic primary pulmonary hypertension. The diagnosis requires a high degree of clinical suspicion and a careful evaluation to rule out causes of secondary pulmonary hypertension. In addition to supportive measures, anticoagulation and vasodilators have been used to treat this disorder, although sufficient data regarding long-term results with these therapies are lacking. PMID:9799910

  9. Arrhenius parameters for primary thermal injury in human tonsillar tissue

    NASA Astrophysics Data System (ADS)

    McMillan, Kathleen; Radabaugh, Rebecca; Coad, James E.

    2011-03-01

    Clinical implementation of a thermal therapy requires the ability to predict tissue injury following exposures to specific thermal histories. As part of an effort to develop a nonexcisional alternative to tonsillectomy, the degree of primary hyperthermic tissue injury in human tonsil was characterized. Fifteen fresh pediatric hypertrophic tonsillectomy specimens were sectioned and treated in a NIST-calibrated saline bath at temperatures of 40 to 70°C with hold times of one to seven minutes. The treated tissues were subsequently nitroblue tetrazolium (NBT) stained to assess for thermal respiratory enzyme inactivation as a marker of cellular injury/death. The NBT stains were quantitatively image analyzed and used to calculate Arrhenius parameters for primary thermal injury in human tonsils.

  10. Teaching students to read the primary literature using POGIL activities.

    PubMed

    Murray, Tracey Arnold

    2014-01-01

    The ability to read, interpret, and evaluate articles in the primary literature are important skills that science majors will use in graduate school and professional life. Because of this, it is important that students are not only exposed to the primary literature in undergraduate education, but also taught how to read and interpret these articles. To achieve this objective, POGIL activities were designed to use the primary literature in a majors biochemistry sequence. Data show that students were able to learn content from the literature without separate activities or lecture. Students also reported an increase in comfort and confidence in approaching the literature as a result of the activities.

  11. Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32.

    PubMed

    Schmid, Mattia; Gemperle, Claudio; Rimann, Nicole; Hersberger, Martin

    2016-04-15

    Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of inflammation, but its mechanism of action in humans is not clear. We show that the RvD1 receptor GPR32 is present on resting, proinflammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-β and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1β and IL-8; abolished chemotaxis to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4) macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-β, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans.

  12. Activity Based Astronomy for Primary Science Programs.

    ERIC Educational Resources Information Center

    Ginns, Ian

    Print materials in astronomy such as books, journals, charts, and posters are typically the sources of information for teachers and children about the moon, the sun, lunar and solar eclipses, planetary sizes, distances of planets from the sun, planetary atmospheres, and so on. This paper describes and analyzes a number of activities designed to…

  13. HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes

    PubMed Central

    Brinzevich, Daria; Young, George R.; Sebra, Robert; Ayllon, Juan; Maio, Susan M.; Deikus, Gintaras; Chen, Benjamin K.; Fernandez-Sesma, Ana; Simon, Viviana

    2014-01-01

    ABSTRACT Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication. IMPORTANCE Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins

  14. Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation

    PubMed Central

    Melton, Andrew C.; Melrose, Jennifer; Alajoki, Liisa; Privat, Sylvie; Cho, Hannah; Brown, Naomi; Plavec, Ana Marija; Nguyen, Dat; Johnston, Elijah D.; Yang, Jian; Polokoff, Mark A.; Plavec, Ivan; Berg, Ellen L.; O'Mahony, Alison

    2013-01-01

    Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F. PMID:23505568

  15. Transcriptional comparison of human induced and primary midbrain dopaminergic neurons

    PubMed Central

    Xia, Ninuo; Zhang, Pengbo; Fang, Fang; Wang, Zhengyuan; Rothstein, Megan; Angulo, Benjamin; Chiang, Rosaria; Taylor, James; Reijo Pera, Renee A.

    2016-01-01

    Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson’s disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson’s Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression, especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies, including in vitro modeling of PD. However, further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. PMID:26842779

  16. Amylase activity in human bile.

    PubMed

    Donaldson, L A; Joffe, S N; McIntosh, W; Brodie, M J

    1979-03-01

    The mean amylase level in 42 human bile samples was 154 IU/l and there was no significant difference in the amylase activity of 32 paired serum and bile samples. Estimation of the amylase thermolability of bile showed it to be similar to that of serum. This suggests that the amylase activity in bile may have filtered through the liver from the hepatic circulation rather than refluxed from the pancreatic duct. The presence of amylase in human bile provides further evidence that the liver might have a role in the regulation of serum amylase.

  17. Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

    PubMed Central

    Sivalingam, Jaichandran; Kenanov, Dimitar; Han, Hao; Nirmal, Ajit Johnson; Ng, Wai Har; Lee, Sze Sing; Masilamani, Jeyakumar; Phan, Toan Thang; Maurer-Stroh, Sebastian; Kon, Oi Lian

    2016-01-01

    Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. PMID:26689265

  18. Human Bocavirus 1 Primary Infection and Shedding in Infants

    PubMed Central

    Martin, Emily T.; Kuypers, Jane; McRoberts, John P.; Englund, Janet A.; Zerr, Danielle M.

    2015-01-01

    Background. Human bocavirus 1 (HBoV-1) is frequently detected in young children. The role of HBoV-1 in respiratory illness is unclear, owing to frequent detection in asymptomatic children. Methods. Weekly oral fluid samples from a longitudinal cohort of infants were tested by quantitative polymerase chain reaction for HBoV-1 DNA. Symptoms during HBoV-1 primary shedding events were compared to those during 14-day control periods occurring 1 month prior to and following the primary event. Eight single-nucleotide polymorphisms were analyzed to assess HBoV-1 variants. Results. Sixty-six of 87 children (76%), followed for at least 18 months from birth, had a primary HBoV-1 infection. HBoV-1 was consistently detected for >1 month (maximum duration, 402 days) following 42 of 66 primary shedding events. Children were more likely to experience new cough symptoms (odds ratio [OR], 2.7; 95% confidence interval [CI], 1.4–5.5) and to visit a healthcare provider (OR, 2.8; 95% CI, 1.02–7.7) during the 14 days surrounding the time of initial detection of HBoV-1. Recurrent HBoV-1 shedding events were found in 33 children (50%). Twelve of 48 children with HBoV-1 variant data had multiple viral allelic patterns over time. Conclusions. HBoV-1 primary shedding events are associated with mild respiratory illness with subsequent prolonged detection of HBoV-1 DNA for up to a year. HBoV-1 reinfection contributes to long-term shedding. PMID:25632039

  19. Expression of androgen and progesterone receptors in primary human meningiomas.

    PubMed

    Maxwell, M; Galanopoulos, T; Neville-Golden, J; Antoniades, H N

    1993-03-01

    Meningiomas are common brain tumors that show a predilection for females and become more aggressive during pregnancy and menses. The existence of gender-specific hormone receptors in meningiomas has long been a matter of controversy; the recent cloning of androgen, estrogen, and progesterone receptors has facilitated their direct evaluation. The authors have demonstrated the expression of androgen and progesterone receptor messenger ribonucleic acid and protein product in nine primary human meningiomas by Northern blot analysis. Cellular localization was achieved by in situ hybridization analysis. Estrogen receptor expression was not detected. Normal adult meninges were shown to express very low levels of both androgen and progesterone receptors.

  20. Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.

    PubMed

    Johnson, M Brittany; Criss, Alison K

    2013-08-01

    Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  1. Science Activities That Work: Perceptions of Primary School Teachers

    NASA Astrophysics Data System (ADS)

    Appleton, Ken

    2002-06-01

    Many primary school teachers in Australia tend to be reluctant to teach science, partly because they are not confident in science and have limited science background knowledge. However, quite a number of primary school teachers still manage to teach some science. When they plan to teach science, many of them use the term science activities that work. Such activities seem to be related to science pedagogical content knowledge for some primary teachers. In order to better understand what the term activities that work means, twenty teachers from several schools were interviewed and asked what they understood by this expression. Themes that emerged suggest that activities that work are hands on, are interesting and motivating for the children, have a clear outcome or result, are manageable in the classroom, use equipment that is readily available, and are preferably used in a context where science is integrated into themes. Implications for curriculum and for preservice teacher education are considered.

  2. Intrinsic Patterns of Human Activity

    NASA Astrophysics Data System (ADS)

    Hu, Kun; Ivanov, Plamen Ch.; Chen, Zhi; Hilton, Michael; Stanley, H. Eugene; Shea, Steven

    2003-03-01

    Activity is one of the defining features of life. Control of human activity is complex, being influenced by many factors both extrinsic and intrinsic to the body. The most obvious extrinsic factors that affect activity are the daily schedule of planned events, such as work and recreation, as well as reactions to unforeseen or random events. These extrinsic factors may account for the apparently random fluctuations in human motion observed over short time scales. The most obvious intrinsic factors are the body clocks including the circadian pacemaker that influences our sleep/wake cycle and ultradian oscillators with shorter time scales [2, 3]. These intrinsic rhythms may account for the underlying regularity in average activity level over longer periods of up to 24 h. Here we ask if the known extrinsic and intrinsic factors fully account for all complex features observed in recordings of human activity. To this end, we measure activity over two weeks from forearm motion in subjects undergoing their regular daily routine. Utilizing concepts from statistical physics, we demonstrate that during wakefulness human activity possesses previously unrecognized complex dynamic patterns. These patterns of activity are characterized by robust fractal and nonlinear dynamics including a universal probability distribution and long-range power-law correlations that are stable over a wide range of time scales (from minutes to hours). Surprisingly, we find that these dynamic patterns are unaffected by changes in the average activity level that occur within individual subjects throughout the day and on different days of the week, and between subjects. Moreover, we find that these patterns persist when the same subjects undergo time-isolation laboratory experiments designed to account for the phase of the circadian pacemaker, and control the known extrinsic factors by restricting behaviors and manipulating scheduled events including the sleep/wake cycle. We attribute these newly

  3. Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts.

    PubMed

    van der Meijden, K; Bakker, A D; van Essen, H W; Heijboer, A C; Schulten, E A J M; Lips, P; Bravenboer, N

    2016-02-01

    The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading. Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400 nM) or 1,25(OH)2D3 (100 nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400 nM). We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3. In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect

  4. Primary human bronchial epithelial cells grown from explants.

    PubMed

    Yaghi, Asma; Zaman, Aisha; Dolovich, Myrna

    2010-01-01

    Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly

  5. Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat

    PubMed Central

    Seiler, Sarah E; Xu, Dan; Ho, Jia-Pei; Lo, Kinyui Alice; Buehrer, Benjamin M; Ludlow, Y John W; Kovalik, Jean-Paul; Sun, Lei

    2015-01-01

    Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system. Likewise, the lack of a well-characterized human model has limited the discovery of novel targets for the activation of human brown fat. To address this current need, we aimed to identify and describe the first primary brown adipocyte cell culture system from human fetal interscapular brown adipose tissue. Pre-adipocytes isolated from non-viable human fetal interscapular tissue were expanded and cryopreserved. Cells were then thawed and plated alongside adult human subcutaneous and omental pre-adipocytes for subsequent differentiation and phenotypic characterization. Interscapular pre-adipocytes in cell culture differentiated into mature adipocytes that were morphologically indistinguishable from the adult white depots. Throughout differentiation, cultured human fetal interscapular adipocytes demonstrated increased expression of classical brown fat markers compared to subcutaneous and omental cells. Further, functional analysis revealed an elevation in fatty acid oxidation as well as maximal and uncoupled oxygen consumption in interscapular brown adipocytes compared to white control cells. These data collectively identify the brown phenotype of these cells. Thus, our primary cell culture system derived from non-viable human fetal interscapular brown adipose tissue provides a valuable tool for the study of human brown adipocyte biology and for the development of anti-obesity therapeutics. PMID:26451287

  6. Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood.

    PubMed

    Hall, J Perry; Kurdi, Yahya; Hsu, Sang; Cuozzo, John; Liu, Julie; Telliez, Jean-Baptiste; Seidl, Katherine J; Winkler, Aaron; Hu, Yonghan; Green, Neal; Askew, G Roger; Tam, Steve; Clark, James D; Lin, Lih-Ling

    2007-11-16

    Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases. PMID:17848581

  7. Primary human monocyte differentiation regulated by Nigella sativa pressed oil

    PubMed Central

    2011-01-01

    Background Oxidized low density lipoprotein plays an important role in development of foam cells in atherosclerosis. The study was focused on regulation of primary human monocyte growth and CD11b expression in presence of Nigella sativa oil. Methods Primary human monocytes were isolated from whole blood and grown at 37°C and 5% CO2 saturation for five days prior to treatment with Nigella sativa oil. The cells were plated and washed before treatment with ox-LDL (10 μg/ml) as positive control and combined treatment of ox-LDL (10 μg/ml) and (140 ng/ml) Nigella sativa oil. The growth progression was monitored every 24 hours for 3 days. Results Macrophages showed reduced growth in comparison to monocytes 24 hours after treatment with Nigella sativa oil. The mean cell diameter was significantly different between untreated and treated condition in monocytes and macrophages (p < 0.001). Similarly, intracellular lipid accumulation was hindered in combined treatment with Nigella sativa oil. This was further supported by cell surface expression analysis, where CD11b was markedly reduced in cells treated with combination oxLDL and Nigella sativa oil compared to oxLDL alone. More cells differentiated into macrophage-like cells when monocytes were supplemented with oxidized LDL alone. Conclusions The finding provides preliminary evidence on regulation of cell growth and differentiation in monocyte and monocyte-derived macrophages by Nigella sativa oil. Further investigations need to be conducted to explain its mechanism in human monocyte. PMID:22104447

  8. Active support system for 1-m SONG primary mirror

    NASA Astrophysics Data System (ADS)

    Niu, Dongsheng; Wang, Guomin; Gu, Bozhong

    2012-05-01

    Chinese-node telescope of Stellar Observations Network Group (SONG) has a primary mirror 1m in diameter with flat back, which will be supported actively. The performance evaluation of the telescope's active optics system is conducted. Finite element analysis (FEA) is employed to analyze the optical surface figures of the primary mirror, and two optimizations are carried out by using ANSYS: (1) the locations and forces of axial supports are optimized with the telescope pointing to zenith; (2) the lateral support forces are calculated with the telescope pointing to horizon. Axial support force sensitivities are calculated in a case that a single axial support has a force error of 0.5N. The correction ability of the active support system is analyzed when an arbitrary axial support is failure. Several low order Zernike modes are modeled with MATLAB procedure, and active optics corrections are applied to these modes. Thermal deformation of the mirror is also corrected using active support system.

  9. Repeating spatial activations in human entorhinal cortex.

    PubMed

    Miller, Jonathan F; Fried, Itzhak; Suthana, Nanthia; Jacobs, Joshua

    2015-04-20

    The ability to remember and navigate spatial environments is critical for everyday life. A primary mechanism by which the brain represents space is through hippocampal place cells, which indicate when an animal is at a particular location. An important issue is understanding how the hippocampal place-cell network represents specific properties of the environment, such as signifying that a particular position is near a doorway or that another position is near the end of a corridor. The entorhinal cortex (EC), as the main input to the hippocampus, may play a key role in coding these properties because it contains neurons that activate at multiple related positions per environment. We examined the diversity of spatial coding across the human medial temporal lobe by recording neuronal activity during virtual navigation of an environment containing four similar paths. Neurosurgical patients performed this task as we recorded from implanted microelectrodes, allowing us to compare the human neuronal representation of space with that of animals. EC neurons activated in a repeating manner across the environment, with individual cells spiking at the same relative location across multiple paths. This finding indicates that EC cells represent non-specific information about location relative to an environment's geometry, unlike hippocampal place cells, which activate at particular random locations. Given that spatial navigation is considered to be a model of how the brain supports non-spatial episodic memory, these findings suggest that EC neuronal activity is used by the hippocampus to represent the properties of different memory episodes.

  10. Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

    SciTech Connect

    Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko; Hiratsuka, Masaharu; Sano, Akiko; Osawa, Kanako; Okazaki, Akiyo; Katoh, Motonobu; Kazuki, Yasuhiro; Oshimura, Mitsuo; Tomizuka, Kazuma

    2008-05-09

    Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-{beta}-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.

  11. Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

    EPA Science Inventory

    The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, ...

  12. Youth who sexual offended: primary human goods and offense pathways.

    PubMed

    Chu, Chi Meng; Koh, Li Lian; Zeng, Gerald; Teoh, Jennifer

    2015-04-01

    There has been an increased focus on understanding youth sexual offending in recent years, but there has been limited empirical research on the causes, pathways, and treatment of youth who have sexually offended-especially within a non-Western context. The Good Lives and Self-Regulation Models have often been used to understand and rehabilitate adult sexual offenders, but (unfortunately) there is scant research on youth who sexually offended using these models. The present study aims to describe the different primary goods that are associated with youth sexual offending behaviors in an Asian context. In addition, the study sought to explore whether the age of victim (child vs. nonchild) and nature of sexual offense (penetrative vs. nonpenetrative) influenced the youth's engagement in offense pathways. The results suggest that pleasure, relatedness, and inner peace were the primary human goods that were most sought after by a sample of 168 youth who sexually offended in Singapore. In addition, offender classification (in relation to the age of victim and nature of sexual offense) influenced the pathways to sexual offending. Therefore, these findings have important clinical implications for assessment, management, and intervention planning for youth who sexually offended. PMID:24048701

  13. Youth who sexual offended: primary human goods and offense pathways.

    PubMed

    Chu, Chi Meng; Koh, Li Lian; Zeng, Gerald; Teoh, Jennifer

    2015-04-01

    There has been an increased focus on understanding youth sexual offending in recent years, but there has been limited empirical research on the causes, pathways, and treatment of youth who have sexually offended-especially within a non-Western context. The Good Lives and Self-Regulation Models have often been used to understand and rehabilitate adult sexual offenders, but (unfortunately) there is scant research on youth who sexually offended using these models. The present study aims to describe the different primary goods that are associated with youth sexual offending behaviors in an Asian context. In addition, the study sought to explore whether the age of victim (child vs. nonchild) and nature of sexual offense (penetrative vs. nonpenetrative) influenced the youth's engagement in offense pathways. The results suggest that pleasure, relatedness, and inner peace were the primary human goods that were most sought after by a sample of 168 youth who sexually offended in Singapore. In addition, offender classification (in relation to the age of victim and nature of sexual offense) influenced the pathways to sexual offending. Therefore, these findings have important clinical implications for assessment, management, and intervention planning for youth who sexually offended.

  14. Commercial Activities in Primary Schools: A Quantitative Study

    ERIC Educational Resources Information Center

    Raine, Gary

    2007-01-01

    The commercialisation of schools is a controversial issue, but very little is known about the actual situation in UK schools. The aim of this study was to investigate, with particular reference to health education and health promotion, commercial activities and their regulation in primary schools in the Yorkshire and Humber region of the UK. A…

  15. Active Classroom Participation in a Group Scribbles Primary Science Classroom

    ERIC Educational Resources Information Center

    Chen, Wenli; Looi, Chee-Kit

    2011-01-01

    A key stimulus of learning efficacy for students in the classroom is active participation and engagement in the learning process. This study examines the nature of teacher-student and student-student discourse when leveraged by an interactive technology--Group Scribbles (GS) in a Primary 5 Science classroom in Singapore which supports rapid…

  16. Compulsory "Foreign Language Activities" in Japanese Primary Schools

    ERIC Educational Resources Information Center

    Hashimoto, Kayoko

    2011-01-01

    From 2011, the new curriculum for introducing English to Japanese primary schools will be fully implemented in the form of "foreign language activities". This innovation forms part of the government's plan to cultivate "Japanese with English abilities", a development based on the awareness, particularly in the business sector, that equipping…

  17. Human Rights Texts: Converting Human Rights Primary Source Documents into Data

    PubMed Central

    Fariss, Christopher J.; Linder, Fridolin J.; Jones, Zachary M.; Crabtree, Charles D.; Biek, Megan A.; Ross, Ana-Sophia M.; Kaur, Taranamol; Tsai, Michael

    2015-01-01

    We introduce and make publicly available a large corpus of digitized primary source human rights documents which are published annually by monitoring agencies that include Amnesty International, Human Rights Watch, the Lawyers Committee for Human Rights, and the United States Department of State. In addition to the digitized text, we also make available and describe document-term matrices, which are datasets that systematically organize the word counts from each unique document by each unique term within the corpus of human rights documents. To contextualize the importance of this corpus, we describe the development of coding procedures in the human rights community and several existing categorical indicators that have been created by human coding of the human rights documents contained in the corpus. We then discuss how the new human rights corpus and the existing human rights datasets can be used with a variety of statistical analyses and machine learning algorithms to help scholars understand how human rights practices and reporting have evolved over time. We close with a discussion of our plans for dataset maintenance, updating, and availability. PMID:26418817

  18. Human Rights Texts: Converting Human Rights Primary Source Documents into Data.

    PubMed

    Fariss, Christopher J; Linder, Fridolin J; Jones, Zachary M; Crabtree, Charles D; Biek, Megan A; Ross, Ana-Sophia M; Kaur, Taranamol; Tsai, Michael

    2015-01-01

    We introduce and make publicly available a large corpus of digitized primary source human rights documents which are published annually by monitoring agencies that include Amnesty International, Human Rights Watch, the Lawyers Committee for Human Rights, and the United States Department of State. In addition to the digitized text, we also make available and describe document-term matrices, which are datasets that systematically organize the word counts from each unique document by each unique term within the corpus of human rights documents. To contextualize the importance of this corpus, we describe the development of coding procedures in the human rights community and several existing categorical indicators that have been created by human coding of the human rights documents contained in the corpus. We then discuss how the new human rights corpus and the existing human rights datasets can be used with a variety of statistical analyses and machine learning algorithms to help scholars understand how human rights practices and reporting have evolved over time. We close with a discussion of our plans for dataset maintenance, updating, and availability.

  19. The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells

    PubMed Central

    Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

    2016-01-01

    N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.

  20. The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells.

    PubMed

    Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

    2016-01-01

    N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.

  1. The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells

    PubMed Central

    Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

    2016-01-01

    N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain. PMID:27683542

  2. The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells.

    PubMed

    Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

    2016-01-01

    N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain. PMID:27683542

  3. The LATT way towards large active primaries for space telescopes

    NASA Astrophysics Data System (ADS)

    Briguglio, Runa; Arcidiacono, Carmelo; Xompero, Marco; Lisi, Franco; Riccardi, Armando; Biasi, Roberto; Patauner, Christian; Gallieni, Daniele; Lazzarini, Paolo; Tintori, Matteo; d'Amato, Francesco; Pucci, Mauro; Duò, Fabrizio; Vettore, Christian; Zuccaro Marchi, Alessandro

    2016-07-01

    The Large Aperture Telescope Technology (LATT) goes beyond the current paradigm of future space telescopes, based on a deformable mirror in the pupil relay. Through the LATT project we demonstrated the concept of a low-weight active primary mirror, whose working principle and control strategy benefit from two decades of advances in adaptive optics for ground-based telescopes. We developed a forty centimeter spherical mirror prototype, with an areal density lower than 17 kg/m2, controlled through contactless voice coil actuators with co-located capacitive position sensors. The prototype was subjected to thermo-vacuum, vibration and optical tests, to push its technical readiness toward level 5. In this paper we present the background and the outcomes of the LATT activities under ESA contract (TRP programme), exploring the concept of a lightweight active primary mirror for space telescopes. Active primaries will open the way to very large segmented apertures, actively shaped, which can be lightweight, deployable and accurately phased once in flight.

  4. Comparison of the cytotoxic activity of melphalan with L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine in human tumour cell lines and primary cultures of tumour cells from patients.

    PubMed Central

    Larsson, R.; Dhar, S.; Ehrsson, H.; Nygren, P.; Lewensohn, R.

    1998-01-01

    m-L-sarcolysin (m-L-SL) is an isomer of melphalan (Mel) with the di(2-chloroethyl) amino group being substituted in the meta position of phenylalanine. By covalent conjugation of amino acids to m-L-SL, a peptide complex consisting of six m-L-SL-based oligopeptides known as peptichemio (PTC) was developed previously. In the present study, the cytotoxic activity pattern of the different oligopeptides of PTC was investigated in ten human tumour cell lines representing different mechanisms of cytotoxic drug resistance using the fluorometric microculture cytotoxicity assay (FMCA). In the cell line panel, L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine (P2) was the most active oligopeptide, showing slightly lower mean IC50 values (2.6 vs 3.9 and 4.1 microg ml(-1)) than Mel and m-L-SL. The other five oligopeptides were less active than Mel. All active oligopeptides showed mechanistic similarity to Mel as judged by the correlation analysis of the cell line panel log IC50 values (R > or = 0.90), although P2 appeared to be less sensitive to GSH-mediated drug resistance. The relative activity of Mel and P2 was found to be related to degree of proliferation, P2 being more active towards low-proliferating cell lines. P2 and Mel were then further characterized in 49 fresh human tumour samples. In these samples P2 was considerably more active than Mel and showed a higher relative solid tumour activity (2.7 to 4.5-fold). However, the correlation of log IC50s between P2 and Mel in patient cells was high (R = 0.79), indicating a similar mechanism of action in this tumour model too. Cross-resistance with other standard drugs was lower for P2 than Mel. The results show that P2 is the most potent component of PTC and demonstrates a favourable activity profile compared with Mel. These data suggest that further investigation of P2 as a potential anti-tumour agent is warranted. PMID:9703278

  5. Active thermal figure control for the TOPS II primary mirror

    NASA Astrophysics Data System (ADS)

    Angel, Roger; Kang, Tae; Cuerden, Brian; Guyon, Olivier; Stahl, Phil

    2007-09-01

    TOPS (Telescope to Observe Planetary Systems) is the first coronagraphic telescope concept designed specifically to take advantage of Guyon's method of Phase Induced Amplitude Apodization PIAA).1 The TOPS primary mirror may incorporates active figure control to help achieve the desired wavefront control to approximately 1 angstrom RMS accurate across the spectral bandwidth. Direct correction of the primary figure avoids the need for a separate small deformable mirror. Because of Fresnel propagation, correction at a separate surface can introduce serious chromatic errors unless it is precisely conjugated to the primary. Active primary control also reduces complexity and mass and increases system throughput, and will likely enable a full system test to the 10-10 level in the 1 g environment before launch. We plan to use thermal actuators with no mechanical disturbance, using radiative heating or cooling fingers distributed inside the cells of a honeycomb mirror. The glass would have very small but finite coefficient of expansion of ~ 5x10 -8/C. Low order modes would be controlled by front-to-back gradients and high order modes by local rib expansion and contraction. Finite element models indicate that for a mirror with n cells up to n Zernike modes can be corrected to better than 90% fidelity, with still higher accuracy for the lower modes. An initial demonstration has been made with a borosilicate honeycomb mirror. Interferometric measurements show a single cell influence function with 300 nm stroke and ~5 minute time constant.

  6. Human prion protein-induced autophagy flux governs neuron cell damage in primary neuron cells

    PubMed Central

    Moon, Ji-Hong; Lee, Ju-Hee; Nazim, Uddin MD; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-hwa; Park, Sang-Youel

    2016-01-01

    An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease. PMID:27102156

  7. Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

    PubMed

    McLean, Gary R; Cho, Chin-wen; Schrader, John W

    2006-05-01

    The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

  8. Punicalagin promotes autophagy to protect primary human syncytiotrophoblasts from apoptosis.

    PubMed

    Wang, Ying; Chen, Baosheng; Longtine, Mark S; Nelson, D Michael

    2016-02-01

    Punicalagin is a prominent polyphenol in pomegranate juice that protects cultured syncytiotrophoblasts from stress-induced apoptosis. Here, we test the hypothesis that punicalagin has this effect by inhibiting the mTOR kinase pathway to enhance autophagic turnover and limit apoptosis in cultured primary human syncytiotrophoblasts. In syncytiotrophoblasts, starvation, rapamycin, or punicalagin all decreased the expression of phosphorylated ribosomal protein S6, a downstream target of the mTOR kinase, and of the autophagy markers, LC3-II and p62. In contrast, in the presence of bafilomycin, an inhibitor of late stages of autophagy and degradation in the autophagolysosome, syncytiotrophoblasts exposed to starvation, rapamycin, or punicalagin all showed increased levels of LC3-II and p62. The number of LC3-II punctae also increased in punicalagin-treated syncytiotrophoblasts exposed to chloroquine, another inhibitor of autophagic degradation, and punicalagin increased the number of lysosomes. The apoptosis-reducing effect of punicalagin was attenuated by inhibition of autophagy using bafilomycin or knockdown of the autophagy related gene, ATG16L1. Collectively, these data support the hypothesis that punicalagin modulates the crosstalk between autophagy and apoptosis to promote survival in cultured syncytiotrophoblasts. PMID:26659860

  9. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  10. 3D Cultivation Techniques for Primary Human Hepatocytes

    PubMed Central

    Bachmann, Anastasia; Moll, Matthias; Gottwald, Eric; Nies, Cordula; Zantl, Roman; Wagner, Helga; Burkhardt, Britta; Sánchez, Juan J. Martínez; Ladurner, Ruth; Thasler, Wolfgang; Damm, Georg; Nussler, Andreas K.

    2015-01-01

    One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  11. Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

    PubMed Central

    Ghosh, Santosh K.; Yohannes, Elizabeth; Bebek, Gurkan; Weinberg, Aaron; Jiang, Bin; Willard, Belinda; Chance, Mark R.; Kinter, Michael T.; McCormick, Thomas S.

    2012-01-01

    Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the inter-individual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes. PMID:23035736

  12. Dynamical activities of primary somatosensory cortices studied by magnetoencephalography

    NASA Astrophysics Data System (ADS)

    Kishida, Kuniharu

    2009-11-01

    A blind identification method of transfer functions in feedback systems is introduced for examination of dynamical activities of cortices by magnetoencephalography study. Somatosensory activities are examined in 5 Hz periodical median nerve stimulus. In the present paper, we will try two careful preprocessing procedures for the identification method to obtain impulse responses between primary somatosensory cortices. Time series data of the somatosensory evoked field are obtained by using a blind source separation of the T/k type (fractional) decorrelation method. Time series data of current dipoles of primary somatosensory cortices are transformed from the time series data of the somatosensory evoked field by the inverse problem. Fluctuations of current dipoles of them are obtained after elimination of deterministic periodical evoked waveforms. An identification method based on feedback system theory is used for estimation of transfer functions in a feedback model from obtained fluctuations of currents dipoles of primary somatosensory cortices. Dynamical activities between them are presented by Bode diagrams of transfer functions and their impulse responses: the time delay of about 30 ms via corpus callosum is found in the impulse response of identified transfer function.

  13. Houttuynia cordata Thunb extract inhibits cell growth and induces apoptosis in human primary colorectal cancer cells.

    PubMed

    Lai, Kuang-Chi; Chiu, Yu-Jen; Tang, Yih-Jing; Lin, Kuei-Li; Chiang, Jo-Hua; Jiang, Yi-Lin; Jen, Hsiu-Fang; Kuo, Yueh-Hsiung; Agamaya, Sakae; Chung, Jing-Gung; Yang, Jai-Sing

    2010-09-01

    It is reported that Houttuynia cordata Thunb. (HCT), a traditional Chinese herbal medicine, has many biological properties such as antiviral, antibacterial and antileukemic activities. However, the molecular mechanisms of cytotoxicity and apoptosis in human primary colorectal cancer cells are not clear. In this study, whether HCT induced cytotoxicity in primary colorectal cancer cells obtained from three patients was investigated. The results indicated that HCT inhibited growth of cancer cells in a dose-dependent manner. After treatment with HCT (250 μg/ml) for 24 h, cells exhibited chromatin condensation (an apoptotic characteristic). HCT increased reactive oxygen species (ROS) production and decreased the mitochondrial membrane potential (ΔΨ(m)) in examined cells. Mitochondria-dependent apoptotic signaling pathway was shown to be involved as determined by increase in the levels of cytochrome c, Apaf-1, and caspase-3 and -9. The decrease in the level of ΔΨ(m) was associated with an increase in the BAX/BCL-2 ratio which led to activation of caspase-9 and -3. Based on our results, HCT induced apoptotic cell death in human primary colorectal cancer cells through a mitochondria-dependent signaling pathway. PMID:20944136

  14. Telomerase activity in human cancer

    SciTech Connect

    Griffith, J.

    2000-10-01

    The overall goal of this collaborative project was to investigate the role in malignant cells of both chromosome telomeres, and telomerase, the enzyme that replicates telomeres. Telomeres are highly conserved nucleoprotein complexes located at the ends of eucaryotic chromosomes. Telomere length in somatic cells is reduced by 40--50 nucleotide pairs with every cell division due to incomplete replication of terminal DNA sequences and the absence of telomerase, the ribonucleoprotein that adds telomere DNA to chromosome ends. Although telomerase is active in cells with extended proliferative capacities, including more than 85% of tumors, work performed under this contract demonstrated that the telomeres of human cancer cells are shorter than those of paired normal cells, and that the length of the telomeres is characteristic of particular types of cancers. The extent of telomere shortening ostensibly is related to the number of cell divisions the tumor has undergone. It is believed that ongoing cell proliferation leads to the accumulation and fixation of new mutations in tumor cell lineages.Therefore, it is not unreasonable to assume that the degree of phenotypic variability is related to the proliferative history of the tumor, and therefore to telomere length, implying a correlation with prognosis. In some human tumors, short telomeres are also correlated with genomic instabilities, including interstitial chromosome translocation, loss of heterozygosity, and aneuoploidy. Moreover, unprotected chromosome ends are highly recombinogenic and telomere shortening in cultured human cells correlates with the formation of dicentric chromosomes, suggesting that critically short telomeres not only identify, but also predispose, cells to genomic instability, again implying a correlation with prognosis. Therefore, telomere length or content could be an important predictor of metastatic potential or responsiveness to various therapeutic modalities.

  15. Bisphenol S Induces Adipogenesis in Primary Human Preadipocytes From Female Donors.

    PubMed

    Boucher, Jonathan G; Ahmed, Shaimaa; Atlas, Ella

    2016-04-01

    Human exposure to bisphenol A has been associated with negative health outcomes in humans and its use is now regulated in a number of countries. Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A; however, its effects on cellular metabolism and potential role as an endocrine disruptor have not been fully characterized. In the current study, we evaluated the effect of BPS on adipogenesis in primary human preadipocytes. The effect of BPS on the differentiation of human preadipocytes was determined after treatment with BPS at concentrations ranging from 0.1 nM to 25 μM by quantifying lipid accumulation and mRNA and protein levels of key adipogenic markers. Treatment of preadipocytes with 25 μM BPS induced lipid accumulation and increased the mRNA and protein levels of several adipogenic markers including lipoprotein lipase and adipocyte protein 2 (aP2). Cotreatment of cells with the estrogen receptor antagonist ICI-182,780 significantly inhibited BPS-induced lipid accumulation and affected aP2 but not lipoprotein lipase protein levels. Cotreatment of cells with the glucocorticoid receptor antagonist RU486 had no effect on BPS-induced lipid accumulation or protein levels. Furthermore, reporter gene assays using a synthetic promoter containing peroxisome proliferator-activated receptor-γ (PPARG)-response elements and a PPARG-responsive human aP2 promoter region showed that BPS was able to activate PPARG. To our knowledge, this study is the first to show that BPS induces lipid accumulation and differentiation of primary human preadipocytes, and this effect may be mediated through a PPARG pathway.

  16. Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans.

    PubMed

    Mambula, S S; Simons, E R; Hastey, R; Selsted, M E; Levitz, S M

    2000-11-01

    It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules. PMID:11035733

  17. Reverse sequencing syllables of spoken words activates primary visual cortex.

    PubMed

    Ino, Tadashi; Asada, Tomohiko; Hirose, Syuichi; Ito, Jin; Fukuyama, Hidenao

    2003-10-27

    Using fMRI, we investigated the neural correlates for sequencing the individual syllables of spoken words in reverse order. The comparison of this task to a control task requiring subjects to repeat identical syllables given acoustically revealed the activation of the primary visual cortex. Because one syllable is generally expressed by one kana character (Japanese phonogram), most subjects used a strategy in which the kana character string corresponding to the word was imagined visually and then read mentally in reverse order to perform the task effectively. Such strategy was not used during a control condition. These results suggest that the primary visual cortex plays a role in the generation of an imagined string.

  18. Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain.

    PubMed

    Kim, Yu Shin; Anderson, Michael; Park, Kyoungsook; Zheng, Qin; Agarwal, Amit; Gong, Catherine; Saijilafu; Young, LeAnne; He, Shaoqiu; LaVinka, Pamela Colleen; Zhou, Fengquan; Bergles, Dwight; Hanani, Menachem; Guan, Yun; Spray, David C; Dong, Xinzhong

    2016-09-01

    Primary sensory neurons in the DRG play an essential role in initiating pain by detecting painful stimuli in the periphery. Tissue injury can sensitize DRG neurons, causing heightened pain sensitivity, often leading to chronic pain. Despite the functional importance, how DRG neurons function at a population level is unclear due to the lack of suitable tools. Here we developed an imaging technique that allowed us to simultaneously monitor the activities of >1,600 neurons/DRG in live mice and discovered a striking neuronal coupling phenomenon that adjacent neurons tend to activate together following tissue injury. This coupled activation occurs among various neurons and is mediated by an injury-induced upregulation of gap junctions in glial cells surrounding DRG neurons. Blocking gap junctions attenuated neuronal coupling and mechanical hyperalgesia. Therefore, neuronal coupling represents a new form of neuronal plasticity in the DRG and contributes to pain hypersensitivity by "hijacking" neighboring neurons through gap junctions. PMID:27568517

  19. Two methods for establishing primary human endometrial stromal cells from hysterectomy specimens.

    PubMed

    Jividen, Kasey; Movassagh, Mercedeh Javanbakht; Jazaeri, Amir; Li, Hui

    2014-01-01

    Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as "the scraping method" and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed "the trypsin method." This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF). PMID:24894444

  20. Characterizing Primary Care Visit Activities at Veterans Health Administration Clinics.

    PubMed

    Gutierrez, Jennifer C; Terwiesch, Christian; Pelak, Mary; Pettit, Amy R; Marcus, Steven C

    2015-01-01

    Medical home models seek to increase efficiency and maximize the use of resources by ensuring that all care team members work at the top of their licenses. We sought to break down primary care office visits into measurable activities to better under stand how primary care providers (PCPs) currently spend visit time and to provide insight into potential opportunities for revision or redistribution of healthcare tasks. We videotaped 27 PCPs during office visits with 121 patients at four Veterans Health Administration medical centers. Based on patterns emerging from the data, we identified a taxonomy of 12 provider activity categories that enabled us to quantify the frequency and duration of activities occurring during routine primary care visits. We conducted descriptive and multivariate analyses to examine associations between visit characteristics and provider and clinic characteristics. We found that PCPs spent the greatest percentage of their visit time discussing existing conditions (20%), discussing new conditions (18%), record keeping (13%), and examining patients (13%). Providers spent the smallest percentage of time on preventive care and coordination of care. Mean visit length was 22.9 minutes (range 7.9-58.0 minutes). Site-level ratings of medical home implementation were not associated with differences in how visit time was spent. These data provide a window into how PCPs are spending face-to-face time with patients. The methodology and taxonomy presented here may prove useful for future quality improvement and research endeavors, particularly those focused on opportunities to increase nonappointment care and to ensure that team members work at the top of their skill level.

  1. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links.

    PubMed

    Clingen, Peter H; Arlett, Colin F; Hartley, John A; Parris, Christopher N

    2007-02-15

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity. PMID:17188678

  2. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

    SciTech Connect

    Clingen, Peter H. . E-mail: p.clingen@ucl.ac.uk; Arlett, Colin F.; Hartley, John A.; Parris, Christopher N.

    2007-02-15

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.

  3. Attachment of human primary osteoblast cells to modified polyethylene surfaces.

    PubMed

    Poulsson, Alexandra H C; Mitchell, Stephen A; Davidson, Marcus R; Johnstone, Alan J; Emmison, Neil; Bradley, Robert H

    2009-04-01

    Ultra-high-molecular-weight polyethylene (UHMWPE) has a long history of use in medical devices, primarily for articulating surfaces due to its inherent low surface energy which limits tissue integration. To widen the applications of UHMWPE, the surface energy can be increased. The increase in surface energy would improve the adsorption of proteins and attachment of cells to allow tissue integration, thereby allowing UHMWPE to potentially be used for a wider range of implants. The attachment and function of human primary osteoblast-like (HOB) cells to surfaces of UHMWPE with various levels of incorporated surface oxygen have been investigated. The surface modification of the UHMWPE was produced by exposure to a UV/ozone treatment. The resulting surface chemistry was studied using X-ray photoelectron spectroscopy (XPS), and the topography and surface structure were probed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which showed an increase in surface oxygen from 11 to 26 atom % with no significant change to the surface topography. The absolute root mean square roughness of both untreated and UV/ozone-treated surfaces was within 350-450 nm, and the water contact angles decreased with increasing oxygen incorporation, i.e., showing an increase in surface hydrophilicity. Cell attachment and functionality were assessed over a 21 day period for each cell-surface combination studied; these were performed using SEM and the alamarBlue assay to study cell attachment and proliferation and energy-dispersive X-ray (EDX) analysis to confirm extracellular mineral deposits, and total protein assay to examine the intra- and extracellular protein expressed by the cells. HOB cells cultured for 21 days on the modified UHMWPE surfaces with 19 and 26 atom % oxygen incorporated showed significantly higher cell densities compared to cells cultured on tissue culture polystyrene (TCPS) from day 3 onward. This indicated that the cells attached and proliferated more

  4. Inquiry-Based Learning in Teacher Education: A Primary Humanities Example

    ERIC Educational Resources Information Center

    Preston, Lou; Harvie, Kate; Wallace, Heather

    2015-01-01

    Inquiry-based learning features strongly in the new Australian Humanities and Social Sciences curriculum and increasingly in primary school practice. Yet, there is little research into, and few exemplars of, inquiry approaches in the primary humanities context. In this article, we outline and explain the implementation of a place-based simulation…

  5. Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

    PubMed

    Strittmatter, Gerhard E; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena; Beer, Hans-Dietmar

    2016-01-01

    Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1. PMID:27665556

  6. The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells

    PubMed Central

    Willumeit-Römer, Regine; Laipple, Daniel; Luthringer, Bérengère; Feyerabend, Frank

    2016-01-01

    Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity. PMID:27327435

  7. Particle resuspension via human activity

    NASA Astrophysics Data System (ADS)

    Qian, Jing

    This dissertation consists of three correlated parts that are related to particle resuspension from floorings in indoor environment. The term resuspension in this dissertation refers the re-entrainment of deposited particles into atmosphere via mechanic disturbances by human activity indoors, except where it is specified. The first part reviews the literature related to particle resuspension. Fundamental concepts and kinetics of resuspension of particles were extracted from previous studies. Suggestions for future research on indoor particle resuspension have been given based on the literature reviews and the findings of part 2 and part 3. The second part involved 54 resuspension experiments conducted in a room-scale environmental chamber. Three floorings types and two ventilation configurations were tested. Air exchange rate were fixed during the experiments, and the temperature/RH were monitored. The airborne particle concentration was measured by an array of optical particle counters (OPCs) in the chamber. Resuspension rates were estimated in size ranges of 0.8--1, 1.0--2.0, 2.0--5.0, and 5.0--10 mum ranging from 10-5--10 -2 hr-1, with higher resuspension rates associated with larger particles. Resuspension via walking activity varied from experiment to experiment. A "heavy and fast" walking style was associated with a higher resuspension rate than a less active style. Given the same floor loading of the test particles, resuspension rates for the carpeted floor were on the same order of magnitude but significantly higher than those for the hard floor. In the third part, an image analysis method (IAM) was adapted to characterize the particle distribution on fabric floorings. The IAM results showed the variability of particles loading on various carpets. The dust particles on fibers from ten carpets vary in sizes. The normal dust loading varies from house to house from 3.6x106 particles/cm2 to 8.2x106 particles/cm2. The dust particle number distribution for size

  8. Human norovirus in untreated sewage and effluents from primary, secondary and tertiary treatment processes.

    PubMed

    Campos, Carlos J A; Avant, Justin; Lowther, James; Till, Dale; Lees, David N

    2016-10-15

    Wastewater treatments are considered important means to control the environmental transmission of human norovirus (NoV). Information about NoV concentrations in untreated and treated effluents, their seasonality and typical removal rates achieved by different treatment processes is required to assess the effectiveness of sewage treatment processes in reducing human exposure to NoV. This paper reports on a characterisation of concentrations of NoV (genogroups I and II) in untreated sewage (screened influent) and treated effluents from five full scale wastewater treatment works (WwTW) in England. Results are shown for effluent samples characteristic of primary- (primary settlement, storm tank overflows), secondary- (activated sludge, trickling filters, humus tanks) and tertiary (UV disinfection) treatments. NoV occurrence in untreated sewage varied between years. This variation was consistent with the annual variation of the virus in the community as indicated by outbreak laboratory reports. Significant differences were found between mean NoV concentrations in effluents subject to different levels of treatment. Primary settlement achieved approximately 1 log10 removal for both genogroups. Concentrations of NoV and Escherichia coli in untreated sewage were of the same order of magnitude of those in storm tank overflows. Of the secondary treatments studied, activated sludge was the most effective in removing NoV with mean log10 removals of 3.11 and 2.34 for GI and GII, respectively. The results of this study provide evidence that monitoring of NoV in raw sewage or treated effluents could provide early warning of an elevated risk for NoV and potentially help prevent outbreaks through environmental exposure. They also provide evidence that elimination of stormwater discharges and improvement of the efficiency of activated sludge for NoV removal would be effective for reducing the risk of environmental transmission. PMID:27470292

  9. Hyperoside protects human primary melanocytes against H2O2-induced oxidative damage

    PubMed Central

    YANG, BIN; YANG, QIN; YANG, XIN; YAN, HONG-BO; LU, QI-PING

    2016-01-01

    Cuscutae semen has been shown to have beneficial effects in the treatment of vitiligo, recorded in the Chinese Pharmacopoeia, whereas the effects of its constituent compounds remains to be elucidated. Using a tetrazolium bromide assay, the present study found that hyperoside (0.5–200 µg/ml) significantly increased the viability of human melanocytes in a time- and dose-dependent manner. The present study used a cell model of hydrogen peroxide (H2O2)-induced oxidative damage to examine the effect of hyperoside on human primary melanocytes. The results demonstrated that hyperoside pretreatment for 2 h decreased cell apoptosis from 54.03±9.11 to 17.46±3.10% in the H2O2-injured melanocytes. The levels of oxidative stress in the mitochondrial membrane potential of the melanocytes increased following hyperoside pretreatment. The mRNA and protein levels of B-cell lymphoma-2/Bcl-2-associated X protein and caspase 3 were regulated by hyperoside, and phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase signaling were also mediated by hyperoside. In conclusion, the results of the present study demonstrated that hyperoside protected the human primary melanocytes against oxidative damage. PMID:27082158

  10. Oxidative Stress Kills Human Primary Oligodendrocytes Via Neutral Sphingomyelinase: Implications for Multiple Sclerosis

    PubMed Central

    Jana, Arundhati

    2007-01-01

    Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system where oxidative stress has been proposed to play an important role in oligodendroglial death. However, molecular mechanisms that couple oxidative stress to the loss of oligodendrocytes are poorly understood. This study underlines the importance of neutral sphingomyelinase–ceramide pathway in mediating oxidative stress-induced apoptosis and cell death of human primary oligodendrocytes. Various oxidative stress-inducing agents, such as, superoxide radical produced by hypoxanthine and xanthine oxidase, hydrogen peroxide, aminotriazole capable of inhibiting catalase and increasing intracellular level of H2O2, or reduced glutathione-depleting diamide induced the activation of neutral sphingomyelinase and the production of ceramide. It is interesting to note that antisense knockdown of neutral but not acidic sphingomyelinase ablated oxidative stress-induced apoptosis and cell death in human primary oligodendrocytes. This study identifies neutral but not acidic sphingomyelinase as a target for possible therapeutic intervention in MS. PMID:18040843

  11. Predicting hand orientation in reach-to-grasp tasks using neural activities from primary motor cortex.

    PubMed

    Zhang, Peng; Ma, Xuan; Huang, Hailong; He, Jiping

    2014-01-01

    Hand orientation is an important control parameter during reach-to-grasp task. In this paper, we presented a study for predicting hand orientation of non-human primate by decoding neural activities from primary motor cortex (M1). A non-human primate subject was guided to do reaching and grasping tasks meanwhile neural activities were acquired by chronically implanted microelectrode arrays. A Support Vector Machines (SVMs) classifier has been trained for predicting three different hand orientations using these M1 neural activities. Different number of neurons were selected and analyzed; the classifying accuracy was 94.1% with 2 neurons and was 100% with 8 neurons. Data from highly event related neuron units contribute a lot to the accuracy of hand orientation prediction. These results indicate that three different hand orientations can be predicted accurately and effectively before the actual movements occurring with a small number of related neurons in M1.

  12. Bisphenol A and Bisphenol S Induce Distinct Transcriptional Profiles in Differentiating Human Primary Preadipocytes

    PubMed Central

    Boucher, Jonathan G.; Gagné, Rémi; Rowan-Carroll, Andrea; Boudreau, Adèle; Yauk, Carole L.; Atlas, Ella

    2016-01-01

    Bisphenol S (BPS) is increasingly used as a replacement plasticizer for bisphenol A (BPA) but its effects on human health have not been thoroughly examined. Recent evidence indicates that both BPA and BPS induce adipogenesis, although the mechanisms leading to this effect are unclear. In an effort to identify common and distinct mechanisms of action in inducing adipogenesis, transcriptional profiles of differentiating human preadipocytes exposed to BPA or BPS were compared. Human subcutaneous primary preadipocytes were differentiated in the presence of either 25 μM BPA or BPS for 2 and 4 days. Poly-A RNA-sequencing was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis. BPA-treatment resulted in 472 and 176 DEGs on days 2 and 4, respectively, affecting pathways such as liver X receptor (LXR)/retinoid X receptor (RXR) activation, hepatic fibrosis and cholestasis. BPS-treatment resulted in 195 and 51 DEGs on days 2 and 4, respectively, revealing enrichment of genes associated with adipogenesis and lipid metabolism including the adipogenesis pathway and cholesterol biosynthesis. Interestingly, the transcription repressor N-CoR was identified as a negative upstream regulator in both BPA- and BPS-treated cells. This study presents the first comparison of BPA- and BPS-induced transcriptional profiles in human differentiating preadipocytes. While we previously showed that BPA and BPS both induce adipogenesis, the results from this study show that BPS affects adipose specific transcriptional changes earlier than BPA, and alters the expression of genes specifically related to adipogenesis and lipid metabolism. The findings provide insight into potential BPS and BPA-mediated mechanisms of action in inducing adipogenesis in human primary preadipocytes. PMID:27685785

  13. Primary human chorionic gonadotropin secreting germinoma of the corpus callosum

    PubMed Central

    Chuan Aaron, Foo Song; Dawn, Chong Q. Q.; Kenneth, Chang T. E.; Hoe, Ng Wai; Yen, Soh Shui; Chee Kian, Tham

    2013-01-01

    Background: Primary intracranial germinomas are a rare subset of intracranial tumors derived from mis-incorporated germ cells within the folding neural plate during embryogenesis. Though known to arise from midline structures in the central nervous system (CNS), occurrence within the corpus callosum is exceedingly rare. Case Description: We present a rare case of secreting primary intracranial germinoma with extensive intraventricular metastasis presenting as a multi-cystic butterfly lesion in the genu of the corpus callosum in a young boy. Conclusion: Intracranial germ cell tumors must be considered for any multi-cystic lesion arising from midline structures in the CNS in the preadult population. PMID:24233184

  14. Direction of Movement Is Encoded in the Human Primary Motor Cortex

    PubMed Central

    Toxopeus, Carolien M.; de Jong, Bauke M.; Valsan, Gopal; Conway, Bernard A.; Leenders, Klaus L.; Maurits, Natasha M.

    2011-01-01

    The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1. PMID:22110768

  15. Direction of movement is encoded in the human primary motor cortex.

    PubMed

    Toxopeus, Carolien M; de Jong, Bauke M; Valsan, Gopal; Conway, Bernard A; Leenders, Klaus L; Maurits, Natasha M

    2011-01-01

    The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1.

  16. [Physical activity in basic and primary prevention of cardiovascular disease].

    PubMed

    Sobieszczańska, Małgorzata; Kałka, Dariusz; Pilecki, Witold; Adamus, Jerzy

    2009-06-01

    On account of the frequency of appearing and character of atherosclerosis cardiac vascular disease, one of the most crucial elements of effective fight against it is preparation of complex preventive programs including as vast number of population as possible. Consequently, Benjamin and Smitch suggested attaching the notion of basic prevention to the standard division into primary and secondary one. The basic prevention, carrying out in the general population, should concern genetic predisposition, psychosocial factors, keeping up proper body weight, healthy eating and physical activity. Especially high hopes are connected with high efficiency, simplicity and low money-consumption of preventive activities associated with physical activity modification, which has a crucial influence on reducing negative impact of atherosclerosis hazard. The results of numerous scientific research, carried out in many countries and on various, large groups, proved undoubtedly that at the healthy adult people of both sex the systematic physical activity of moderate intensification plays an essential part in preventing CVD and decreasing the death risk because of that reason as well. Moreover, systematic physical exercises show many other health-oriented actions, thanks to which they have an influence on decreasing premature and total death rate. The risk of incidence of civilization-related diseases such as diabetes type II, hypertension, obesity, osteoporosis, tumors (of large intestine, breast, prostatic gland) and depression has decreased significantly. Unequivocally positive influence has been proved at many observations dedicated to health recreational physical activity and physical activity connected with professional work based on aerobe effort. The positive effects have been also observed at children population and senior population which is more and more numerous and the most at risk. The beneficial action of physical activity is connected with direct effect on organism

  17. Serodiagnosis of primary infections with human parvovirus 4, Finland.

    PubMed

    Lahtinen, Anne; Kivelä, Pia; Hedman, Lea; Kumar, Arun; Kantele, Anu; Lappalainen, Maija; Liitsola, Kirsi; Ristola, Matti; Delwart, Eric; Sharp, Colin; Simmonds, Peter; Söderlund-Venermo, Maria; Hedman, Klaus

    2011-01-01

    To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle-based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus-infected injection drug users.

  18. [Human ecology and interdisciplinary cooperation for primary prevention of environmental risk factors for public health].

    PubMed

    Dobrowolski, Jan W

    2007-01-01

    training activity in ecologically-based primary prevention. Training in this important field is not adequate in medical, technological, and also natural subjects of studies. There is not enough opportunity for education of the students and graduates toward the application of integrated system approach of new achievements in different sciences and technologies. Interesting are experiences connected with long-term case studies in highly polluted regions in Poland, Japan, India, as well as exchange of methodological experiences during the series of International Summer Schools on the Human Environment from 1972, as well as during series of 11 International Conferences on Sustainable Development organized at AGH-UST from 1989 to 2006 and Polish Conferences in 2004 and 2007. It seems necessary not only to develop a training of experts that would be adequate to present needs, but also education of the whole society (including formal activities at all levels of education) as well as informal education (e.g. at Open Universities and Distance Education, based on the Internet) to achieve the integration of activity of scientists, practitioners and the whole society. It would be useful to focus this activity on crucial problems and selected regions. Let me propose as the top priority for inhabitants of Tarnow region as well as pilot projects for Poland; utilization of all possible achievements of science and technology for primary prevention of health hazard for inhabitants of Gmina Szczucin that is very polluted by asbestos, and also model management reducing risk factors for the natural environment and health of inhabitants in the regions of new motor-ways, as well as better primary prevention against flood accidents and connected with their effects (higher humidity of housing environment and its contamination by toxinogenic moulds) risk factors for health of communities living in rivers regions. For the purpose of optimisation of preventive action, it is necessary not only to

  19. [Human ecology and interdisciplinary cooperation for primary prevention of environmental risk factors for public health].

    PubMed

    Dobrowolski, Jan W

    2007-01-01

    training activity in ecologically-based primary prevention. Training in this important field is not adequate in medical, technological, and also natural subjects of studies. There is not enough opportunity for education of the students and graduates toward the application of integrated system approach of new achievements in different sciences and technologies. Interesting are experiences connected with long-term case studies in highly polluted regions in Poland, Japan, India, as well as exchange of methodological experiences during the series of International Summer Schools on the Human Environment from 1972, as well as during series of 11 International Conferences on Sustainable Development organized at AGH-UST from 1989 to 2006 and Polish Conferences in 2004 and 2007. It seems necessary not only to develop a training of experts that would be adequate to present needs, but also education of the whole society (including formal activities at all levels of education) as well as informal education (e.g. at Open Universities and Distance Education, based on the Internet) to achieve the integration of activity of scientists, practitioners and the whole society. It would be useful to focus this activity on crucial problems and selected regions. Let me propose as the top priority for inhabitants of Tarnow region as well as pilot projects for Poland; utilization of all possible achievements of science and technology for primary prevention of health hazard for inhabitants of Gmina Szczucin that is very polluted by asbestos, and also model management reducing risk factors for the natural environment and health of inhabitants in the regions of new motor-ways, as well as better primary prevention against flood accidents and connected with their effects (higher humidity of housing environment and its contamination by toxinogenic moulds) risk factors for health of communities living in rivers regions. For the purpose of optimisation of preventive action, it is necessary not only to

  20. Active supports and force optimization for the MMT primary mirror

    NASA Astrophysics Data System (ADS)

    Martin, Hubert M.; Callahan, Shawn P.; Cuerden, Brian; Davison, Warren B.; Derigne, S. T.; Dettmann, Lee R.; Parodi, G.; Trebisky, T. J.; West, Steve C.; Williams, Joseph T.

    1998-08-01

    We describe the active support system and optimization of support forces for the 6.5 m primary mirror for the Multiple Mirror Telescope Conversion. The mirror was figured to an accuracy of 26 nm rms surface error, excluding certain flexible bending modes that will be controlled by support forces in the telescope. On installation of the mirror into its telescope support cell, an initial optimization of support forces is needed because of minor differences between the support used during fabrication and that in the telescope cell. The optimization is based on figure measurements made interferometrically in the vibration- isolated test tower of the Steward Observatory Mirror Lab. Actuator influence functions were determined by finite- element analysis and verified by measurement. The optimization is performed by singular value decomposition of the influence functions into normal modes. Preliminary results give a wavefront accuracy better than that of the atmosphere in 0.11 arcsecond seeing.

  1. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies.

  2. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  3. Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

    PubMed

    Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

    2015-01-01

    We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

  4. Characterization of primary human keratinocytes transformed by human papillomavirus type 18

    SciTech Connect

    Kaur, P.; McDougall, J.K. )

    1988-06-01

    Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.

  5. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells.

    PubMed

    Haye, Kester; Burmakina, Svetlana; Moran, Thomas; García-Sastre, Adolfo; Fernandez-Sesma, Ana

    2009-07-01

    The NS1 protein of the influenza A virus is a potent virulence factor that inhibits type I interferon (IFN) synthesis, allowing the virus to overcome host defenses and replicate efficiently. However, limited studies have been conducted on NS1 function using human virus strains and primary human cells. We used NS1 truncated mutant influenza viruses derived from the human isolate influenza A/TX/91 (TX WT, where WT is wild type) to study the functions of NS1 in infected primary cells. Infection of primary differentiated human tracheo-bronchial epithelial cells with an NS1 truncated mutant demonstrated limited viral replication and enhanced type I IFN induction. Additionally, human dendritic cells (DCs) infected with human NS1 mutant viruses showed higher levels of activation and stimulated naïve T-cells better than TX WT virus-infected DCs. We also compared infections of DCs with TX WT and our previously characterized laboratory strain A/PR/8/34 (PR8) and its NS1 knockout strain, deltaNS1. TX WT-infected DCs displayed higher viral replication than PR8 but had decreased antiviral gene expression at late time points and reduced naïve T-cell stimulation compared to PR8 infections, suggesting an augmented inhibition of IFN production and human DC activation. Our findings show that human-derived influenza A viruses have a high capacity to inhibit the antiviral state in a human system, and here we have evaluated the possible mechanism of this inhibition. Lastly, C-terminal truncations in the NS1 protein of human influenza virus are sufficient to make the virus attenuated and more immunogenic, supporting its use as a live attenuated influenza vaccine in humans.

  6. Simulation of Human Plasma Concentrations of Thalidomide and Primary 5-Hydroxylated Metabolites Explored with Pharmacokinetic Data in Humanized TK-NOG Mice.

    PubMed

    Nishiyama, Sayako; Suemizu, Hiroshi; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2015-11-16

    Plasma concentrations of thalidomide and primary 5-hydroxylated metabolites including 5,6-dihydroxythalidomide and glutathione (GSH) conjugate(s) were investigated in chimeric mice with highly "humanized" liver cells harboring cytochrome P450 3A5*1. Following oral administration of thalidomide (100 mg/kg), plasma concentrations of GSH conjugate(s) of 5-hydroxythalidomide were higher in humanized mice than in controls. Simulation of human plasma concentrations of thalidomide were achieved with a simplified physiologically based pharmacokinetic model in accordance with reported thalidomide concentrations. The results indicate that the pharmacokinetics in humans of GSH conjugate and/or catechol primary 5-hydroxylated thalidomide contributing in vivo activation can be estimated for the first time. PMID:26492539

  7. Simulation of Human Plasma Concentrations of Thalidomide and Primary 5-Hydroxylated Metabolites Explored with Pharmacokinetic Data in Humanized TK-NOG Mice.

    PubMed

    Nishiyama, Sayako; Suemizu, Hiroshi; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

    2015-11-16

    Plasma concentrations of thalidomide and primary 5-hydroxylated metabolites including 5,6-dihydroxythalidomide and glutathione (GSH) conjugate(s) were investigated in chimeric mice with highly "humanized" liver cells harboring cytochrome P450 3A5*1. Following oral administration of thalidomide (100 mg/kg), plasma concentrations of GSH conjugate(s) of 5-hydroxythalidomide were higher in humanized mice than in controls. Simulation of human plasma concentrations of thalidomide were achieved with a simplified physiologically based pharmacokinetic model in accordance with reported thalidomide concentrations. The results indicate that the pharmacokinetics in humans of GSH conjugate and/or catechol primary 5-hydroxylated thalidomide contributing in vivo activation can be estimated for the first time.

  8. Phenotypic differentiation does not affect tumorigenicity of primary human colon cancer initiating cells.

    PubMed

    Dubash, Taronish D; Hoffmann, Christopher M; Oppel, Felix; Giessler, Klara M; Weber, Sarah; Dieter, Sebastian M; Hüllein, Jennifer; Zenz, Thorsten; Herbst, Friederike; Scholl, Claudia; Weichert, Wilko; Werft, Wiebke; Benner, Axel; Schmidt, Manfred; Schneider, Martin; Glimm, Hanno; Ball, Claudia R

    2016-02-28

    Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions. Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.

  9. Integrin activation controls metastasis in human breast cancer

    NASA Astrophysics Data System (ADS)

    Felding-Habermann, Brunhilde; O'Toole, Timothy E.; Smith, Jeffrey W.; Fransvea, Emilia; Ruggeri, Zaverio M.; Ginsberg, Mark H.; Hughes, Paul E.; Pampori, Nisar; Shattil, Sanford J.; Saven, Alan; Mueller, Barbara M.

    2001-02-01

    Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin v3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin v3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

  10. Chromosomal translocation engineering to recapitulate primary events of human cancer.

    PubMed

    Forster, A; Pannell, R; Drynan, L; Cano, F; Chan, N; Codrington, R; Daser, A; Lobato, N; Metzler, M; Nam, C-H; Rodriguez, S; Tanaka, T; Rabbitts, T

    2005-01-01

    Mouse models of human cancers are important for understanding determinants of overt disease and for "preclinical" development of rational therapeutic strategies; for instance, based on macrodrugs. Chromosomal translocations underlie many human leukemias, sarcomas, and epithelial tumors. We have developed three technologies based on homologous recombination in mouse ES cells to mimic human chromosome translocations. The first, called the knockin method, allows creation of fusion genes like those typical of translocations of human leukemias and sarcomas. Two new conditional chromosomal translocation mimics have been developed. The first is a method for generating reciprocal chromosomal translocations de novo using Cre-loxP recombination (translocator mice). In some cases, there is incompatible gene orientation and the translocator model cannot be applied. We have developed a different model (invertor mice) for these situations. This method consists of introducing an inverted cDNA cassette into the intron of a target gene and bringing the cassette into the correct transcriptional orientation by Cre-loxP recombination. We describe experiments using the translocator model to generate MLL-mediated neoplasias and the invertor method to generate EWS-ERG-mediated cancer. These methods mimic the situation found in human chromosome translocations and provide the framework for design and study of human chromosomal translocations in mice.

  11. Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

    PubMed

    Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

    2015-10-23

    The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells.

  12. Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture.

    PubMed

    Weidner, Maria; Lenczyk, Marlies; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2013-03-18

    The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times. PMID:23363530

  13. Rapid Reprogramming of Primary Human Astrocytes into Potent Tumor-Initiating Cells with Defined Genetic Factors.

    PubMed

    Li, Fang; Liu, Xinjian; Sampson, John H; Bigner, Darell D; Li, Chuan-Yuan

    2016-09-01

    Cancer stem-like cells (CSC) are thought to drive brain cancer, but their cellular and molecular origins remain uncertain. Here, we report the successful generation of induced CSC (iCSC) from primary human astrocytes through the expression of defined genetic factors. Combined transduction of four factors, Myc, Oct-4, p53DD, and Ras, induced efficient transformation of primary human astrocytes into malignant cells with powerful tumor-initiating capabilities. Notably, transplantation of 100 transduced cells into nude mice was sufficient for tumor formation. The cells showed unlimited self-renewal ability with robust telomerase activities. In addition, they expressed typical glioma stem-like cell markers, such as CD133, CD15, and CD90. Moreover, these cells could form spheres in culture and differentiate into neuron-like, astrocyte-like, and oligodendrocyte-like cells. Finally, they also displayed resistance to the widely used brain cancer drug temozolomide. These iCSCs could provide important tools for studies of glioma biology and therapeutics development. Cancer Res; 76(17); 5143-50. ©2016 AACR. PMID:27364552

  14. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.

    PubMed

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-08-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  15. Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection▿

    PubMed Central

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B.; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L.; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-01-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  16. Lost and Found: Music Activities Delivered by Primary Classroom Generalists

    ERIC Educational Resources Information Center

    King, Fiona

    2015-01-01

    Primary classroom teachers can play a vital role in the music education of primary school students, providing a basis for lifelong learning in music and the arts. Research shows that not all Victorian primary school students have equitable access to music education and that the role of the classroom teacher becomes valuable in supplying or…

  17. Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

    SciTech Connect

    Xie, Yuchao; McGill, Mitchell R.; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Jaeschke, Hartmut

    2014-09-15

    Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to

  18. Antimicrobial activity of filling materials used in primary teeth pulpotomy.

    PubMed

    Pimenta, Hévelin Couto; Borges, Álvaro Henrique; Bandeca, Matheus Coelho; Neves, Ana Thereza Sabóia; Fontes, Rodrigo Gusmão; da Silva, Priscila Vieira; Aranha, Andreza Maria Fábio

    2015-04-01

    The aim of this study was to investigate the antibacterial activity of pulp capping materials used in primary teeth (formocresol [FC], zinc oxide and eugenol cement [ZOE], ZOE mixed with FC [ZOEFC], mineral trioxide aggregate [MTA] and calcium hydroxide [CH]) against cariogenic bacteria. The agar plate diffusion test was used for the cultures, including saline solution as a negative control. A base layer of 15 mL of brain heart infusion agar was inoculated with 300 mL of each inoculum. Twelve wells were made and completely filled with one of the testing materials for each bacteria strain. The plates were incubated at 37°C for 48 h. Zones of microbial inhibition and material diffusion were measured and photographed. The results obtained were analyzed by Kruskal-Wallis and Mann-Whitney non-parametric tests. Respectively, the medium zones of bacteria inhibition of FC, ZOE, ZOEFC, MTA and CH against Streptococcus mutans growth were 28.5, 15.2, 20.8, 9.3 and 11.6; against Lactobacillus acidophilus growth were 28.7, 14.8, 15.3, 15.2 and 20.0, and against Actinomyces viscosus growth were 13.6, 13.5, 14.7, 10.0 and 13.6. We might confirmed the high antibacterial activity of FC solution, especially against S. mutans and L. acidophilus, as wells as, the low inhibitory effect of MTA cement on the cariogenic bacteria studied.

  19. Antimicrobial Activity of Filling Materials Used in Primary Teeth Pulpotomy

    PubMed Central

    Pimenta, Hévelin Couto; Borges, Álvaro Henrique; Bandeca, Matheus Coelho; Neves, Ana Thereza Sabóia; Fontes, Rodrigo Gusmão; da Silva, Priscila Vieira; Aranha, Andreza Maria Fábio

    2015-01-01

    The aim of this study was to investigate the antibacterial activity of pulp capping materials used in primary teeth (formocresol [FC], zinc oxide and eugenol cement [ZOE], ZOE mixed with FC [ZOEFC], mineral trioxide aggregate [MTA] and calcium hydroxide [CH]) against cariogenic bacteria. The agar plate diffusion test was used for the cultures, including saline solution as a negative control. A base layer of 15 mL of brain heart infusion agar was inoculated with 300 mL of each inoculum. Twelve wells were made and completely filled with one of the testing materials for each bacteria strain. The plates were incubated at 37°C for 48 h. Zones of microbial inhibition and material diffusion were measured and photographed. The results obtained were analyzed by Kruskal–Wallis and Mann–Whitney non-parametric tests. Respectively, the medium zones of bacteria inhibition of FC, ZOE, ZOEFC, MTA and CH against Streptococcus mutans growth were 28.5, 15.2, 20.8, 9.3 and 11.6; against Lactobacillus acidophilus growth were 28.7, 14.8, 15.3, 15.2 and 20.0, and against Actinomyces viscosus growth were 13.6, 13.5, 14.7, 10.0 and 13.6. We might confirmed the high antibacterial activity of FC solution, especially against S. mutans and L. acidophilus, as wells as, the low inhibitory effect of MTA cement on the cariogenic bacteria studied. PMID:25954072

  20. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  1. Learning and Recognition of a Non-conscious Sequence of Events in Human Primary Visual Cortex

    PubMed Central

    Rosenthal, Clive R.; Andrews, Samantha K.; Antoniades, Chrystalina A.; Kennard, Christopher; Soto, David

    2016-01-01

    Summary Human primary visual cortex (V1) has long been associated with learning simple low-level visual discriminations [1] and is classically considered outside of neural systems that support high-level cognitive behavior in contexts that differ from the original conditions of learning, such as recognition memory [2, 3]. Here, we used a novel fMRI-based dichoptic masking protocol—designed to induce activity in V1, without modulation from visual awareness—to test whether human V1 is implicated in human observers rapidly learning and then later (15–20 min) recognizing a non-conscious and complex (second-order) visuospatial sequence. Learning was associated with a change in V1 activity, as part of a temporo-occipital and basal ganglia network, which is at variance with the cortico-cerebellar network identified in prior studies of “implicit” sequence learning that involved motor responses and visible stimuli (e.g., [4]). Recognition memory was associated with V1 activity, as part of a temporo-occipital network involving the hippocampus, under conditions that were not imputable to mechanisms associated with conscious retrieval. Notably, the V1 responses during learning and recognition separately predicted non-conscious recognition memory, and functional coupling between V1 and the hippocampus was enhanced for old retrieval cues. The results provide a basis for novel hypotheses about the signals that can drive recognition memory, because these data (1) identify human V1 with a memory network that can code complex associative serial visuospatial information and support later non-conscious recognition memory-guided behavior (cf. [5]) and (2) align with mouse models of experience-dependent V1 plasticity in learning and memory [6]. PMID:26948883

  2. Learning and Recognition of a Non-conscious Sequence of Events in Human Primary Visual Cortex.

    PubMed

    Rosenthal, Clive R; Andrews, Samantha K; Antoniades, Chrystalina A; Kennard, Christopher; Soto, David

    2016-03-21

    Human primary visual cortex (V1) has long been associated with learning simple low-level visual discriminations [1] and is classically considered outside of neural systems that support high-level cognitive behavior in contexts that differ from the original conditions of learning, such as recognition memory [2, 3]. Here, we used a novel fMRI-based dichoptic masking protocol-designed to induce activity in V1, without modulation from visual awareness-to test whether human V1 is implicated in human observers rapidly learning and then later (15-20 min) recognizing a non-conscious and complex (second-order) visuospatial sequence. Learning was associated with a change in V1 activity, as part of a temporo-occipital and basal ganglia network, which is at variance with the cortico-cerebellar network identified in prior studies of "implicit" sequence learning that involved motor responses and visible stimuli (e.g., [4]). Recognition memory was associated with V1 activity, as part of a temporo-occipital network involving the hippocampus, under conditions that were not imputable to mechanisms associated with conscious retrieval. Notably, the V1 responses during learning and recognition separately predicted non-conscious recognition memory, and functional coupling between V1 and the hippocampus was enhanced for old retrieval cues. The results provide a basis for novel hypotheses about the signals that can drive recognition memory, because these data (1) identify human V1 with a memory network that can code complex associative serial visuospatial information and support later non-conscious recognition memory-guided behavior (cf. [5]) and (2) align with mouse models of experience-dependent V1 plasticity in learning and memory [6]. PMID:26948883

  3. Development of an Advanced Primary Human In Vitro Model of the Small Intestine.

    PubMed

    Schweinlin, Matthias; Wilhelm, Sabine; Schwedhelm, Ivo; Hansmann, Jan; Rietscher, Rene; Jurowich, Christian; Walles, Heike; Metzger, Marco

    2016-09-01

    Intestinal in vitro models are valuable tools in drug discovery and infection research. Despite several advantages, the standard cell line-based Transwell(®) models based for example on colonic epithelial Caco-2 cells, lack the cellular complexity and transport activity associated with native small intestinal tissue. An additional experimental set-back arises from the most commonly used synthetic membranes, on which the cells are routinely cultured. These can lead to an additional barrier activity during in vitro testing. To overcome these limitations, we developed an alternative primary human small intestinal tissue model. This novel approach combines previously established gut organoid technology with a natural extracellular matrix (ECM) based on porcine small intestinal scaffold (SIS). Intestinal crypts from healthy human small intestine were expanded as gut organoids and seeded as single cells on SIS in a standardized Transwell-like setting. After only 7 days on the ECM scaffold, the primary cells formed an epithelial barrier while a subpopulation differentiated into intestinal specific cell types such as mucus-producing goblet cells or hormone-secreting enteroendocrine cells. Furthermore, we tested the influence of subepithelial fibroblasts and dynamic culture conditions on epithelial barrier function. The barrier integrity was stabilized by coculture in the presence of gut-derived fibroblasts. Compared to static or dynamic culture on an orbital shaker, dynamic culture in a defined perfusion bioreactor had an additional significant impact on epithelial cell differentiation, indicated by high prismatic cell morphology and upregulation of CYP3A4 enzyme and Mdr1 transporter activity. In summary, more physiological tissue models as presented in our study might be useful tools in preclinical research and development. PMID:27481569

  4. Electrical Stimulation Modulates the Expression of Multiple Wound Healing Genes in Primary Human Dermal Fibroblasts.

    PubMed

    Park, Hyun Jin; Rouabhia, Mahmoud; Lavertu, Denis; Zhang, Ze

    2015-07-01

    This study profiled multiple human dermal fibroblast wound-healing genes in response to electrical stimulation (ES) by using an RT(2) profiler PCR-Array system. Primary human skin fibroblasts were seeded on heparin (HE)-bioactivated polypyrrole (PPy)/poly(l-lactic acid) (PLLA) conductive membranes, cultured, and subsequently exposed to ES of 50 or 200 mV/mm for 6 h. Following ES, the cells were used to extract RNA for gene profiling, and culture supernatants were used to measure the level of the different wound healing mediators. A total of 57 genes were affected (activated/repressed) by ES; among these, 49 were upregulated and 8 were downregulated. ES intensities at 50 and 200 mV/mm activated/repressed different genes. The ES-modulated genes are involved in cell adhesion, remodeling and spreading, cytoskeletal activity, extracellular matrix metabolism, production of inflammatory cytokines/chemokines and growth factors, as well as signal transduction. The expression of several genes was supported by protein production. Protein analyses showed that ES increased CCL7, KGF, and TIMP2, but reduced MMP2. This study demonstrated that ES modulates the expression of a variety of genes involved in the wound healing process, confirming that ES is a useful tool in regenerative medicine. PMID:25873313

  5. Dengue-2 infection and the induction of apoptosis in human primary monocytes.

    PubMed

    Torrentes-Carvalho, Amanda; Azeredo, Elzinandes L; Reis, Sonia R I; Miranda, Alessandro S; Gandini, Mariana; Barbosa, Luciana S; Kubelka, Claire F

    2009-12-01

    Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-alpha and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

  6. Functional expression of voltage-gated sodium channels in primary cultures of human cervical cancer.

    PubMed

    Diaz, Daniel; Delgadillo, Dulce Maria; Hernández-Gallegos, Elizabeth; Ramírez-Domínguez, Martha Eugenia; Hinojosa, Luz María; Ortiz, Cindy Sharon; Berumen, Jaime; Camacho, Javier; Gomora, Juan Carlos

    2007-02-01

    Cervical cancer (CaC) is the third most frequent cause of death from cancer among women in the world and the first in females of developing countries. Several ion channels are upregulated in cancer, actually potassium channels have been suggested as tumor markers and therapeutic targets for CaC. Voltage-gated sodium channels (VGSC) activity is involved in proliferation, motility, and invasion of prostate and breast cancer cells; however, the participation of this type of channels in CaC has not been explored. In the present study, we identified both at the molecular and electrophysiological level VGSC in primary cultures from human cervical carcinoma biopsies. With the whole cell patch clamp technique, we isolated and identified a voltage-gated Na(+) current as the main component of the inward current in all investigated cells. Sodium current was characterized by its kinetics, voltage dependence, sensitivity to tetrodotoxin (TTX) block and dependence to [Na(+)](o). By analyzing the expression of mRNAs encoding TTX-sensitive Na(+) channel alpha subunits with standard RT-PCR and specific primers, we detected Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 transcripts in total RNA obtained from primary cultures and biopsies of CaC. Restriction enzyme analysis of PCR products was consistent with the molecular nature of the corresponding genes. Notably, only transcripts for Na(v)1.4 sodium channels were detected in biopsies from normal cervix. The results show for the first time the functional expression of VGSC in primary cultures from human CaC, and suggest that these channels might be considered as potential molecular markers for this type of cancer.

  7. Primary Sources of Cognate Research in Human Performance Technology

    ERIC Educational Resources Information Center

    Johnsen, Liz V.; Huglin, Linda M.; Marker, Anthony

    2008-01-01

    This article is third in a series written to address questions regarding the need for more empirical research in the field of human performance technology (HPT) and the need to determine the future direction of HPT research. The call for more empirical research has been published in journals such as "Performance Improvement Quarterly" and…

  8. Physical Activity of Malaysian Primary School Children: Comparison by Sociodemographic Variables and Activity Domains.

    PubMed

    Wong, Jyh Eiin; Parikh, Panam; Poh, Bee Koon; Deurenberg, Paul

    2016-07-01

    This study describes the physical activity of primary school children according to sociodemographic characteristics and activity domains. Using the Malaysian South East Asian Nutrition Surveys data, 1702 children aged 7 to 12 years were included in the analysis. Physical activity was reported as a total score and categorized into low, medium, and high levels based on Physical Activity Questionnaire for Older Children. Higher overall activity scores were found in boys, younger age, non-Chinese ethnicity, and normal body mass index category. Sex, age, and ethnicity differences were found in structured or organized, physical education, and outside-of-school domain scores. Transport-related scores differed by age group, ethnicity, household income, and residential areas but not among the three physical activity levels. Participation of girls, Chinese, and older children were low in overall and almost all activity domains. Sociodemographic characteristics are important factors to consider in increasing the different domains of physical activity among Malaysian children. PMID:27257293

  9. RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans

    PubMed Central

    Shamseldin, Hanan; Alazami, Anas M.; Manning, Melanie; Hashem, Amal; Caluseiu, Oana; Tabarki, Brahim; Esplin, Edward; Schelley, Susan; Innes, A. Micheil; Parboosingh, Jillian S.; Lamont, Ryan; Majewski, Jacek; Bernier, Francois P.; Alkuraya, Fowzan S.

    2015-01-01

    Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963∗] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration. PMID:26608784

  10. Effects of Simulated Microgravity on Primary Human NK Cells

    PubMed Central

    Li, Qi; Mei, Qibing; Huyan, Ting; Xie, Li; Che, Su; Yang, Hui; Zhang, Mingjie

    2013-01-01

    Abstract The deleterious effects of microgravity on lymphocytes have been demonstrated in previous studies. However, research on the effects of microgravity on human natural killer (NK) cells remains exceedingly limited. In this study, we demonstrated that NK cell cytotoxicity was significantly decreased under simulated microgravity (SMG) conditions (p<0.05). Several processes, including apoptosis, receptor expression, and cytokine secretion, were investigated in human NK cells under SMG. We observed decreased cytotoxicity, concurrent with increased apoptosis and necrosis, in NK cells after exposure to SMG (p<0.05). Additionally, interferon (IFN)-γ and perforin expression decreased significantly, and the expression of granzyme-B was only slightly reduced. Meanwhile, SMG selectively inhibited the expression of certain surface receptors on NK cells. Specifically, the expression of NKG2A and NKG2D were significantly downregulated under SMG, but the expression of NKp30 and NKp44 was not affected. We also found that interleukin (IL)–15 alone or in combination with IL-12 could counteract the inhibition of NK cell cytotoxicity under SMG. Our findings indicate that human NK cells were sensitive to SMG, as reflected by their decreased cytotoxicity. Factors such as increased early apoptosis and late apoptosis/necrosis and the decreased expression of INF-γ, cytolytic proteins, and cell surface receptors may be responsible for the loss of cytotoxicity in human NK cells under SMG. A combination of IL-12 and IL-15 may be useful as a therapeutic strategy for overcoming the effects of microgravity on human NK cells during long space missions. Key Words: Simulated microgravity (SMG)—Natural killer (NK) cells—Cytotoxicity. Astrobiology 13, 703–714. PMID:23919749

  11. Devolution and human resources in primary healthcare in rural Mali

    PubMed Central

    2011-01-01

    Devolution, as other types of decentralization (e.g. deconcentration, delegation, privatization), profoundly changes governance relations in the health system. Devolution is meant to affect performance of the health system by transferring responsibilities and authority to locally elected governments. The key question of this article is: what does devolution mean for human resources for health in Mali? This article assesses the key advantages and dilemmas associated with devolution such as responsiveness to local needs, downward accountability and health worker retention. Challenges of politics and capacities are also addressed in relation to human resources for health at the local level. Examples are derived from experiences in Mali with a capacity development programme and from case studies of other countries. It is not research findings that are presented, but highlights of key issues at stake aimed at inspiring the debate in Mali and elsewhere. A first lesson from the discussion suggests that in the context of human resources for health, decentralization of authority and resources is not the main issue. The challenge is to develop or strengthen accountability of those who decide and act, whether they are local politicians, bureaucrats or community representatives. If decentralization policies do not address public accountability, they will not fundamentally change human resource management, quality and equity of staffing. A second lesson is that successful devolution requires innovations in capacity development of all actors involved and in designing effective incentive measures. A final key conclusion is that the topic of devolution policy and its effects on human resources for health, and vice versa, merit more attention. A better understanding may lead to more appropriate policy designs and better preparation for the actors involved in countries that are embarking on decentralization, as is the case in Mali. PMID:21651817

  12. Devolution and human resources in primary healthcare in rural Mali.

    PubMed

    Lodenstein, Elsbet; Dao, Dramane

    2011-06-08

    Devolution, as other types of decentralization (e.g. deconcentration, delegation, privatization), profoundly changes governance relations in the health system. Devolution is meant to affect performance of the health system by transferring responsibilities and authority to locally elected governments. The key question of this article is: what does devolution mean for human resources for health in Mali?This article assesses the key advantages and dilemmas associated with devolution such as responsiveness to local needs, downward accountability and health worker retention. Challenges of politics and capacities are also addressed in relation to human resources for health at the local level. Examples are derived from experiences in Mali with a capacity development programme and from case studies of other countries. It is not research findings that are presented, but highlights of key issues at stake aimed at inspiring the debate in Mali and elsewhere.A first lesson from the discussion suggests that in the context of human resources for health, decentralization of authority and resources is not the main issue. The challenge is to develop or strengthen accountability of those who decide and act, whether they are local politicians, bureaucrats or community representatives. If decentralization policies do not address public accountability, they will not fundamentally change human resource management, quality and equity of staffing. A second lesson is that successful devolution requires innovations in capacity development of all actors involved and in designing effective incentive measures. A final key conclusion is that the topic of devolution policy and its effects on human resources for health, and vice versa, merit more attention. A better understanding may lead to more appropriate policy designs and better preparation for the actors involved in countries that are embarking on decentralization, as is the case in Mali.

  13. Patterns of invasive growth in vitro. Human decidua graviditatis confronted with established human cell lines and primary human explants.

    PubMed

    Schleich, A B; Frick, M; Mayer, A

    1976-02-01

    Human decidua graviditatis was used as a receptor tissue to demonstrate invasive properties of human cells in vitro. In the confrontation between decidua and established cell lines of tumors and of normal origin as well as primary explanted cells, we noted differences in the cells' interactions with decidual tissue. The two original tumor cell lines, HeLa (carcinoma) and AFi (sarcoma), and a spontaneously transformed lymphoblastoid cell line showed aggressiveness, i.e., invasion, rapid proliferation, injury, and destruction of the decidual tissue. Strain HeLa S3 and two established cell lines, FL (amnion) and Girardi Heart, which are regarded as transformed cells because of their increased mitotic rate and polyploidy, exhibited various degrees of aggressiveness but did not invade. Freshly explanted fetal lung and endometrium caused no injury to the receptor tissue and were included in the decidual tissue.

  14. Human activity recognition based on human shape dynamics

    NASA Astrophysics Data System (ADS)

    Cheng, Zhiqing; Mosher, Stephen; Cheng, Huaining; Webb, Timothy

    2013-05-01

    Human activity recognition based on human shape dynamics was investigated in this paper. The shape dynamics describe the spatial-temporal shape deformation of a human body during its movement and thus provide important information about the identity of a human subject and the motions performed by the subject. The dynamic shapes of four subjects in five activities (digging, jogging, limping, throwing, and walking) were created via 3-D motion replication. The Paquet Shape Descriptor (PSD) was used to describe subject shapes in each frame. The principal component analysis was performed on the calculated PSDs and principal components (PCs) were used to characterize PSDs. The PSD calculation was then reasonably approximated by its significant projections in the eigen-space formed by PCs and represented by the corresponding projection coefficients. As such, the dynamic human shapes for each activity were described by these projection coefficients, which in turn, along with their derivatives were used to form the feature vectors (attribute sets) for activity classification. Data mining technology was employed with six classification methods used. Seven attribute sets were evaluated with high classification accuracy attained for most of them. The results from this investigation illustrate the great potential of human shape dynamics for activity recognition.

  15. Transcriptome Patterns from Primary Cutaneous Leishmania braziliensis Infections Associate with Eventual Development of Mucosal Disease in Humans

    PubMed Central

    Maretti-Mira, Ana Claudia; Bittner, Jaime; Oliveira-Neto, Manoel Paes; Liu, Minghsun; Kang, Dezhi; Li, Huiying; Pirmez, Claude; Craft, Noah

    2012-01-01

    Introduction Localized Cutaneous Leishmaniasis (LCL) and Mucosal Leishmaniasis (ML) are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not. Methods Using RNA-seq, we analyzed both the human and Leishmania transcriptomes in primary cutaneous lesions. Results Limited number of reads mapping to Leishmania transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome. Conclusions We were able to simultaneously sequence both human and Leishmania mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of L. braziliensis infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity. PMID:23029578

  16. Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes.

    PubMed Central

    Schubert, U; Clouse, K A; Strebel, K

    1995-01-01

    The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunodeficiency virus type 1: a primary isolate, AD8+, and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8+ or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4+ T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used. PMID:7494279

  17. Sensing Human Activity: GPS Tracking

    PubMed Central

    van der Spek, Stefan; van Schaick, Jeroen; de Bois, Peter; de Haan, Remco

    2009-01-01

    The enhancement of GPS technology enables the use of GPS devices not only as navigation and orientation tools, but also as instruments used to capture travelled routes: as sensors that measure activity on a city scale or the regional scale. TU Delft developed a process and database architecture for collecting data on pedestrian movement in three European city centres, Norwich, Rouen and Koblenz, and in another experiment for collecting activity data of 13 families in Almere (The Netherlands) for one week. The question posed in this paper is: what is the value of GPS as ‘sensor technology’ measuring activities of people? The conclusion is that GPS offers a widely useable instrument to collect invaluable spatial-temporal data on different scales and in different settings adding new layers of knowledge to urban studies, but the use of GPS-technology and deployment of GPS-devices still offers significant challenges for future research. PMID:22574061

  18. Expression of GD2 ganglioside by untreated primary human neuroblastomas.

    PubMed

    Wu, Z L; Schwartz, E; Seeger, R; Ladisch, S

    1986-01-01

    Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the frequency of tumors expressing a specific glycosphingolipid, GD2 ganglioside. Total tissue gangliosides were purified by a new partition method, quantitated, and analyzed by high-performance thin-layer chromatography. All 36 neuroblastoma tumors, representing all clinical stages, contained GD2 ganglioside. The mean relative and absolute concentrations of GD2 were substantial (12% of the total tissue gangliosides and 50 nmol/g of tissue) and were independent of the clinical stage of the tumor. In contrast, 6 samples of related but more differentiated tumors (ganglioneuroblastoma and ganglioneuroma) had little or no detectable GD2 (less than or equal to 1.5% of total gangliosides and less than or equal to 4 nmol/g of tissue). These results suggest that GD2 is a sensitive marker for neuroblastoma tissue and may be an excellent target antigen for immunotherapy of this tumor.

  19. Interferon Alpha as a Primary Pathogenic Factor in Human Lupus

    PubMed Central

    2011-01-01

    Interferon alpha (IFN-α) is a critical mediator of human systemic lupus erythematosus (SLE). This review will summarize evidence supporting the role for IFN-α in the initiation of human SLE. IFN-α functions in viral immunity at the interface of innate and adaptive immunity, a position well suited to setting thresholds for autoimmunity. Some individuals treated with IFN-α for chronic viral infections develop de novo SLE, which frequently resolves when IFN-α is withdrawn, supporting the idea that IFN-α was causal. Abnormally high IFN-α levels are clustered within SLE families, suggesting that high serum IFN-α is a heritable risk factor for SLE. Additionally, SLE-risk genetic variants in the IFN-α pathway are gain of function in nature, resulting in either higher circulating IFN-α levels or greater sensitivity to IFN-α signaling in SLE patients. A recent genome-wide association study has identified additional novel genetic loci associated with high serum IFN-α in SLE patients. These data support the idea that genetically determined endogenous elevations in IFN-α predispose to human SLE. It is possible that some of these gain-of-function polymorphisms in the IFN-α pathway are useful in viral defense, and that risk of SLE is a burden we have taken on in the fight to defend ourselves against viral infection. PMID:21923413

  20. Frequency preference and attention effects across cortical depths in the human primary auditory cortex

    PubMed Central

    De Martino, Federico; Moerel, Michelle; Ugurbil, Kamil; Goebel, Rainer; Yacoub, Essa; Formisano, Elia

    2015-01-01

    Columnar arrangements of neurons with similar preference have been suggested as the fundamental processing units of the cerebral cortex. Within these columnar arrangements, feed-forward information enters at middle cortical layers whereas feedback information arrives at superficial and deep layers. This interplay of feed-forward and feedback processing is at the core of perception and behavior. Here we provide in vivo evidence consistent with a columnar organization of the processing of sound frequency in the human auditory cortex. We measure submillimeter functional responses to sound frequency sweeps at high magnetic fields (7 tesla) and show that frequency preference is stable through cortical depth in primary auditory cortex. Furthermore, we demonstrate that—in this highly columnar cortex—task demands sharpen the frequency tuning in superficial cortical layers more than in middle or deep layers. These findings are pivotal to understanding mechanisms of neural information processing and flow during the active perception of sounds. PMID:26668397

  1. Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

    PubMed

    Hare, Ian; Evans, Rebecca; Fortney, James; Moses, Blake; Piktel, Debbie; Slone, William; Gibson, Laura F

    2016-10-01

    Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted. PMID:27586146

  2. Nanoparticle-mediated conversion of primary human astrocytes into neurons and oligodendrocytes.

    PubMed

    Li, Xiaowei; Kozielski, Kristen; Cheng, Yu-Hao; Liu, Huanhuan; Zamboni, Camila Gadens; Green, Jordan; Mao, Hai-Quan

    2016-06-21

    Central nervous system (CNS) diseases and injuries are accompanied by reactive gliosis and scarring involving the activation and proliferation of astrocytes to form hypertrophic and dense structures, which present a significant barrier to neural regeneration. Engineering astrocytes to functional neurons or oligodendrocytes may constitute a novel therapeutic strategy for CNS diseases and injuries. Such direct cellular programming has been successfully demonstrated using viral vectors via the transduction of transcriptional factors, such as Sox2, which could program resident astrocytes into neurons in the adult brain and spinal cord, albeit the efficiency was low. Here we report a non-viral nanoparticle-based transfection method to deliver Sox2 or Olig2 into primary human astrocytes and demonstrate the effective conversion of the astrocytes into neurons and oligodendrocyte progenitors following the transgene expression of Sox2 and Olig2, respectively. This approach is highly translatable for engineering astrocytes to repair injured CNS tissues. PMID:27328202

  3. Discharge Chamber Primary Electron Modeling Activities in Three-Dimensions

    NASA Technical Reports Server (NTRS)

    Steuber, Thomas J.

    2004-01-01

    Designing discharge chambers for ion thrusters involves many geometric configuration decisions. Various decisions will impact discharge chamber performance with respect to propellant utilization efficiency, ion production costs, and grid lifetime. These hardware design decisions can benefit from the assistance of computational modeling. Computational modeling for discharge chambers has been limited to two-dimensional codes that leveraged symmetry for interpretation into three-dimensional analysis. This paper presents model development activities towards a three-dimensional discharge chamber simulation to aid discharge chamber design decisions. Specifically, of the many geometric configuration decisions toward attainment of a worthy discharge chamber, this paper focuses on addressing magnetic circuit considerations with a three-dimensional discharge chamber simulation as a tool. With this tool, candidate discharge chamber magnetic circuit designs can be analyzed computationally to gain insight into factors that may influence discharge chamber performance such as: primary electron loss width in magnetic cusps, cathode tip position with respect to the low magnetic field volume, definition of a low magnetic field region, and maintenance of a low magnetic field region across the grid span. Corroborating experimental data will be obtained from mockup hardware tests. Initially, simulated candidate magnetic circuit designs will resemble previous successful thruster designs. To provide opportunity to improve beyond previous performance benchmarks, off-design modifications will be simulated and experimentally tested.

  4. UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

    PubMed Central

    Kraemer, Anne; Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Volkmer, Beate; Atkinson, Michael J.; Moertl, Simone; Greinert, Ruediger

    2013-01-01

    MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. PMID:24391759

  5. Primary human adult lung epithelial cells in vitro: response to interferon-gamma and cytomegalovirus.

    PubMed Central

    Ibrahim, L; Dominguez, M; Yacoub, M

    1993-01-01

    Primary human adult lung epithelial cells (ALEC) were established in culture using the most distal parts of the lung to avoid the airways. Immunocytochemical peroxidase staining and semiquantitative flow cytometry were used to characterize the cells in conjunction with a panel of monoclonal antibodies (mAb). The cells showed a constitutive expression of major histocompatibility complex (MHC) class I antigens, patchy expression of intercellular adhesion molecule-1 (ICAM-1) and a weak patchy expression of MHC class II antigens (detected using immunocytochemical staining). Incubation of the primary ALEC with interferon-gamma (IFN-gamma) (250 U/ml) stimulated an up-regulation of the expression of these three antigens to varying degrees; expression of MHC class I antigens and ICAM-1 molecules showed an up-regulation at 10 hr after the start of the treatment, reaching a peak at 48 hr, maintaining it for the next 24 hr and then, steadily and progressively, losing it towards the end of the experiment at 96 hr. Expression of HLA-DR showed an up-regulation at 17 hr after the start of the treatment, reaching a peak at 72 hr and maintaining it for the next 24 hr. Cytomegalovirus (CMV) infection of ALEC in culture caused an up-regulation of expression of class I antigens and ICAM-1, but not DR. However, when the infected cells were incubated with IFN-gamma, an up-regulation in the expression of DR took place. Therefore, within the micro-environment of the transplanted lung the presence of cytokines (IFN-gamma) produced by infiltrating activated mononuclear cells, may render the lung epithelial cells capable of acting as antigen-presenting cells, expressing high levels of class I antigens, ICAM-1 and class II antigens, activating CD8 and CD4 cells thus playing a major part in the process of rejection of the lung allograft; themselves becoming a primary target in the process. Images Figure 1 Figure 2 PMID:8099565

  6. Biocompatibility of Polypyrrole with Human Primary Osteoblasts and the Effect of Dopants

    PubMed Central

    Fahlgren, Anna; Bratengeier, Cornelia; Gelmi, Amy; Semeins, Cornelis M.; Klein-Nulend, Jenneke; Jager, Edwin W. H.; Bakker, Astrid D.

    2015-01-01

    Polypyrrole (PPy) is a conducting polymer that enables controlled drug release upon electrical stimulation. We characterized the biocompatibility of PPy with human primary osteoblasts, and the effect of dopants. We investigated the biocompatibility of PPy comprising various dopants, i.e. p-toluene sulfonate (PPy-pTS), chondroitin sulfate (PPy-CS), or dodecylbenzenesulfonate (PPy-DBS), with human primary osteoblasts. PPy-DBS showed the roughest appearance of all surfaces tested, and its wettability was similar to the gold-coated control. The average number of attached cells was 45% higher on PPy-DBS than on PPy-CS or PPy-pTS, although gene expression of the proliferation marker Ki-67 was similar in osteoblasts on all surfaces tested. Osteoblasts seeded on PPy-DBS or gold showed similar vinculin attachment points, vinculin area per cell area, actin filament structure, and Feret’s diameter, while cells seeded on PPY-CS or PPY-pTS showed disturbed focal adhesions and were enlarged with disorganized actin filaments. Osteoblasts grown on PPy-DBS or gold showed enhanced alkaline phosphatase activity and osteocalcin gene expression, but reduced osteopontin gene expression compared to cells grown on PPy-pTS and PPy-CS. In conclusion, PPy doped with DBS showed excellent biocompatibility, which resulted in maintaining focal adhesions, cell morphology, cell number, alkaline phosphatase activity, and osteocalcin gene expression. Taken together, conducting polymers doped with DBS are well tolerated by osteoblasts. Our results could provide a basis for the development of novel orthopedic or dental implants with controlled release of antibiotics and pharmaceutics that fight infections or focally enhance bone formation in a tightly controlled manner. PMID:26225862

  7. Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells.

    PubMed

    Tylden, Garth D; Hirsch, Hans H; Rinaldo, Christine Hanssen

    2015-01-01

    BK polyomavirus (BKPyV)-associated hemorrhagic cystitis (PyVHC) complicates 5 to 15% of allogeneic hematopoietic stem cell transplantations. Targeted antivirals are still unavailable. Brincidofovir (BCV; previously CMX001) has shown inhibitory activity against diverse viruses, including BKPyV in a primary human renal tubule cell culture model of polyomavirus-associated nephropathy. We investigated the effects of BCV in BKPyV-infected and uninfected primary human urothelial cells (HUCs), the target cells of BKPyV in PyVHC. The BCV concentrations causing 50 and 90% reductions (EC50 and EC90) in the number of intracellular BKPyV genome equivalents per cell (icBKPyV) were 0.27 μM and 0.59 μM, respectively. At 0.63 μM, BCV reduced viral late gene expression by 90% and halted progeny release. Preinfection treatment for only 24 h reduced icBKPyV similarly to treatment from 2 to 72 h postinfection, while combined pre- and postinfection treatment suppressed icBKPyV completely. After investigating BCV's effects on HUC viability, mean selectivity indices at 50 and 90% inhibition (SI50 and SI90) calculated for cellular DNA replication were 2.7 and 2.9, respectively, those for mitochondrial activity were 8.9 and 10.4, those for total ATP were 8.6 and 8.2, and those for membrane integrity were 25.9 and 16.7. The antiviral and cytostatic effects, but less so the cytotoxic effects, were inversely related to cell density. The cytotoxic effects at concentrations of ≥10 μM were rapid and likely related to BCV's lipid moiety. After carefully defining the antiviral, cytostatic, and cytotoxic properties of BCV in HUCs, we conclude that a preemptive or prophylactic approach in PyVHC is likely to give the best results.

  8. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    PubMed

    Blewett, Megan M; Xie, Jiji; Zaro, Balyn W; Backus, Keriann M; Altman, Amnon; Teijaro, John R; Cravatt, Benjamin F

    2016-01-01

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology. PMID:27625306

  9. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    PubMed

    Blewett, Megan M; Xie, Jiji; Zaro, Balyn W; Backus, Keriann M; Altman, Amnon; Teijaro, John R; Cravatt, Benjamin F

    2016-09-13

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.

  10. Chemical proteomic map of dimethyl fumarate–sensitive cysteines in primary human T cells

    PubMed Central

    Blewett, Megan M.; Xie, Jiji; Zaro, Balyn W.; Backus, Keriann M.; Altman, Amnon; Teijaro, John R.; Cravatt, Benjamin F.

    2016-01-01

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear, but may involve the covalent modification of proteins or DMF serving as a pro-drug that is converted to monomethyl fumarate (MMF). Here, we found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF-sensitivity of > 2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase C θ (PKCθ). Furthermore, DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology. PMID:27625306

  11. Teaching Students to Read the Primary Literature Using POGIL Activities

    ERIC Educational Resources Information Center

    Murray, Tracey Arnold

    2014-01-01

    The ability to read, interpret, and evaluate articles in the primary literature are important skills that science majors will use in graduate school and professional life. Because of this, it is important that students are not only exposed to the primary literature in undergraduate education, but also taught how to read and interpret these…

  12. All reovirus subtypes show oncolytic potential in primary cells of human high-grade glioma.

    PubMed

    Alloussi, S H; Alkassar, M; Urbschat, S; Graf, N; Gärtner, B

    2011-09-01

    Reoviridae are non-human pathogenic viruses. The family of reoviridae consists of 4 different subtypes. Many studies have proven that the Dearing subtype 3 has oncolytic potential. This potential is related to the RAS protein expression in tumour cells. The aim of this study, was to investigate whether all reovirus subtypes have oncolytic potential and whether there are differences in their efficacy, in particular for high-grade glioma. To evaluate the oncolytic potential, we performed an in vitro head-to-head study for all reovirus subtypes in 5 primary cell cultures of high-grade gliomas. The oncolytic activity was determined using end-point titration with observation of the cytopathogenic effect. For measurement of RAS activity, we performed an immunofluorescent detection stain on all cell cultures. For quantification of the virus, an RT-PCR measurement for all subtypes was performed. All reovirus subtypes showed oncolytic activity in the observed glioma biopsies. These observations correlated with RAS overexpression in the observed cells. All glioma biopsies overexpressed the RAS protein. The quantitative oncolytic potential differed in relation to the single observed cell culture and in relation to the chosen reovirus subtype. To our knowledge, this is the first study showing oncolytic activity for all reovirus subtypes. We show the relationship and correlation between RAS protein overexpression and vulnerability of cells to reovirus. Efficacy of the different subtypes is interindividually different and cannot be forecast.

  13. Molecular genetics of human primary microcephaly: an overview.

    PubMed

    Faheem, Muhammad; Naseer, Muhammad Imran; Rasool, Mahmood; Chaudhary, Adeel G; Kumosani, Taha A; Ilyas, Asad Muhammad; Pushparaj, Peter; Ahmed, Farid; Algahtani, Hussain A; Al-Qahtani, Mohammad H; Saleh Jamal, Hasan

    2015-01-01

    Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder that is characterised by microcephaly present at birth and non-progressive mental retardation. Microcephaly is the outcome of a smaller but architecturally normal brain; the cerebral cortex exhibits a significant decrease in size. MCPH is a neurogenic mitotic disorder, though affected patients demonstrate normal neuronal migration, neuronal apoptosis and neural function. Twelve MCPH loci (MCPH1-MCPH12) have been mapped to date from various populations around the world and contain the following genes: Microcephalin, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1 and CDK6. It is predicted that MCPH gene mutations may lead to the disease phenotype due to a disturbed mitotic spindle orientation, premature chromosomal condensation, signalling response as a result of damaged DNA, microtubule dynamics, transcriptional control or a few other hidden centrosomal mechanisms that can regulate the number of neurons produced by neuronal precursor cells. Additional findings have further elucidated the microcephaly aetiology and pathophysiology, which has informed the clinical management of families suffering from MCPH. The provision of molecular diagnosis and genetic counselling may help to decrease the frequency of this disorder. PMID:25951892

  14. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes

    PubMed Central

    Resseguie, Mary; Song, Jiannan; Niculescu, Mihai D.; da Costa, Kerry-Ann; Randall, Thomas A.; Zeisel, Steven H.

    2008-01-01

    Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-β-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) ∼7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.—Resseguie, M., Song, J., Niculescu, M. D., da Costa, K., Randall, T. A., Zeisel, S. H. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. PMID:17456783

  15. SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    Exposure of humans to PM results in increased mortality and morbidity. Recent toxicology studies have shown a number of pathophysiological pulmonary and car...

  16. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  17. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  18. Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

    PubMed

    Tamarozzi, F; Wright, H L; Johnston, K L; Edwards, S W; Turner, J D; Taylor, M J

    2014-10-01

    The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism. PMID:24909063

  19. Human body contour data based activity recognition.

    PubMed

    Myagmarbayar, Nergui; Yuki, Yoshida; Imamoglu, Nevrez; Gonzalez, Jose; Otake, Mihoko; Yu, Wenwei

    2013-01-01

    This research work is aimed to develop autonomous bio-monitoring mobile robots, which are capable of tracking and measuring patients' motions, recognizing the patients' behavior based on observation data, and providing calling for medical personnel in emergency situations in home environment. The robots to be developed will bring about cost-effective, safe and easier at-home rehabilitation to most motor-function impaired patients (MIPs). In our previous research, a full framework was established towards this research goal. In this research, we aimed at improving the human activity recognition by using contour data of the tracked human subject extracted from the depth images as the signal source, instead of the lower limb joint angle data used in the previous research, which are more likely to be affected by the motion of the robot and human subjects. Several geometric parameters, such as, the ratio of height to weight of the tracked human subject, and distance (pixels) between centroid points of upper and lower parts of human body, were calculated from the contour data, and used as the features for the activity recognition. A Hidden Markov Model (HMM) is employed to classify different human activities from the features. Experimental results showed that the human activity recognition could be achieved with a high correct rate. PMID:24111015

  20. Human body contour data based activity recognition.

    PubMed

    Myagmarbayar, Nergui; Yuki, Yoshida; Imamoglu, Nevrez; Gonzalez, Jose; Otake, Mihoko; Yu, Wenwei

    2013-01-01

    This research work is aimed to develop autonomous bio-monitoring mobile robots, which are capable of tracking and measuring patients' motions, recognizing the patients' behavior based on observation data, and providing calling for medical personnel in emergency situations in home environment. The robots to be developed will bring about cost-effective, safe and easier at-home rehabilitation to most motor-function impaired patients (MIPs). In our previous research, a full framework was established towards this research goal. In this research, we aimed at improving the human activity recognition by using contour data of the tracked human subject extracted from the depth images as the signal source, instead of the lower limb joint angle data used in the previous research, which are more likely to be affected by the motion of the robot and human subjects. Several geometric parameters, such as, the ratio of height to weight of the tracked human subject, and distance (pixels) between centroid points of upper and lower parts of human body, were calculated from the contour data, and used as the features for the activity recognition. A Hidden Markov Model (HMM) is employed to classify different human activities from the features. Experimental results showed that the human activity recognition could be achieved with a high correct rate.

  1. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  2. Experience in primary culture of human peritoneal mesothelial cell.

    PubMed

    Chen, Kuo-Su; Chen, Wen-Shiang

    2012-08-31

    To compare the growth condition between different sources and different culture environments, mesothelial cells were isolated from omentum and peritoneal dialysate effluent (PDE), seeded at different densities (5 × 10⁵, 1 × 10⁵, 5 × 10⁴, 1 × 10⁴, 5 × 10³, 1 × 10³ and 5 × 10² cells/cm², respectively), supported with different fetal calf serum (FCS) concentrations (3%, 6%, 10% and 15%) and grown in dishes with and without gelatin pre-coating. Growth condition was evaluated by simple morphological observation. Cells phenotype was examined by immunofluorescent staining. The results showed that omentum-derived mesothelial cells generally showed a uniform growth pattern with good quality. Alternatively, there was a wide patient-to-patient variation in PDE-derived culture. Heterogeneous colonies composed of a mixture of large, small or abortive mesothelial colonies as well as fibroblastoid colonies were frequently observed. A minimum seeding density of 5 × 10³ cells/cm² is required for the omentum-derived mesothelial cells to grow to confluent monolayer (1-5 × 10⁴ cells/cm² for initial culture from fresh PDE). Appropriate seeding density is always associated with successful culture in omentumbased culture, but not in PDE-based culture. Mesothelial cells could grow to confluency regardless of FCS concentration and gelatin pre-coating. However, growth rate was slower in lower FCS concentrations and on dishes without gelatin coating. Most cells in culture expressed cytokeratin and vimentin, but not VWF. Alpha-smooth muscle actin frequently appeared in cytokeratin+ mesothelial cells, especially in higher FCS concentrations and in PDE-derived culture. Our data demonstrate that PDE, in contrast to omentum, provides a source of mesothelial cells with poor and unstable quality for primary culture. Healthy cell quality and sufficient seeding density seem to be the most important factors for successful culture of mesothelial cells. The frequent occurrence

  3. Enhanced uptake of multiple sclerosis-derived myelin by THP-1 macrophages and primary human microglia

    PubMed Central

    2014-01-01

    Background The pathological hallmark of multiple sclerosis (MS) is myelin phagocytosis. It remains unclear why microglia and macrophages demyelinate axons in MS, but previously found or yet-unknown changes in the myelin of MS patients could contribute to this process. We therefore studied whether myelin from normal-appearing white matter (NAWM) of MS donors is phagocytosed more efficiently than myelin from control donors. Methods Myelin was isolated from 11 MS and 12 control brain donors and labeled with the pH-sensitive fluorescent dye pHrodo to quantify uptake in lysosomes. Phagocytosis by differentiated THP-1 macrophages and by primary human microglia was quantified with flow cytometry. Whereas myelin uptake by THP-1 macrophages reached a plateau after approximately 24 hours, uptake by primary human microglia showed an almost linear increase over a 72–hour period. Data were statistically analyzed with the Mann–Whitney U test. Results MS-derived myelin was phagocytosed more efficiently by THP-1 macrophages after 6-hour incubation (P = 0.001 for the percentage of myelin-phagocytosing cells and P = 0.0005 for total myelin uptake) and after 24-hour incubation (P = 0.0006 and P = 0.0001, respectively), and by microglia after 24-hour incubation (P = 0.0106 for total myelin uptake). This enhanced uptake was not due to differences in the oxidation status of the myelin. Interestingly, myelin phagocytosis correlated negatively with the age of myelin donors, whereas the age of microglia donors showed a positive trend with myelin phagocytosis. Conclusions Myelin isolated from normal-appearing white matter of MS donors was phagocytosed more efficiently than was myelin isolated from control brain donors by both THP-1 macrophages and primary human microglia. These data indicate that changes in MS myelin might precede phagocyte activation and subsequent demyelination in MS. Identifying these myelin changes responsible for enhancing phagocytic ability

  4. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo.

    PubMed

    Greening, David W; Ji, Hong; Chen, Maoshan; Robinson, Bruce W S; Dick, Ian M; Creaney, Jenette; Simpson, Richard J

    2016-01-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets.

  5. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo

    NASA Astrophysics Data System (ADS)

    Greening, David W.; Ji, Hong; Chen, Maoshan; Robinson, Bruce W. S.; Dick, Ian M.; Creaney, Jenette; Simpson, Richard J.

    2016-09-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets.

  6. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo

    PubMed Central

    Greening, David W.; Ji, Hong; Chen, Maoshan; Robinson, Bruce W. S.; Dick, Ian M.; Creaney, Jenette; Simpson, Richard J.

    2016-01-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets. PMID:27605433

  7. Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo.

    PubMed

    Greening, David W; Ji, Hong; Chen, Maoshan; Robinson, Bruce W S; Dick, Ian M; Creaney, Jenette; Simpson, Richard J

    2016-01-01

    Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets. PMID:27605433

  8. The role of arginine vasopressin in electroacupuncture treatment of primary sciatica in human.

    PubMed

    Zhao, Xue-Yan; Zhang, Qi-Shun; Yang, Jun; Sun, Fang-Jie; Wang, Da-Xin; Wang, Chang-Hong; He, Wei-Ya

    2015-08-01

    It has been implicated that electroacupuncture can relieve the symptoms of sciatica with the increase of pain threshold in human, and arginine vasopressin (AVP) in the brain rather than the spinal cord and blood circulation participates in antinociception. Our previous study has proven that AVP in the brain played a role in the process of electroacupuncture analgesia in rat. The goal of the present study was to investigate the role of AVP in electroacupuncture in treating primary sciatica in human. The results showed that (1) AVP concentration of cerebrospinal fluid (CSF) (7.5 ± 2.5 pg/ml), not plasma (13.2 ± 4.2 pg/ml) in primary sciatica patients was lower than that in health volunteers (16.1 ± 3.8 pg/ml and 12.3 ± 3.4 pg/ml), although the osmotic pressure in CSF and plasma did not change; (2) electroacupuncture of the bilateral "Zusanli" points (St. 36) for 60 min relieved the pain sensation in primary sciatica patients; (3) electroacupuncture increased the AVP level of CSF, not plasma in primary sciatica patients; and (4) there was the positive correlation between the effect of electroacupuncture relieving the pain and the AVP level of CSF in the primary sciatica patients. The data suggested that central AVP, not peripheral AVP might improve the effect of electroacupuncture treatment of primary sciatica in human, i.e., central AVP might take part in the electroacupuncture relieving the pain sensation in primary sciatica patients.

  9. Effects of Rosmarinus officinalis extract on human primary omental preadipocytes and adipocytes

    PubMed Central

    Stefanon, Bruno; Pomari, Elena

    2015-01-01

    The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement. PMID:25710930

  10. Orientation anisotropies in human primary visual cortex depend on contrast.

    PubMed

    Maloney, Ryan T; Clifford, Colin W G

    2015-10-01

    Orientation processing in visual cortex appears matched to the environment, such that larger neural populations are tuned to cardinal (horizontal/vertical) than oblique orientations. This may be manifested perceptually as a cardinal bias: poorer sensitivity to oblique compared to cardinal orientations (the "oblique effect"). However, a growing body of psychophysical data reveals the opposite pattern of anisotropy: a bias towards the oblique over the cardinal orientations (the "horizontal effect"), something matched by recent functional magnetic resonance imaging (fMRI) studies that have found an increased response to the oblique over the cardinal orientations in early visual cortex. This may reveal the operation of an efficient coding strategy optimised to the diet of orientations encountered during natural viewing. From consideration of coding efficiency, it might be expected that the anisotropies would change as the quality/strength of the oriented stimulus changes. In two experiments, fMRI response modulations were measured in retinotopically-defined human early visual cortex as a function of the contrast and orientation of sinusoidal gratings. Both experiments revealed a marked change in the V1 response from a cardinal (vertical) bias at low contrast to an oblique bias at high contrast. In Experiment 2, this was also apparent in areas V2 and V3. On average, there was no systematic "radial bias" (a preference for orientations aligned with the visual field meridian) in V1, although it was present in some individual subjects. The change in orientation anisotropies with contrast is consistent with an adaptive stimulus coding strategy in cortex that shifts according to the strength of the sensory inputs. PMID:26093331

  11. Matrigel improves functional properties of primary human salivary gland cells.

    PubMed

    Maria, Ola M; Zeitouni, Anthony; Gologan, Olga; Tran, Simon D

    2011-05-01

    Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to <5% of huSG cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

  12. IFNL3 genotype is associated with differential induction of IFNL3 in primary human hepatocytes

    PubMed Central

    Kurbanov, Fuat; Kim, Yonghak; Latanich, Rachel; Chaudhari, Pooja; El-Diwany, Ramy; Knabel, Matt; Kandathil, Abraham J; Cameron, Andrew; Cox, Andrea; Jang, Yoon-Young; Thomas, David L; Balagopal, Ashwin

    2016-01-01

    Background Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive. Methods IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3. Results PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07). Conclusions Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection. PMID:26109548

  13. Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes

    PubMed Central

    Grosse, Susann; Stenvik, Jørgen; Nilsen, Asbjørn M

    2016-01-01

    Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response. PMID:27695322

  14. Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes

    PubMed Central

    Grosse, Susann; Stenvik, Jørgen; Nilsen, Asbjørn M

    2016-01-01

    Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response.

  15. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed Central

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-01-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  16. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-11-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  17. Activated human platelets induce factor XIIa-mediated contact activation.

    PubMed

    Bäck, Jennie; Sanchez, Javier; Elgue, Graciela; Ekdahl, Kristina Nilsson; Nilsson, Bo

    2010-01-01

    Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation. PMID:19878657

  18. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells.

    PubMed

    Lugini, Luana; Matarrese, Paola; Tinari, Antonella; Lozupone, Francesco; Federici, Cristina; Iessi, Elisabetta; Gentile, Massimo; Luciani, Francesca; Parmiani, Giorgio; Rivoltini, Licia; Malorni, Walter; Fais, Stefano

    2006-04-01

    The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. PMID:16585188

  19. Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture.

    PubMed

    Gauthier, B; Robb, M; McPherson, R

    1999-02-01

    The expression pattern of the CETP gene in relationship to that of LPL, adipsin, PPARgamma, C/EBPalpha, ADD1/SREBPI and actin was examined by RT-PCR during differentiation of human fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were isolated from subcutaneous fat obtained from healthy female subjects undergoing mammary reduction procedures, and induced to differentiate in culture. Morphologically, adipogenesis was confirmed by the accumulation of lipid droplets in cells. We show that the gene encoding CETP is expressed in preadipocytes and is present throughout differentiation as compared to LPL and adipsin which were detected in the majority of samples by day 2 or 3 of adipogenesis. The transcription factors, PPARgamma, ADD1/SREBP1 and C/EBPalpha were expressed by day 2, concomitant with the appearance of LPL and adipsin but subsequent to the appearance of CETP. CETP mRNA was not detectable in human skin fibroblasts. These studies demonstrate that CETP. expression is induced at an early stage of commitment to the adipocyte lineage and may be activated by transcription factor(s), which are not members of the PPAR, ADD1/SREBP1 or C/EBP families. PMID:10030381

  20. Membrane damage and repair in primary monocytes exposed to human β-defensin-3

    PubMed Central

    Lioi, Anthony B.; Reyes Rodriguez, Angel L.; Funderburg, Nicholas T.; Feng, Zhimin; Weinberg, Aaron; Sieg, Scott F.

    2012-01-01

    Interactions of AMPs with plasma membranes of primary human immune cells are poorly characterized. Analysis of PI exclusion as a measure of membrane integrity indicated that hBD-3 caused membrane perturbations in monocytes but not T or B cells at concentrations typically used to kill bacteria or to induce activation of APCs. Bleb-like structures were observed in monocytes exposed to hBD-3. These cells also increased surface expression of LAMP1, a membrane repair marker after exposure to hBD-3. Furthermore, cell death was enhanced by adding an inhibitor of membrane repair. Removal of cholesterol from membranes resulted in greater susceptibility of cells to hBD-3, but cholesterol content was not different between the cell types, as assessed by filipin staining. Freshly isolated monocytes expressed higher levels of the negatively charged phospholipid, PS, on their outer leaflet compared with B or T cells. Preincubation of monocytes with molecules that bind PS protected these cells from hBD-3-induced membrane damage, suggesting that outer-membrane PS expression can at least partially explain monocyte susceptibility to hBD-3. The potential for membrane disruption caused by AMPs should be evaluated in various cell types when considering these molecules for therapeutic applications in humans. PMID:22837529

  1. Active stochastic stress fluctuations in the cell cytoskeleton stir the cell and activate primary cilia

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.; Fakhri, Nikta; Battle, Christopher; Ott, Carolyn M.; Wessel, Alok D.; Lippincott-Schwartz, Jennifer; Mackintosh, Frederick C.

    2015-03-01

    Cells are active systems with molecular force generation that drives complex dynamics at the supramolecular scale. Much of cellular dynamics is driven by myosin motors interacting with the actin cytoskeleton. We discovered active random ``stirring'' driven by cytoplasmic myosin as an intermediate mode of transport, different from both thermal diffusion and directed motor activity. We found a further manifestation of cytoskeletal dynamics in the active motion patterns of primary cilia generated by epithelial cells. These cilia were thought to be immotile due to the absence of dynein motors, but it turns out that their anchoring deeper inside the cell in combination with the strongly fluctuating cortex results in clearly measurable non-equilibrium fluctuations.

  2. Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study

    PubMed Central

    Bernik, Maria B.; Kwaan, Hau C.

    1969-01-01

    Human tissues and cells from pre- and postnatal life were cultivated and studied for plasminogen activator activity. Cultures were obtained from kidney, renal blood vessels, ureter, bladder, lung, and heart. Local activator activity of cells was demonstrated by histochemical techniques. Activator released by cells into the supernatant culture media was assayed by fibrin plate techniques and was investigated for immunological identity using specific antisera to an activator of human origin, urokinase (UK). Plasminogen activator was produced in primary cultures where cells retain specific functions and generally reflect the enzyme pattern of the tissues of origin. Cells from fetal and adult sources were found to yield activator antigenically identical to UK, as well as activator activity which differed from that of UK in immunoassays and which may represent tissue type activator. Such activity was released after injury or death of cells while UK was produced in cultures containing live, metabolizing cells. Primary cultures of kidney confirmed that this organ is a rich source of UK and demonstrated, in addition, that UK is produced from the early stages of gestation and in increasing amounts thereafter. However, primary cultures also demonstrated that the ability to produce UK is not limited to the kidney but is a function of cells which are distributed widely in body tissues. Thus, activator antigenically identical to UK accumulated progressively after many refeedings in culture supernates of fetal lung and ureter, as well as in supernates of renal blood vessels of adults. These findings indicate continuous formation of UK by the cultured cells and, furthermore, provide evidence of UK production in blood vessels. In cultures from other tissues, particularly those from fetal heart and adult lung and bladder, investigation of activator was hindered by inhibitory activity which accumulated in the supernates. Such activity was derived from cells in culture and was

  3. Deep Human Parsing with Active Template Regression.

    PubMed

    Liang, Xiaodan; Liu, Si; Shen, Xiaohui; Yang, Jianchao; Liu, Luoqi; Dong, Jian; Lin, Liang; Yan, Shuicheng

    2015-12-01

    In this work, the human parsing task, namely decomposing a human image into semantic fashion/body regions, is formulated as an active template regression (ATR) problem, where the normalized mask of each fashion/body item is expressed as the linear combination of the learned mask templates, and then morphed to a more precise mask with the active shape parameters, including position, scale and visibility of each semantic region. The mask template coefficients and the active shape parameters together can generate the human parsing results, and are thus called the structure outputs for human parsing. The deep Convolutional Neural Network (CNN) is utilized to build the end-to-end relation between the input human image and the structure outputs for human parsing. More specifically, the structure outputs are predicted by two separate networks. The first CNN network is with max-pooling, and designed to predict the template coefficients for each label mask, while the second CNN network is without max-pooling to preserve sensitivity to label mask position and accurately predict the active shape parameters. For a new image, the structure outputs of the two networks are fused to generate the probability of each label for each pixel, and super-pixel smoothing is finally used to refine the human parsing result. Comprehensive evaluations on a large dataset well demonstrate the significant superiority of the ATR framework over other state-of-the-arts for human parsing. In particular, the F1-score reaches 64.38 percent by our ATR framework, significantly higher than 44.76 percent based on the state-of-the-art algorithm [28]. PMID:26539846

  4. Deep Human Parsing with Active Template Regression.

    PubMed

    Liang, Xiaodan; Liu, Si; Shen, Xiaohui; Yang, Jianchao; Liu, Luoqi; Dong, Jian; Lin, Liang; Yan, Shuicheng

    2015-12-01

    In this work, the human parsing task, namely decomposing a human image into semantic fashion/body regions, is formulated as an active template regression (ATR) problem, where the normalized mask of each fashion/body item is expressed as the linear combination of the learned mask templates, and then morphed to a more precise mask with the active shape parameters, including position, scale and visibility of each semantic region. The mask template coefficients and the active shape parameters together can generate the human parsing results, and are thus called the structure outputs for human parsing. The deep Convolutional Neural Network (CNN) is utilized to build the end-to-end relation between the input human image and the structure outputs for human parsing. More specifically, the structure outputs are predicted by two separate networks. The first CNN network is with max-pooling, and designed to predict the template coefficients for each label mask, while the second CNN network is without max-pooling to preserve sensitivity to label mask position and accurately predict the active shape parameters. For a new image, the structure outputs of the two networks are fused to generate the probability of each label for each pixel, and super-pixel smoothing is finally used to refine the human parsing result. Comprehensive evaluations on a large dataset well demonstrate the significant superiority of the ATR framework over other state-of-the-arts for human parsing. In particular, the F1-score reaches 64.38 percent by our ATR framework, significantly higher than 44.76 percent based on the state-of-the-art algorithm [28].

  5. Primary adult human astrocytes as an ex vivo vehicle for beta-glucuronidase delivery in the brain.

    PubMed

    Serguera, C; Sarkis, C; Ridet, J L; Colin, P; Moullier, P; Mallet, J

    2001-06-01

    Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.

  6. BOLD signal change and contrast reversing frequency: an event-related fMRI study in human primary visual cortex.

    PubMed

    Sun, Pei; Guo, Jianfei; Guo, Shichun; Chen, Jingyi; He, Le; Fu, Shimin

    2014-01-01

    It is believed that human primary visual cortex (V1) increases activity with increasing temporal frequency of a visual stimulus. Two kinds of visual stimulus were used in the previous studies, one is patterned-flash stimulus with a fixed onset period and an increasing average luminance with the increase of temporal frequency, the other is contrast reversing flickering checkerboard or grating with a constant average luminance across different temporal frequencies. That hemodynamic responses change as a function of reversal frequency of contrast reversing checkerboard is at odds with neurophysiological studies in animals and neuroimaging studies in humans. In the present study, we addressed the relationship between reversal frequency of contrast reversing checkerboard and hemodynamic response in human V1 using an event-related experimental paradigm and found that the transient characteristics of blood oxygenation level dependent response in human V1 depended very little on the reversal frequency of a contrast reversing checkerboard.

  7. Primary in vitro generation of cytotoxic cells specific for human minor histocompatibility antigens between HLA-identical siblings.

    PubMed

    Tekolf, W A; Shaw, S

    1984-04-01

    A limiting dilution culture system was developed for the primary in vitro detection of human minor histocompatibility antigens by cytotoxic T lymphocytes (CTL). CTL were generated in primary in vitro culture between two HLA-identical sibling pairs and propagated as stable CTL lines. Population and family studies indicate that these CTL lines recognize minor histocompatibility antigens in an HLA-restricted manner. The antigen recognized by one CTL line is detected on six (out of 37) HLA-B7-positive donors but not on 32 HLA-B7-negative donors. The cytotoxicity of this CTL line is mediated by T3+, T8+ effector cells. The antigen detected by this CTL population is different from all known human minor histocompatibility antigens. The data of this study, like those in the mouse system, suggest that a suppressor cell is diluted out in a limiting dilution culture, which allows the activation of the CTL precursors.

  8. Human enamel veneer restoration: an alternative technique to restore anterior primary teeth.

    PubMed

    Oliveira, Luciana Butini; Tamay, Tereza Keiko; Oliveira, Marta Dutra Machado; Rodrigues, Célia Martins Delgado; Wanderley, Marcia Turolla

    2006-01-01

    Restoration of severely decayed primary teeth is a clinical challenge in Pediatric Dentistry. Among the restorative treatment options, the use of prefabricated crowns and resin composite restorations, either by means of direct or indirect techniques is mentioned in the literature. The purpose of this article is to describe the rehabilitation of primary anterior teeth in a 5-year-old patient. Dental treatment consisted on an anterior space maintainer prosthesis made with natural primary teeth, plus human dental enamel veneer (facet) restorations. The advantages of this technique are better esthetics and the natural enamel has physiologic wear and offers superficial smoothness and cervical adaptation compatible with those of the surrounding teeth.

  9. Human enamel veneer restoration: an alternative technique to restore anterior primary teeth.

    PubMed

    Oliveira, Luciana Butini; Tamay, Tereza Keiko; Oliveira, Marta Dutra Machado; Rodrigues, Célia Martins Delgado; Wanderley, Marcia Turolla

    2006-01-01

    Restoration of severely decayed primary teeth is a clinical challenge in Pediatric Dentistry. Among the restorative treatment options, the use of prefabricated crowns and resin composite restorations, either by means of direct or indirect techniques is mentioned in the literature. The purpose of this article is to describe the rehabilitation of primary anterior teeth in a 5-year-old patient. Dental treatment consisted on an anterior space maintainer prosthesis made with natural primary teeth, plus human dental enamel veneer (facet) restorations. The advantages of this technique are better esthetics and the natural enamel has physiologic wear and offers superficial smoothness and cervical adaptation compatible with those of the surrounding teeth. PMID:16937849

  10. Integrated Extravehicular Activity Human Research Plan: 2016

    NASA Technical Reports Server (NTRS)

    Abercromby, Andrew F. J.; Ross, Amy J.; Cupples, J. Scott; Rajulu, Sudhakar; Norcross, Jason R.; Chappell, Steven P.

    2016-01-01

    Multiple organizations within NASA and outside of NASA fund and participate in research related to extravehicular activity (EVA). In October 2015, representatives of the EVA Office, the Crew and Thermal Systems Division (CTSD), and the Human Research Program (HRP) at NASA Johnson Space Center agreed on a formal framework to improve multi-year coordination and collaboration in EVA research. At the core of the framework is an Integrated EVA Human Research Plan and a process by which it will be annually reviewed and updated. The over-arching objective of the collaborative framework is to conduct multi-disciplinary cost-effective research that will enable humans to perform EVAs safely, effectively, comfortably, and efficiently, as needed to enable and enhance human space exploration missions. Research activities must be defined, prioritized, planned and executed to comprehensively address the right questions, avoid duplication, leverage other complementary activities where possible, and ultimately provide actionable evidence-based results in time to inform subsequent tests, developments and/or research activities. Representation of all appropriate stakeholders in the definition, prioritization, planning and execution of research activities is essential to accomplishing the over-arching objective. A formal review of the Integrated EVA Human Research Plan will be conducted annually. External peer review of all HRP EVA research activities including compilation and review of published literature in the EVA Evidence Report is will also continue at a frequency determined by HRP management. Coordination with stakeholders outside of the EVA Office, CTSD, and HRP is already in effect on a study-by-study basis; closer coordination on multi-year planning with other EVA stakeholders including academia is being actively pursued. Details of the current Integrated EVA Human Research Plan are presented including description of ongoing and planned research activities in the areas of

  11. Human telomerase: biogenesis, trafficking, recruitment, and activation.

    PubMed

    Schmidt, Jens C; Cech, Thomas R

    2015-06-01

    Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species.

  12. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    SciTech Connect

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  13. Resveratrol induces cell death and inhibits human herpesvirus 8 replication in primary effusion lymphoma cells.

    PubMed

    Tang, Feng-Yi; Chen, Chang-Yu; Shyu, Huey-Wen; Hong, Shin; Chen, Hung-Ming; Chiou, Yee-Hsuan; Lin, Kuan-Hua; Chou, Miao-Chen; Wang, Lin-Yu; Wang, Yi-Fen

    2015-12-01

    Resveratrol (3,4',5-trihydroxy-trans-stilbene) has been reported to inhibit proliferation of various cancer cells. However, the effects of resveratrol on the human herpesvirus 8 (HHV8) harboring primary effusion lymphoma (PEL) cells remains unclear. The anti-proliferation effects and possible mechanisms of resveratrol in the HHV8 harboring PEL cells were examined in this study. Results showed that resveratrol induced caspase-3 activation and the formation of acidic vacuoles in the HHV8 harboring PEL cells, indicating resveratrol treatment could cause apoptosis and autophagy in PEL cells. In addition, resveratrol treatment increased ROS generation but did not lead to HHV8 reactivation. ROS scavenger (N-acetyl cysteine, NAC) could attenuate both the resveratrol induced caspase-3 activity and the formation of acidic vacuoles, but failed to attenuate resveratrol induced PEL cell death. Caspase inhibitor, autophagy inhibitors and necroptosis inhibitor could not block resveratrol induced PEL cell death. Moreover, resveratrol disrupted HHV8 latent infection, inhibited HHV8 lytic gene expression and decreased virus progeny production. Overexpression of HHV8-encoded viral FLICE inhibitory protein (vFLIP) could partially block resveratrol induced cell death in PEL cells. These data suggest that resveratrol-induced cell death in PEL cells may be mediated by disruption of HHV8 replication. Resveratrol may be a potential anti-HHV8 drug and an effective treatment for HHV8-related tumors.

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2014-04-01

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

  16. Segmentally arranged somatotopy within the face representation of human primary somatosensory cortex.

    PubMed

    Moulton, Eric A; Pendse, Gautam; Morris, Susie; Aiello-Lammens, Matthew; Becerra, Lino; Borsook, David

    2009-03-01

    Though somatotypic representation within the face in human primary somatosensory cortex (S1) to innocuous stimuli is controversial; previous work suggests that painful heat is represented based on an "onion-skin" or segmental pattern on the face. The aim of this study was to determine if face somatotopy for brush stimuli in S1 also follows this segmental representation model. Twelve healthy subjects (nine men: three women) underwent functional magnetic resonance imaging to measure blood oxygen level dependent signals during brush (1 Hz, 15 s) applied to their faces. Separate functional scans were collected for brush stimuli repetitively applied to each of five separate stimulation sites on the right side of the face. These sites were arranged in a vertical, horizontal, and circular manner encompassing the three divisions of the trigeminal nerve. To minimize inter-individual morphological differences in the post-central gyrus across subjects, cortical surface-based registration was implemented before group statistical image analysis. Based on activation foci, somatotopic activation in the post-central gyrus was detected for brush, consistent with the segmental face representation model. PMID:18266215

  17. Clouds caused by human activities: the anthropoclouds

    NASA Astrophysics Data System (ADS)

    Mazon, Jordi; Costa, Marcel; Pino, David; Lorente, Jeroni

    2013-04-01

    The classification of clouds is based on the pioneering classification carried out by Howard (1804). In this classification, and also in the successive editions of the International Classification of Clouds published by the World Meteorological Organization (WMO, 1975, 1987) 10 basic cloud genera are included and described. In all cases, the cause that leads to the formation of clouds remains as a secondary issue. It is assumed that all of them are exclusively produced by natural mechanisms without any human intervention. However, aerosol and water vapour emissions produced by human activity may increase cloud formation having an increasing importance in the atmospheric energy budget and consequently in the earth's climate. Effectively, since the end of the Nineteenth century, human activity has been injecting large amounts of water vapour into the atmosphere, cloud condensation nuclei and hot air mainly generated in the combustion processes that under certain spatial and temporal conditions can enhance cloud formation. These anthropogenic aerosols are linked to the climate and the water cycle (Kaufman et al, 2002). The aim of this communication is to point out the anthropic origin of some clouds in the cloud classification. Several cases of the 7 basic genera cloud caused by human activities will be shown to discuss the importance of differentiating the origin of clouds in weather observations. This differentiation would improve the understanding the contribution of these clouds to climate change. To differentiate the clouds formed by human activity, we propose to use the prefix anthropo- before the scientific name (and a- before the abbreviation) in some of the 10 basic clouds defined by the International Classification of Clouds, those which could have an anthropic origin, and thus begin new data of cloud observations that could help future research to improve the effect of human activity in the troposphere.

  18. Flying the "Active School Flag": Physical Activity Promotion through Self-Evaluation in Primary Schools in Ireland

    ERIC Educational Resources Information Center

    Chroinin, Deirdre Ni; Murtagh, Elaine; Bowles, Richard

    2012-01-01

    Primary schools are key sites where children can be active, advance their knowledge and understanding of how to participate in physical activity (PA) and develop an appreciation of its importance in their lives. This study explored the role of schools in promoting PA asking: how do primary schools approach the promotion of whole-school PA? Data…

  19. Scaling behavior of online human activity

    NASA Astrophysics Data System (ADS)

    Zhao, Zhi-Dan; Cai, Shi-Min; Huang, Junming; Fu, Yan; Zhou, Tao

    2012-11-01

    The rapid development of the Internet technology enables humans to explore the web and record the traces of online activities. From the analysis of these large-scale data sets (i.e., traces), we can get insights about the dynamic behavior of human activity. In this letter, the scaling behavior and complexity of human activity in the e-commerce, such as music, books, and movies rating, are comprehensively investigated by using the detrended fluctuation analysis technique and the multiscale entropy method. Firstly, the interevent time series of rating behaviors of these three types of media show similar scaling properties with exponents ranging from 0.53 to 0.58, which implies that the collective behaviors of rating media follow a process embodying self-similarity and long-range correlation. Meanwhile, by dividing the users into three groups based on their activities (i.e., rating per unit time), we find that the scaling exponents of the interevent time series in the three groups are different. Hence, these results suggest that a stronger long-range correlations exist in these collective behaviors. Furthermore, their information complexities vary in the three groups. To explain the differences of the collective behaviors restricted to the three groups, we study the dynamic behavior of human activity at the individual level, and find that the dynamic behaviors of a few users have extremely small scaling exponents associated with long-range anticorrelations. By comparing the interevent time distributions of four representative users, we can find that the bimodal distributions may bring forth the extraordinary scaling behaviors. These results of the analysis of the online human activity in the e-commerce may not only provide insight into its dynamic behaviors but may also be applied to acquire potential economic interest.

  20. Active tectonics and human survival strategies

    NASA Astrophysics Data System (ADS)

    King, Geoffrey; Bailey, Geoffrey; Sturdy, Derek

    1994-10-01

    Tectonic movements continuously remould the surface of Earth in response to plate motion. Yet such deformation is rarely taken into account when assessing landscape change and its impact on human land use, except perhaps as an occasional hazard to human life or a temporary disruption in the longer term patterns of human history. However, active tectonics also create and sustain landscapes that can be beneficial to human survival, forming a complex topography of potentially fertile sedimentary basins enclosed by mountain barriers that can facilitate the control and explotation of food resources, especially animal prey. We discuss the tectonic history of northwest Greece and show how the Paleolithic sites of the region are located to take advantage of tectonically created features at both a local and a regional scale. We suggest that the association of significant concentrations of early Paleolithic sites with tectonically acitve regions is not coincidental and that on the longer time spans of human biological evolution, active tectonics has been an important selective agent contributing to the development of the human species as an intelligent predator.

  1. Schedule-induced locomotor activity in humans.

    PubMed

    Muller, P G; Crow, R E; Cheney, C D

    1979-01-01

    In two experiments, humans received tokens either on a fixed-interval schedule for plunger pulling or various response-nondependent fixed-time schedules ranging from 16 to 140 seconds. Locomotor activity such as walking, shifting weight, or pacing was recorded in quarters of the interreinforcement interval to examine the induced characteristics of that behavior in humans. While performance was variable, several characteristics were present that have counterparts in experiments with nonhumans during periodic schedules of food reinforcement: (a) first quarter rates, and sometimes overall rates, of locomotor activity were greater during intervals that terminated in a visual stimulus and token delivery than those without: (b) overall rates of locomotor activity were greater during fixed-time 16-second schedules than during fixed-time 80- or 140-second schedules; (c) rates of locomotor activity decreased during the interreinforcement intervals; (d) locomotor activity was induced by response-dependent and response-nondependent token delivery. These results showed that the rate and temporal pattern of locomotor activity can be schedule-induced in humans. PMID:429959

  2. Differential UGT1A1 induction by chrysin in primary human hepatocytes and HepG2 Cells.

    PubMed

    Smith, Cornelia M; Graham, Richard A; Krol, Wojciech L; Silver, Ivin S; Negishi, Masahiko; Wang, Hongbing; Lecluyse, Edward L

    2005-12-01

    Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo. PMID:16135700

  3. The Human Activity of Evaluation Theorizing.

    ERIC Educational Resources Information Center

    Alkin, Marvin C.; Ellett, Frederick, Jr.

    Theorizing about evaluation should be conceptualized as a human activity governed by certain strategies and principles. The theories advanced by various evaluators have changed over the years, thus illustrating ten principles of evaluation. The starting point for theory development or modification is self-reflection and review of one's own…

  4. Food & Fitness. Directory. Human Nutrition Activities.

    ERIC Educational Resources Information Center

    Department of Agriculture, Washington, DC.

    Activities of the following regulatory and food service agencies of the Department of Agriculture are described: (1) Agricultural Research Service; (2) Cooperative State Research Service; (3) Economic Research Service; (4) Human Nutrition Information Service; (5) Office of Grants and Program Systems; (6) Office of International Cooperation and…

  5. MEASURING CHOLINESTERASE ACTIVITY IN HUMAN SALIVA

    EPA Science Inventory

    To assess the potential for using saliva in pesticide biomonitoring, the consistency of cholinesterase activity in human saliva collected over time was examined. In this pilot study, saliva was collected from 20 healthy adults once per week for 5 consecutive weeks using 2 differe...

  6. MEASURING CHOLINESTERASE ACTIVITY IN HUMAN SALIVA.

    EPA Science Inventory

    To assess the potential for using saliva in pesticide biomonitoring, the consistency of cholinesterase activity in human saliva collected over time was examined. In this pilot study, saliva was collected from 20 healthy adults once per week for 5 consecutive weeks using 2 differe...

  7. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  8. Active change detection by pigeons and humans.

    PubMed

    Hagmann, Carl Erick; Cook, Robert G

    2013-10-01

    Detecting change is vital to both human and nonhuman animals' interactions with the environment. Using the go/no-go dynamic change detection task, we examined the capacity of four pigeons to detect changes in brightness of an area on a computer display. In contrast to our prior research, we reversed the response contingencies so that the animals had to actively inhibit pecking upon detecting change in brightness rather than its constancy. Testing eight rates of change revealed that this direct report change detection contingency produced results equivalent to the earlier indirect procedure. Corresponding tests with humans suggested that the temporal dynamics of detecting change were similar for both species. The results indicate the mechanisms of change detection in both pigeons and humans are organized in similar ways, although limitations in the operations of working memory may prevent pigeons from integrating information over the same time scale as humans.

  9. Exploring Global Patterns in Human Appropriation of Net Primary Production Using Earth Observation Satellites and Statistical Data

    NASA Astrophysics Data System (ADS)

    Imhoff, M.; Bounoua, L.

    2004-12-01

    A unique combination of satellite and socio-economic data were used to explore the relationship between human consumption and the carbon cycle. Biophysical models were applied to consumption data to estimate the annual amount of Earth's terrestrial net primary production humans require for food, fiber and fuel using the same modeling architecture as satellite-supported NPP measurements. The amount of Earth's NPP required to support human activities is a powerful measure of the aggregate human impacts on the biosphere and indicator of societal vulnerability to climate change. Equations were developed estimating the amount of landscape-level NPP required to generate all the products consumed by 230 countries including; vegetal foods, meat, milk, eggs, wood, fuel-wood, paper and fiber. The amount of NPP required was calculated on a per capita basis and projected onto a global map of population to create a spatially explicit map of NPP-carbon demand in units of elemental carbon. NPP demand was compared to a map of Earth's average annual net primary production or supply created using 17 years (1982-1998) of AVHRR vegetation index to produce a geographically accurate balance sheet of terrestrial NPP-carbon supply and demand. Globally, humans consume 20 percent of Earth's total net primary production on land. Regionally the NPP-carbon balance percentage varies from 6 to over 70 percent and locally from near 0 to over 30,000 percent in major urban areas. The uneven distribution of NPP-carbon supply and demand, indicate the degree to which various human populations rely on NPP imports, are vulnerable to climate change and suggest policy options for slowing future growth in NPP demand.

  10. Radiation response of chemically derived mitochondrial DNA-deficient AG01522 human primary fibroblasts.

    PubMed

    Nieri, D; Fioramonti, M; Berardinelli, F; Leone, S; Cherubini, R; De Nadal, V; Gerardi, S; Moreno, S; Nardacci, R; Tanzarella, C; Antoccia, A

    2013-08-30

    Mitochondria are the main cellular source of Reactive Oxygen Species (ROS). Alterations of mitochondrial metabolism and consequent loss of mitochondrial membrane potential may lead to redox imbalance and in turn to DNA damage, chromosomal instability and apoptosis. On the other hand, impaired mitochondrial functions may either exacerbate the detrimental effects of geno- and cytotoxic agents or may bring beneficial cellular responses. To study the role of mitochondria within this framework, AG01522 human primary fibroblasts were incubated with the mitochondrial polymerase γ inhibitor 2',3'-dideoxycytidine (ddC), leading to mitochondrial DNA (mtDNA) depletion and to mitochondrial dysfunctions. The successful treatment toward mtDNA depletion was confirmed by Complex-IV subunit I (COX-I) immunofluorescence and western blot assays. mtDNA-depleted cells and their counterparts were ultrastructurally characterized by transmission electron microscopy. mtDNA-depleted cells showed dramatic mitochondrial alterations such as fragmentation and cristae disruption along with a reduction of the mitochondrial membrane potential and elevated levels of ROS. Despite increased ROS levels, we did not find any difference in telomere length between ddC-treated and untreated cells. The spontaneous rate of DNA double-strand breaks (DSBs) and chromosome aberrations was significantly enhanced in mtDNA-depleted cells whereas the induction of DSBs by low-Linear Energy Transfer (LET) (X-rays; 7.7keV/μm protons) and high-LET radiations (28.5keV/μm protons) did not differ when compared with normal cells. However, in irradiated cells impaired mitochondrial functions seemed to bring beneficial cellular responses to the detrimental effect of radiations. In fact, after X-irradiation mtDNA-depleted cells show less remaining unrejoined DSBs than normal cells and furthermore a lower induction of cytogenetic damage. Overall, these data show that active mitochondrial functions are required for the proper

  11. Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma.

    PubMed

    González-Gómez, Pilar; Crecente-Campo, Jose; Zahonero, Cristina; de la Fuente, Maria; Hernández-Laín, Aurelio; Mira, Helena; Sánchez-Gómez, Pilar; Garcia-Fuentes, Marcos

    2015-05-10

    Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133(+), Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy.

  12. Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma.

    PubMed

    González-Gómez, Pilar; Crecente-Campo, Jose; Zahonero, Cristina; de la Fuente, Maria; Hernández-Laín, Aurelio; Mira, Helena; Sánchez-Gómez, Pilar; Garcia-Fuentes, Marcos

    2015-05-10

    Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133(+), Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy. PMID:25860932

  13. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

    SciTech Connect

    Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania; Prevatt, Edward G.; Santin, Alessandro D.; DiMaio, Daniel

    2012-01-05

    Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

  14. Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

    PubMed Central

    Lanitis, Evripidis; Dangaj, Denarda; Hagemann, Ian S.; Song, De-Gang; Best, Andrew; Sandaltzopoulos, Raphael; Coukos, George; Powell, Daniel J.

    2012-01-01

    The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies. PMID:23189165

  15. MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes.

    PubMed

    Polioudakis, Damon; Abell, Nathan S; Iyer, Vishwanath R

    2015-01-01

    miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.

  16. Risk Factors for Primary Middle East Respiratory Syndrome Coronavirus Illness in Humans, Saudi Arabia, 2014

    PubMed Central

    Alraddadi, Basem M.; Watson, John T.; Almarashi, Abdulatif; Abedi, Glen R.; Turkistani, Amal; Sadran, Musallam; Housa, Abeer; Almazroa, Mohammad A.; Alraihan, Naif; Banjar, Ayman; Albalawi, Eman; Alhindi, Hanan; Choudhry, Abdul Jamil; Meiman, Jonathan G.; Paczkowski, Magdalena; Curns, Aaron; Mounts, Anthony; Feikin, Daniel R.; Marano, Nina; Swerdlow, David L.; Gerber, Susan I.; Hajjeh, Rana

    2016-01-01

    Risk factors for primary Middle East respiratory syndrome coronavirus (MERS-CoV) illness in humans are incompletely understood. We identified all primary MERS-CoV cases reported in Saudi Arabia during March–November 2014 by excluding those with history of exposure to other cases of MERS-CoV or acute respiratory illness of unknown cause or exposure to healthcare settings within 14 days before illness onset. Using a case–control design, we assessed differences in underlying medical conditions and environmental exposures among primary case-patients and 2–4 controls matched by age, sex, and neighborhood. Using multivariable analysis, we found that direct exposure to dromedary camels during the 2 weeks before illness onset, as well as diabetes mellitus, heart disease, and smoking, were each independently associated with MERS-CoV illness. Further investigation is needed to better understand animal-to-human transmission of MERS-CoV. PMID:26692185

  17. Risk Factors for Primary Middle East Respiratory Syndrome Coronavirus Illness in Humans, Saudi Arabia, 2014.

    PubMed

    Alraddadi, Basem M; Watson, John T; Almarashi, Abdulatif; Abedi, Glen R; Turkistani, Amal; Sadran, Musallam; Housa, Abeer; Almazroa, Mohammad A; Alraihan, Naif; Banjar, Ayman; Albalawi, Eman; Alhindi, Hanan; Choudhry, Abdul Jamil; Meiman, Jonathan G; Paczkowski, Magdalena; Curns, Aaron; Mounts, Anthony; Feikin, Daniel R; Marano, Nina; Swerdlow, David L; Gerber, Susan I; Hajjeh, Rana; Madani, Tariq A

    2016-01-01

    Risk factors for primary Middle East respiratory syndrome coronavirus (MERS-CoV) illness in humans are incompletely understood. We identified all primary MERS-CoV cases reported in Saudi Arabia during March-November 2014 by excluding those with history of exposure to other cases of MERS-CoV or acute respiratory illness of unknown cause or exposure to healthcare settings within 14 days before illness onset. Using a case-control design, we assessed differences in underlying medical conditions and environmental exposures among primary case-patients and 2-4 controls matched by age, sex, and neighborhood. Using multivariable analysis, we found that direct exposure to dromedary camels during the 2 weeks before illness onset, as well as diabetes mellitus, heart disease, and smoking, were each independently associated with MERS-CoV illness. Further investigation is needed to better understand animal-to-human transmission of MERS-CoV. PMID:26692185

  18. Human neuromelanin: an endogenous microglial activator for dopaminergic neuron death

    PubMed Central

    Zhang, Wei; Zecca, Luigi; Wilson, Belinda; Ren, RW; Wang, Yong-jun; Wang, Xiao-min; Hong, Jau-Shyong

    2013-01-01

    Substantial evidence indicates that neuroinflammation caused by over-activation of microglial in the substantia nigra is critical in the pathogenesis of dopaminergic neurodegeneration in Parkinson’s disease (PD). Increasing data demonstrates that environmental factors such as rotenone, paraquat play pivotal roles in the death of dopaminergic neurons. Here, potential role and mechanism of neuromelanin (NM), a major endogenous component in dopaminergic neurons of the substantia nigra, on microglial activation and associated dopaminergic neurotoxicity were investigated. Using multiple well-established primary mesencephalic cultures, we tested whether human NM (HNM) could activate microglia, thereby provoking dopaminergic neurodegeneration. The results demonstrated that in primary mesencephalic neuron-glia cultures, HNM caused dopaminergic neuronal damage characterized by the decreased dopamine uptake and reduced numbers and shorted dendrites of dopaminergic neurons. HNM-induced degeneration was relatively selective to dopaminergic neurons since the other types of neurons determined by either gamma-aminobutyric acid uptake and total neuronal numbers after staining showed smaller decrease. We demonstrated that HNM produced modest dopaminergic neurotoxicity in neuron-enriched cultures; in contrast, much greater neurotoxicity was observed in the presence of microglia. HNM-induced microglial activation was shown by morphological changes and production of proinflammatory and neurotoxic factors. These results suggest that HNM, once released from damaged dopaminergic neurons, can be an potent endogenous activator involved in the reactivation of microglia, which may mediate disease progression. Thus, inhibition of reactive microglia can be a useful strategy for PD therapy. PMID:23276965

  19. Common multigenic activation in different human neoplasias.

    PubMed Central

    Hanania, N; Shaool, D; Harel, J; Wiels, J; Tursz, T

    1983-01-01

    It was previously shown that a common multigenic component, designated as tumor-specific DNA (tsDNA), was transcriptionally active in human lymphoid neoplasias and only slightly active, if at all, in normal lymphoid cells, including the Priess cell line immortalized by Epstein-Barr virus. In the Burkitt lymphoma-derived Raji cell line, tsDNA corresponded to 2500-3000 distinct transcription units, arbitrarily defined as encoding mRNA chains of 1000 kds each. In the present study, radioactive Raji cell tsDNA was isolated by a recycling procedure which eliminates transcribed DNA sequences common to both the Raji cell and the Priess cell, and was used as a probe for homologous transcripts. The major part of this probe could be hybridized to RNAs from all the human neoplasias studied: cultured cell lines derived from leukemias, malignant lymphomas or sarcomas, leukemic cells or solid tumors (sarcomas and carcinomas) recovered from patients. In contrast, only a minor portion of Raji cell tsDNA could be hybridized to RNAs from non-malignant cells, normal human lymphoid cells or fibroblasts grown in culture, fetal and chorioplacental tissues. It is concluded that a common multigenic set is activated in a wide variety of, and perhaps in all, human neoplasias. PMID:6315395

  20. Human activity discrimination for maritime application

    NASA Astrophysics Data System (ADS)

    Boettcher, Evelyn; Deaver, Dawne M.; Krapels, Keith

    2008-04-01

    The US Army RDECOM CERDEC Night Vision and Electronic Sensors Directorate (NVESD) is investigating how motion affects the target acquisition model (NVThermIP) sensor performance estimates. This paper looks specifically at estimating sensor performance for the task of discriminating human activities on watercraft, and was sponsored by the Office of Naval Research (ONR). Traditionally, sensor models were calibrated using still images. While that approach is sufficient for static targets, video allows one to use motion cues to aid in discerning the type of human activity more quickly and accurately. This, in turn, will affect estimated sensor performance and these effects are measured in order to calibrate current target acquisition models for this task. The study employed an eleven alternative forced choice (11AFC) human perception experiment to measure the task difficulty of discriminating unique human activities on watercrafts. A mid-wave infrared camera was used to collect video at night. A description of the construction of this experiment is given, including: the data collection, image processing, perception testing and how contrast was defined for video. These results are applicable to evaluate sensor field performance for Anti-Terrorism and Force Protection (AT/FP) tasks for the U.S. Navy.

  1. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures.

    PubMed

    Boucher, R C; Cotton, C U; Gatzy, J T; Knowles, M R; Yankaskas, J R

    1988-11-01

    1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl- pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (-27 +/- 4 mV, mean +/- S.E.M.; n = 28) exceeded the PD of normal epithelia (-10 +/- 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/- 9 microA cm-2) as compared with normal epithelia (23 +/- 3 microA cm-2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl-), the apical membrane of normal cells exhibits an apical membrane Cl- conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride-sensitive Ieq and amiloride-sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride-sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl- permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of

  2. Activated Human Mast Cells Induce LOX-1-Specific Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

    PubMed Central

    Alanne-Kinnunen, Mervi; Lappalainen, Jani; Öörni, Katariina; Kovanen, Petri T.

    2014-01-01

    Objective Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs). Results Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell –induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages. Conclusions Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis. PMID:25250731

  3. Whole Language Discovery Activities for the Primary Grades.

    ERIC Educational Resources Information Center

    Riley, Margaret C.; Coe, Donna L.

    For the K-3 teacher, this book presents hundreds of ready-to-use individual and group activities for developing reading, writing, listening, and speaking skills, all correlated with other curriculum areas and organized into nine monthly sections. The book includes teaching strategies, individual and group games, activity sheets, quizzes, writing…

  4. Metastatic Gastrointestinal Adenocarcinoma with Osteoblastic Activity: A Case Report of Esophageal and Colonic Primaries

    PubMed Central

    Shabaik, Ahmed S.

    2016-01-01

    Adenocarcinoma with osteoblastic metastases is classically seen in prostate, breast, and lung primaries. Less common primary sites include thyroid, kidney, and stomach. We present two cases of primary gastrointestinal adenocarcinoma with metastatic osteoblastic activity from two previously unreported sites. The first case represents an esophageal adenocarcinoma arising in a background of intestinal metaplasia that metastasized with osteoblastic activity to the deltoid muscle. The second case demonstrates a Stage IV sigmoid colon adenocarcinoma with osteoblastic metastases to the liver and lymph nodes. The findings indicate that metastases from various gastrointestinal primary adenocarcinomas can have prominent bone formation. PMID:27738541

  5. Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

    PubMed

    Lauber, C; Vieyres, G; Terczyńska-Dyla, E; Anggakusuma; Dijkman, R; Gad, H H; Akhtar, H; Geffers, R; Vondran, F W R; Thiel, V; Kaderali, L; Pietschmann, T; Hartmann, R

    2015-09-01

    The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium. PMID:26066369

  6. Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

    PubMed

    Lauber, C; Vieyres, G; Terczyńska-Dyla, E; Anggakusuma; Dijkman, R; Gad, H H; Akhtar, H; Geffers, R; Vondran, F W R; Thiel, V; Kaderali, L; Pietschmann, T; Hartmann, R

    2015-09-01

    The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

  7. The ubiquitin-proteasome pathway is important for dengue virus infection in primary human endothelial cells.

    PubMed

    Kanlaya, Rattiyaporn; Pattanakitsakul, Sa-nga; Sinchaikul, Supachok; Chen, Shui-Tein; Thongboonkerd, Visith

    2010-10-01

    Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

  8. Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells.

    PubMed

    Monti, Daria Maria; Guarnieri, Daniela; Napolitano, Giuliana; Piccoli, Renata; Netti, Paolo; Fusco, Sabato; Arciello, Angela

    2015-01-10

    Recent years have witnessed an unprecedented growth in the number of applications—such as drug delivery, nutraceuticals and production of improved biocompatible materials—in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44 nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent. PMID:25444875

  9. Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions.

    PubMed

    Etna, Marilena P; Giacomini, Elena; Pardini, Manuela; Severa, Martina; Bottai, Daria; Cruciani, Melania; Rizzo, Fabiana; Calogero, Raffaele; Brosch, Roland; Coccia, Eliana M

    2015-11-25

    Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors--ESAT6 and CFP10--within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection.

  10. Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions

    PubMed Central

    Etna, Marilena P.; Giacomini, Elena; Pardini, Manuela; Severa, Martina; Bottai, Daria; Cruciani, Melania; Rizzo, Fabiana; Calogero, Raffaele; Brosch, Roland; Coccia, Eliana M.

    2015-01-01

    Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors—ESAT6 and CFP10—within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection. PMID:26602835

  11. Measuring cholinesterase activity in human saliva.

    PubMed

    Claus Henn, Birgit; McMaster, Suzanne; Padilla, Stephanie

    2006-10-01

    To assess the potential for using saliva in pesticide biomonitoring, the consistency of cholinesterase activity in human saliva collected over time was examined. In this pilot study, saliva was collected from 20 healthy adults once per week for 5 consecutive weeks using 2 different collection methods: a disposable plastic pipette, and a cotton-wool roll. A brief questionnaire was conducted each week to document changes in exposure to cholinesterase inhibitors for the duration of the sampling. To measure cholinesterase activity, an existing radiometric method was modified to make it suitable for human saliva. Using this method, cholinesterase activity was measurable in saliva, and duplicate samples showed reliable repeatability. Activity in both collection methods ranged from 3 to 265 nmol/h/ml saliva (mean = 52 +/- 37 [SD] nmol/h/ml saliva). For some individuals, enzyme activity was consistent over the five sampling weeks; for others, activity was highly variable. Coefficients of variation (CVs) were calculated to assess variability, and mean CVs were the same for both collection methods (about 35%). Adjusting for protein concentration in the pipette-collected samples did not change results. Both collection methods worked well for collecting between 1 and 3 ml saliva, but at the majority of visits (86%), participants preferred the cotton-wool roll. Results from this study suggest that saliva may be a useful indicator of potential neurotoxic effects from exposure to organophosphorus and carbamate pesticides, but that factors affecting variability should be explored further.

  12. Quantifying and mapping the human appropriation of net primary production in earth's terrestrial ecosystems

    PubMed Central

    Haberl, Helmut; Erb, K. Heinz; Krausmann, Fridolin; Gaube, Veronika; Bondeau, Alberte; Plutzar, Christoph; Gingrich, Simone; Lucht, Wolfgang; Fischer-Kowalski, Marina

    2007-01-01

    Human appropriation of net primary production (HANPP), the aggregate impact of land use on biomass available each year in ecosystems, is a prominent measure of the human domination of the biosphere. We present a comprehensive assessment of global HANPP based on vegetation modeling, agricultural and forestry statistics, and geographical information systems data on land use, land cover, and soil degradation that localizes human impact on ecosystems. We found an aggregate global HANPP value of 15.6 Pg C/yr or 23.8% of potential net primary productivity, of which 53% was contributed by harvest, 40% by land-use-induced productivity changes, and 7% by human-induced fires. This is a remarkable impact on the biosphere caused by just one species. We present maps quantifying human-induced changes in trophic energy flows in ecosystems that illustrate spatial patterns in the human domination of ecosystems, thus emphasizing land use as a pervasive factor of global importance. Land use transforms earth's terrestrial surface, resulting in changes in biogeochemical cycles and in the ability of ecosystems to deliver services critical to human well being. The results suggest that large-scale schemes to substitute biomass for fossil fuels should be viewed cautiously because massive additional pressures on ecosystems might result from increased biomass harvest. PMID:17616580

  13. Quantifying and mapping the human appropriation of net primary production in earth's terrestrial ecosystems.

    PubMed

    Haberl, Helmut; Erb, K Heinz; Krausmann, Fridolin; Gaube, Veronika; Bondeau, Alberte; Plutzar, Christoph; Gingrich, Simone; Lucht, Wolfgang; Fischer-Kowalski, Marina

    2007-07-31

    Human appropriation of net primary production (HANPP), the aggregate impact of land use on biomass available each year in ecosystems, is a prominent measure of the human domination of the biosphere. We present a comprehensive assessment of global HANPP based on vegetation modeling, agricultural and forestry statistics, and geographical information systems data on land use, land cover, and soil degradation that localizes human impact on ecosystems. We found an aggregate global HANPP value of 15.6 Pg C/yr or 23.8% of potential net primary productivity, of which 53% was contributed by harvest, 40% by land-use-induced productivity changes, and 7% by human-induced fires. This is a remarkable impact on the biosphere caused by just one species. We present maps quantifying human-induced changes in trophic energy flows in ecosystems that illustrate spatial patterns in the human domination of ecosystems, thus emphasizing land use as a pervasive factor of global importance. Land use transforms earth's terrestrial surface, resulting in changes in biogeochemical cycles and in the ability of ecosystems to deliver services critical to human well being. The results suggest that large-scale schemes to substitute biomass for fossil fuels should be viewed cautiously because massive additional pressures on ecosystems might result from increased biomass harvest.

  14. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  15. No evidence for TSLP pathway activity in human breast cancer.

    PubMed

    Ghirelli, Cristina; Sadacca, Benjamin; Reyal, Fabien; Zollinger, Raphaël; Michea, Paula; Sirven, Philémon; Pattarini, Lucia; Martínez-Cingolani, Carolina; Guillot-Delost, Maude; Nicolas, André; Scholer-Dahirel, Alix; Soumelis, Vassili

    2016-08-01

    Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that primes dendritic cells for Th2 induction. It has been implicated in different types of allergic diseases. Recent work suggested that TSLP could play an important role in the tumor microenvironment and influence tumor progression, in particular in breast cancer. In this study we systematically assessed the production of TSLP at the mRNA and protein levels in several human breast cancer cell lines, large-scale public transcriptomics data sets, and primary human breast tumors. We found that TSLP production was marginal, and concerned less than 10% of the tumors, with very low mRNA and protein levels. In most cases TSLP was undetectable and found to be expressed at lower levels in breast cancer as compared to normal breast tissue. Last, we could not detect any functional TSLP receptor (TSLPR) expression neither on hematopoietic cells nor on stromal cells within the primary tumor microenvironment. We conclude that TSLP-TSLPR pathway activity is not significantly detected within human breast cancer. Taken together, these observations do not support TSLP targeting in breast cancer. PMID:27622057

  16. Retrofitting conventional primary clarifiers to activated primary clarifiers to enhance nutrient removal and energy conservation in WWTPs in Beijing, China.

    PubMed

    Wang, Jia-wei; Zhang, Tian-zhu; Chen, Ji-ning; Hu, Zhi-rong

    2011-01-01

    Biological nutrient removal requires sufficient carbon source. Meanwhile, the removal of organic matter in wastewater requires energy consumption in the aeration tank. Carbon source for nutrient removal in most wastewater treatment plants with conventional primary clarifier (CPC) is generally insufficient in China. In order to increase carbon source and to save energy, a part of the CPC may be retrofitted as an activated primary clarifier (APC). In this paper, a pilot scale experiment was conducted to examine the performance of primary sludge fermentation and its effect on nitrogen and phosphorus removal. Results show that the primary sludge fermentation in APC has produced a similar VFA/TP ratio but a higher BOD5/TN ratio compared with those in the CPC effluent, and the TN concentrations in the secondary effluent are at 8.0, 10.8, and 17.4 mg/L, while TP is at 0.45, 1.10, and 2.28 mg/L when the pilot test system was fed with (1) the APC effluent, (2) 50% from the APC effluent and 50% from the CPC effluent, and (3) the CPC effluent, respectively. Results also indicate that the BOD5/TN ratio is a more sensitive factor than the VFA/TP ratio for nutrient removal and energy conservation for the APC fermentation.

  17. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  18. Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

    PubMed Central

    Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

    2013-01-01

    Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

  19. The psychiatric sequelae of human rights violations: a challenge for primary health care.

    PubMed

    Zungu-Dirwayi, Nompumelelo; Kaminer, Debra; Mbanga, Irene; Stein, Dan J

    2004-04-01

    High rates of psychiatric morbidity have been documented in survivors of gross human rights abuses. Nevertheless, there has been relatively little focus on such patients in the context of primary care medicine. A sample of 134 survivors of gross human rights violations was assessed using a structured interview to determine exposure to violations and psychiatric status. In addition, psychiatric treatment history was probed with an open-ended interview. The study found that of the 95 of 134 (72%) participants who were assessed and found to have a current psychiatric diagnosis, only three were receiving treatment for such a disorder. Many subjects had presented to primary care clinics with somatic symptoms and had been prescribed benzodiazepines. Reasons for not reporting trauma or not seeking treatment included issues revolving around fear and mistrust, privacy and confidentiality, re-experiencing the trauma, and lack of awareness. Misdiagnosis and ineffective treatment of survivors of human rights abuses are likely to pose a significant drain on primary care resources. Accurate diagnosis and appropriate treatment are important challenges in primary care settings.

  20. Effective Connectivity within Human Primary Visual Cortex Predicts Interindividual Diversity in Illusory Perception

    PubMed Central

    Schwarzkopf, D. Samuel; Lutti, Antoine; Li, Baojuan; Kanai, Ryota; Rees, Geraint

    2013-01-01

    Visual perception depends strongly on spatial context. A classic example is the tilt illusion where the perceived orientation of a central stimulus differs from its physical orientation when surrounded by tilted spatial contexts. Here we show that such contextual modulation of orientation perception exhibits trait-like interindividual diversity that correlates with interindividual differences in effective connectivity within human primary visual cortex. We found that the degree to which spatial contexts induced illusory orientation perception, namely, the magnitude of the tilt illusion, varied across healthy human adults in a trait-like fashion independent of stimulus size or contrast. Parallel to contextual modulation of orientation perception, the presence of spatial contexts affected effective connectivity within human primary visual cortex between peripheral and foveal representations that responded to spatial context and central stimulus, respectively. Importantly, this effective connectivity from peripheral to foveal primary visual cortex correlated with interindividual differences in the magnitude of the tilt illusion. Moreover, this correlation with illusion perception was observed for effective connectivity under tilted contextual stimulation but not for that under iso-oriented contextual stimulation, suggesting that it reflected the impact of orientation-dependent intra-areal connections. Our findings revealed an interindividual correlation between intra-areal connectivity within primary visual cortex and contextual influence on orientation perception. This neurophysiological-perceptual link provides empirical evidence for theoretical proposals that intra-areal connections in early visual cortices are involved in contextual modulation of visual perception. PMID:24285885

  1. Effects of aircraft noise on human activities

    NASA Technical Reports Server (NTRS)

    Arnoult, M. D.; Gilfillan, L. G.

    1983-01-01

    The effects of aircrft noise on human activities was investigated by developing a battery of tasks (1) representative of a range of human activities and (2) sensitive to the disruptive effects of noise. The noise used were recordings of jet aircraft and helicopter sounds at three lvels of loudness--60, 70, and 80 dB(A). Experiment 1 investigated 12 different cognitive tasks, along with two intelligibility tasks included to validate that the noises were being effective. Interference with intelligibility was essentially the same as found in the research literature, but only inconsistent effects were found on either accuracy or latency of performance on the cognitive tasks. When the tasks were grouped into four categories (Intelligibility, Matching, Verbal, and Arithmetic), reliable differences in rated annoyingness of the noises were related to the task category and to the type of noise (jet or helicopter).

  2. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  3. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

    PubMed Central

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  4. ACTIVITIES OF HUMAN GENIOGLOSSUS MOTOR UNITS

    PubMed Central

    Bailey, E. Fiona

    2011-01-01

    Upper airway muscles play an important role in regulating airway lumen and in increasing the ability of the pharynx to remain patent in the face of subatmospheric intraluminal pressures produced during inspiration. Due to the considerable technical challenges associated with recording from muscles of the upper airway, much of the experimental work conducted in human subjects has centered on recording respiratory-related activities of the extrinsic tongue protudor muscle, the genioglossus (GG). The GG is one of eight muscles that invest the human tongue (Abd-El-Malek, 1939). All eight muscles are innervated by the hypoglossal nerve (cranial nerve XII) the cell bodies of which are located in the hypoglossal motor nucleus (HMN) of the caudal medulla. Much of the earlier work on the respiratory-related activity of XII motoneurons was based on recordings obtained from single motor axons dissected from the whole XII nerve or from whole muscle GG EMG recordings. Detailed information regarding respiratory-related GG motor unit activities was lacking until as recently as 2006. This paper examines key findings that have emerged from the last decade of work conducted in human subjects. Wherever appropriate, these results are compared with results obtained from in vitro and in vivo studies conducted in non-human mammals. The review is written with the objective of facilitating some discussion and some new thoughts regarding future research directions. The material is framed around four topics: a) Motor unit type, b) Rate coding and recruitment, c) Motor unit activity patterns, and d) A compartment based view of pharyngeal airway control. PMID:21558022

  5. Regulation of different human NFAT isoforms by neuronal activity.

    PubMed

    Vihma, Hanna; Luhakooder, Mirjam; Pruunsild, Priit; Timmusk, Tõnis

    2016-05-01

    Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear

  6. Activities of azithromycin and amphotericin B against Naegleria fowleri in vitro and in a mouse model of primary amebic meningoencephalitis.

    PubMed

    Goswick, Shannon M; Brenner, George M

    2003-02-01

    Inhalation of fresh water containing the free-living ameba Naegleria fowleri may lead to a potentially fatal infection known as primary amebic meningoencephalitis. Amphotericin B is the only agent with established clinical efficacy in the treatment of primary amebic meningoencephalitis in humans, but therapy with this drug is often associated with adverse effects on the kidneys and other organs, and not all persons treated with amphotericin B have survived. We investigated the in vitro activity and in vivo efficacy of newer therapeutic agents in an attempt to identify other useful agents for treating primary amebic meningoencephalitis. Azithromycin has shown in vitro activity against Acanthamoeba spp. and in vivo activity against experimental toxoplasmosis. In our study, the MIC of azithromycin against N. fowleri was 13.4 micro M (10 micro g/ml), which was 123 times greater than the MIC of amphotericin B, which was 0.108 micro M (0.1 micro g/ml). Azithromycin protected 100% of mice infected with N. fowleri at a dose of 75 mg/kg/day for 5 days, whereas amphotericin B protected only 50% of mice at a dose of 7.5 mg/kg/day for 5 days, and all control mice died during the 28-day observation period. We conclude that azithromycin has both in vitro and in vivo activity versus N. fowleri and may be a useful addition to therapy for primary amebic meningoencephalitis.

  7. Effects of synbiotic fermentation products on primary chemoprevention in human colon cells.

    PubMed

    Stein, Katrin; Borowicki, Anke; Scharlau, Daniel; Schettler, Anika; Scheu, Kerstin; Obst, Ursula; Glei, Michael

    2012-07-01

    The consumption of synbiotics, a mixture of probiotics and indigestible food constituents such as dietary fiber, has been reported to reduce colon cancer risk. We investigated the effects of fermented wheat aleurone enriched with the probiotics Lactobacillus rhamnosus GG/Bifidobacterium animalis supsp. lactis on the gene expression and functional end points related to cellular defence in HT29 and primary human colon cells. Aleurone was digested and fermented in vitro with/without probiotics. The resulting fermentation supernatants (fs) were analyzed for concentrations of deoxycholic acid and ammonia. The cells were treated with the fs, and effects on gene expression of catalase, GSTP1 and SULT2B1, enzyme activity of catalase and glutathione S-transferase as well as H₂O₂-induced DNA damage were examined. Fermentation of aleurone reduced deoxycholic acid concentration by 84%, while the probiotics enhanced this effect. Ammonia was increased by fs aleurone, whereas a reduction occurred by the addition of L. rhamnosus GG/B. animalis supsp. lactis 12. GSTP1 expression tended to result in an increase by the fs aleurone in both cell types, whereas the probiotics could not additionally increase the effect. Catalase was not modulated by fs aleurone enriched with probiotics. Only in HT29 cells, expression of SULT2B1 was enhanced by fs aleurone. Enzyme activity of catalase and glutathione S-transferase was induced (2-3.6 fold, 72 h) in HT29 cells only. Addition of probiotics had no influence on this effect. In HT29 cells, a reduced H₂O₂-induced DNA damage by the fs aleurone after 48 h, enhanced by the addition of probiotics, was detected. The observed effects could improve detoxification of xenobiotics and therefore may lower colon cancer risk. PMID:21840698

  8. Thrombopoietin receptor expression in human cancer cell lines and primary tissues.

    PubMed

    Columbyova, L; Loda, M; Scadden, D T

    1995-08-15

    c-mpl is the receptor for the recently identified megakaryocyte growth and differentiation factor thrombopoietin. Thrombopoietin has been shown to be capable of raising platelet counts in animals and is about to enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of human c-mpl by reverse transcription PCR on 39 human cell lines and 20 primary human tissue samples derived from both normal and malignant sources. c-mpl transcripts were found in all megakaryocytic cell lines tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia cell line KMT-2, and a hepatocellular carcinoma cell line (Hep3B). Among primary tissues, fetal liver cells and brain had detectable levels of c-mpl message, and among primary tumors, none were found to express c-mpl. These data support the conclusion that c-mpl has restricted expression that is primarily, but not exclusively, related to megakaryocytopoiesis. These observations suggest that thrombopoietin is unlikely to have direct effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia. PMID:7627956

  9. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  10. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.

    PubMed Central

    Koup, R A; Safrit, J T; Cao, Y; Andrews, C A; McLeod, G; Borkowsky, W; Farthing, C; Ho, D D

    1994-01-01

    Virologic and immunologic studies were performed on five patients presenting with primary human immunodeficiency virus type 1 (HIV-1) infection. CD8+ cytotoxic T lymphocyte (CTL) precursors specific for cells expressing antigens of HIV-1 Gag, Pol, and Env were detected at or within 3 weeks of presentation in four of the five patients and were detected in all five patients by 3 to 6 months after presentation. The one patient with an absent initial CTL response had prolonged symptoms, persistent viremia, and low CD4+ T-cell count. Neutralizing antibody activity was absent at the time of presentation in all five patients. These findings suggest that cellular immunity is involved in the initial control of virus replication in primary HIV-1 infection and indicate a role for CTL in protective immunity to HIV-1 in vivo. PMID:8207839

  11. Enhancing Primary School Students' Knowledge about Global Warming and Environmental Attitude Using Climate Change Activities

    NASA Astrophysics Data System (ADS)

    Karpudewan, Mageswary; Roth, Wolff-Michael; Abdullah, Mohd Nor Syahrir Bin

    2015-01-01

    Climate change generally and global warming specifically have become a common feature of the daily news. Due to widespread recognition of the adverse consequences of climate change on human lives, concerted societal effort has been taken to address it (e.g. by means of the science curriculum). This study was designed to test the effect that child-centred, 5E learning cycle-based climate change activities would have over more traditional teacher-centred activities on Malaysian Year 5 primary students (11 years). A quasi-experimental design involving a treatment (n = 55) and a group representing typical teaching method (n = 60) was used to measure the effectiveness of these activities on (a) increasing children's knowledge about global warming; (b) changing their attitudes to be more favourable towards the environment and (c) identify the relationship between knowledge and attitude that exist in this study. Statistically significant differences in favour of the treatment group were detected for both knowledge and environmental attitudes. Non-significant relationship was identified between knowledge and attitude in this study. Interviews with randomly selected students from treatment and comparison groups further underscore these findings. Implications are discussed.

  12. Activation of human factor IX (Christmas factor).

    PubMed

    Di Scipio, R G; Kurachi, K; Davie, E W

    1978-06-01

    Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its

  13. Robots, Bulldozers, and Other Map Activities for the Primary Grades.

    ERIC Educational Resources Information Center

    Pritchard, Sandra F.

    1984-01-01

    Described are activities which will increase fluency in the use of north, south, east, west, up, and down; reinforce the proper positioning of the cardinal directions on a map grid; relate map symbols to familiar surface features; give practice in identifying map symbols; and provide an opportunity to construct maps. (RM)

  14. Teacher Feedback during Active Learning: Current Practices in Primary Schools

    ERIC Educational Resources Information Center

    van den Bergh, Linda; Ros, Anje; Beijaard, Douwe

    2013-01-01

    Background: Feedback is one of the most powerful tools, which teachers can use to enhance student learning. It appears dif?cult for teachers to give qualitatively good feedback, especially during active learning. In this context, teachers should provide facilitative feedback that is focused on the development of meta-cognition and social learning.…

  15. Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.

    PubMed

    Blader, Patrick; Plessy, Charles; Strähle, Uwe

    2003-02-01

    The basic Helix-Loop-Helix gene neurogenin1 (ngn1) is expressed in a complex pattern in the neural plate of zebrafish embryos, demarcating the sites of primary neurogenesis. We have dissected the ngn1 locus to identify cis-regulatory regions that control this expression. We have isolated two upstream elements that drive expression in precursors of Rohon-Beard sensory neurons and hindbrain interneurons and in clusters of neuronal precursors in the anterior neural plate, respectively. A third regulatory region mediates later expression. Thus, regulatory sequences with temporally and spatially distinct activities control ngn1 expression in primary neurons of the zebrafish embryo. These regions are highly similar to 5' sequences in the mouse and human ngn1 gene, suggesting that amniote embryos, despite lacking primary neurons, utilize related mechanism to control ngn1 expression. PMID:12559493

  16. Antibacterial activity of human natural killer cells

    PubMed Central

    1989-01-01

    The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass- adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11- enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16- 24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing. PMID:2642532

  17. Look Around You. A Primary Student Activity Book Introducing Basic Environmental Concepts.

    ERIC Educational Resources Information Center

    Starkey, Sharon

    This activity book, designed for student use, introduces environmental concepts to the primary student. The basic concept around which the guide is developed is the idea that the environment contains many interdependent things. Water, wind, clouds, non-living objects, plants, animals, and pollution are dealt with as part of the primary student's…

  18. Inhibition of human natural killer cell functional activity by human aspartyl β-hydroxylase.

    PubMed

    Huyan, Ting; Li, Qi; Ye, Lin-Jie; Yang, Hui; Xue, Xiao-Ping; Zhang, Ming-Jie; Huang, Qing-Sheng; Yin, Da-Chuan; Shang, Peng

    2014-12-01

    Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl β-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.

  19. Developing supplemental activities for primary health care maternity services.

    PubMed

    Panitz, E

    1990-12-01

    Supplemental health care activities are described in the context of the augmented product. The potential benefits of supplemental services to recipients and provider are discussed. The author describes a study that was the basis for (re)developing a supplemental maternity service. The implementation of the results in terms of changes in the marketing mix of this supplemental program is discussed. The effects of the marketing mix changes on program participation are presented.

  20. Primary silver extraction with a high sulphur activated petroleum coke.

    PubMed

    Schouwenaars, R; Durán Moreno, A; Ramírez Zamora, R M

    2004-01-01

    An extended study was performed to determine the mechanisms that are responsible for the significant silver extraction capacity of activated carbons prepared from a high-sulphur petroleum coke that is available as a waste material from Mexican petroleum refineries. Earlier studies had shown the feasibility of the production of these adsorbents but indicated that the mechanisms of metal adsorption in the present carbons are significantly different from what is classically accepted for commercial carbons. Therefore, selective titration, IR-spectroscopy and scanning electron microscopy of carbons were combined with adsorption experiments and the determination of electrochemical parameters of mixtures of carbon-AgNO3 solution to explain the fundamental reasons for the performance of the obtained carbons. This allowed us to determine the identity of the surface functional groups and to distinguish the effect of different activation processes. The experiments permitted us to explain why these activated carbons, which have a low specific area and lack classical surface functional groups, show such a high silver adsorption capacity.

  1. [Lysosomal glycosidase activity in cultured human fibroblasts].

    PubMed

    Beliaeva, I D; Ivleva, T S; Vidershaĭn, G Ia

    1984-11-01

    A study was made of the activity of 3 lysosomal glycosidases -beta-D-galactosidase (K. P. 3.2.1.23), alpha-L-fucosidase (K. P. 3.2.1.51), N-acetyl-beta-D-hexosoaminidase (K. P. 3.2.1.52) depending on the time after subcultivation and duration of the passage of human skin embryonal and postembryonal fibroblasts. It was established that changes in the specific activity of the enzymes should be calculated with reference to the cell rather than to protein whose amount might vary considerably. It was also found that for measuring the specific activity of enzymes, of great importance are the procedures of cell removal from the base layer (by mechanical scraping off or by trypsin solution) and the regimen of the homogenization of cell preparations.

  2. Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells

    PubMed Central

    Jennings, Michael P.; Jen, Freda E.-C.; Roddam, Louise F.; Apicella, Michael A.; Edwards, Jennifer L.

    2013-01-01

    Summary Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis. PMID:21371235

  3. Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells.

    PubMed

    Puck, Alexander; Aigner, Regina; Modak, Madhura; Cejka, Petra; Blaas, Dieter; Stöckl, Johannes

    2015-01-01

    Schlafen (SLFN/Slfn) family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs) and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence. PMID:26623250

  4. Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells.

    PubMed

    Garcia, E; Lacasa, D; Agli, B; Giudicelli, Y; Castelli, D

    2000-04-01

    The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular. PMID:18503465

  5. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  6. Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

    SciTech Connect

    Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C. ); Grzeschik, K.H. )

    1988-02-01

    Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration.

  7. Production and Concentration of Lentivirus for Transduction of Primary Human T Cells.

    PubMed

    Kennedy, Alan; Cribbs, Adam P

    2016-01-01

    Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility. PMID:27317175

  8. Human T Follicular Helper Cells in Primary Immunodeficiency: Quality Just as Important as Quantity.

    PubMed

    Ma, Cindy S

    2016-05-01

    T follicular helper (Tfh) cells are a subset of effector CD4(+) T cells specialised to induce Ab production by B cells. This review highlights some of the recent advances in the field of human Tfh cells that have come from the study of primary immunodeficiencies. In particular it is increasingly evident that the quality of the Tfh cells that are generated, is just as important as the quantity.

  9. Human Immunodeficiency Virus Type 1 Coat Protein Neurotoxicity Mediated by Nitric Oxide in Primary Cortical Cultures

    NASA Astrophysics Data System (ADS)

    Dawson, Valina L.; Dawson, Ted M.; Uhl, George R.; Snyder, Solomon H.

    1993-04-01

    The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

  10. Eight Stars of Gold--The Story of Alaska's Flag. Primary Grade Activities.

    ERIC Educational Resources Information Center

    Alaska State Museum, Juneau.

    This activities booklet focuses on the story of Alaska's flag. The booklet is intended for teachers to use with primary-grade children. Each activity in the booklet contains background information, a summary and time estimate, Alaska state standards, a step-by-step technique for implementing the activity, assessment tips, materials and resource…

  11. Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

    PubMed Central

    Mak, P; Wójcik, K; Thogersen, I B; Dubin, A

    1996-01-01

    Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3. PMID:8890190

  12. Immunosuppressive activity of human neuroblastoma tumor gangliosides.

    PubMed

    Floutsis, G; Ulsh, L; Ladisch, S

    1989-01-15

    Gangliosides are shed in substantial amounts by some tumors, including human neuroblastoma, and these molecules modulate experimental tumor formation in vivo. We now demonstrate that neuroblastoma tumor gangliosides have potent immunoregulatory activity. Gangliosides of every one of 17 tumors studied were highly inhibitory for the normal in vitro human lymphoproliferative responses to the soluble antigen, tetanus toxoid; 30 nmol ganglioside/ml caused 43% to greater than 99% inhibition and the mean concentration causing 50% inhibition was only 17.3 nmol/ml. Furthermore, gangliosides isolated from clinically more aggressive tumors (Stage III or IV) were up to twice as immunosuppressive as those of the generally less aggressive tumors (Stage I or II) (p less than 0.05). Taken together with the lack of immunosuppressive activity of normal plasma gangliosides, the potent activity of neuroblastoma gangliosides supports the hypothesis that one mechanism by which these shed molecules may act to enhance tumor formation in vivo is through abrogation of the host cellular immune response at the site of tumor formation.

  13. Physical Human Activity Recognition Using Wearable Sensors.

    PubMed

    Attal, Ferhat; Mohammed, Samer; Dedabrishvili, Mariam; Chamroukhi, Faicel; Oukhellou, Latifa; Amirat, Yacine

    2015-12-11

    This paper presents a review of different classification techniques used to recognize human activities from wearable inertial sensor data. Three inertial sensor units were used in this study and were worn by healthy subjects at key points of upper/lower body limbs (chest, right thigh and left ankle). Three main steps describe the activity recognition process: sensors' placement, data pre-processing and data classification. Four supervised classification techniques namely, k-Nearest Neighbor (k-NN), Support Vector Machines (SVM), Gaussian Mixture Models (GMM), and Random Forest (RF) as well as three unsupervised classification techniques namely, k-Means, Gaussian mixture models (GMM) and Hidden Markov Model (HMM), are compared in terms of correct classification rate, F-measure, recall, precision, and specificity. Raw data and extracted features are used separately as inputs of each classifier. The feature selection is performed using a wrapper approach based on the RF algorithm. Based on our experiments, the results obtained show that the k-NN classifier provides the best performance compared to other supervised classification algorithms, whereas the HMM classifier is the one that gives the best results among unsupervised classification algorithms. This comparison highlights which approach gives better performance in both supervised and unsupervised contexts. It should be noted that the obtained results are limited to the context of this study, which concerns the classification of the main daily living human activities using three wearable accelerometers placed at the chest, right shank and left ankle of the subject.

  14. Physical Human Activity Recognition Using Wearable Sensors

    PubMed Central

    Attal, Ferhat; Mohammed, Samer; Dedabrishvili, Mariam; Chamroukhi, Faicel; Oukhellou, Latifa; Amirat, Yacine

    2015-01-01

    This paper presents a review of different classification techniques used to recognize human activities from wearable inertial sensor data. Three inertial sensor units were used in this study and were worn by healthy subjects at key points of upper/lower body limbs (chest, right thigh and left ankle). Three main steps describe the activity recognition process: sensors’ placement, data pre-processing and data classification. Four supervised classification techniques namely, k-Nearest Neighbor (k-NN), Support Vector Machines (SVM), Gaussian Mixture Models (GMM), and Random Forest (RF) as well as three unsupervised classification techniques namely, k-Means, Gaussian mixture models (GMM) and Hidden Markov Model (HMM), are compared in terms of correct classification rate, F-measure, recall, precision, and specificity. Raw data and extracted features are used separately as inputs of each classifier. The feature selection is performed using a wrapper approach based on the RF algorithm. Based on our experiments, the results obtained show that the k-NN classifier provides the best performance compared to other supervised classification algorithms, whereas the HMM classifier is the one that gives the best results among unsupervised classification algorithms. This comparison highlights which approach gives better performance in both supervised and unsupervised contexts. It should be noted that the obtained results are limited to the context of this study, which concerns the classification of the main daily living human activities using three wearable accelerometers placed at the chest, right shank and left ankle of the subject. PMID:26690450

  15. Physical Human Activity Recognition Using Wearable Sensors.

    PubMed

    Attal, Ferhat; Mohammed, Samer; Dedabrishvili, Mariam; Chamroukhi, Faicel; Oukhellou, Latifa; Amirat, Yacine

    2015-01-01

    This paper presents a review of different classification techniques used to recognize human activities from wearable inertial sensor data. Three inertial sensor units were used in this study and were worn by healthy subjects at key points of upper/lower body limbs (chest, right thigh and left ankle). Three main steps describe the activity recognition process: sensors' placement, data pre-processing and data classification. Four supervised classification techniques namely, k-Nearest Neighbor (k-NN), Support Vector Machines (SVM), Gaussian Mixture Models (GMM), and Random Forest (RF) as well as three unsupervised classification techniques namely, k-Means, Gaussian mixture models (GMM) and Hidden Markov Model (HMM), are compared in terms of correct classification rate, F-measure, recall, precision, and specificity. Raw data and extracted features are used separately as inputs of each classifier. The feature selection is performed using a wrapper approach based on the RF algorithm. Based on our experiments, the results obtained show that the k-NN classifier provides the best performance compared to other supervised classification algorithms, whereas the HMM classifier is the one that gives the best results among unsupervised classification algorithms. This comparison highlights which approach gives better performance in both supervised and unsupervised contexts. It should be noted that the obtained results are limited to the context of this study, which concerns the classification of the main daily living human activities using three wearable accelerometers placed at the chest, right shank and left ankle of the subject. PMID:26690450

  16. miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

    PubMed Central

    Almeida, Maria Ines; Silva, Andreia Machado; Vasconcelos, Daniel Marques; Almeida, Catarina Rodrigues; Caires, Hugo; Pinto, Marta Teixeira; Calin, George Adrian; Santos, Susana Gomes; Barbosa, Mário Adolfo

    2016-01-01

    Mesenchymal Stromal/Stem Cells (MSC) are currently being explored in diverse clinical applications, including regenerative therapies. Their contribution to regeneration of bone fractures is dependent on their capacity to proliferate, undergo osteogenesis and induce angiogenesis. This study aimed to uncover microRNAs capable of concomitantly regulate these mechanisms. Following microRNA array results, we identified miR-195 and miR-497 as downregulated in human primary MSC under osteogenic differentiation. Overexpression of miR-195 or miR-497 in human primary MSC leads to a decrease in osteogenic differentiation and proliferation rate. Conversely, inhibition of miR-195 increased alkaline phosphatase expression and activity and cells proliferation. Then, miR-195 was used to study MSC capacity to recruit blood vessels in vivo. We provide evidence that the paracrine effect of MSC on angiogenesis is diminishedwhen cells over-express miR-195. VEGF may partially mediate this effect, as its expression and secreted protein levels are reduced by miR-195, while increased by anti-miR-195, in human MSC. Luciferase reporter assays revealed a direct interaction between miR-195 and VEGF 3′-UTR in bone cancer cells. In conclusion, our results suggest that miR-195 regulates important mechanisms for bone regeneration, specifically MSC osteogenic differentiation, proliferation and control of angiogenesis; therefore, it is a potential target for clinical bone regenerative therapies. PMID:26683705

  17. Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression.

    PubMed

    Liu, XinJian; Huang, Qian; Li, Fang; Li, Chuan-Yuan

    2014-09-01

    Dopaminergic (DA) neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson's disease. A significant obstacle is the low efficiency of conversion during the reprogramming process. Here, we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons. In particular, blocking p53 activity using a dominant-negative p53 (p53-DN) in IMR90 cells increases the conversion efficiency by 5-20 fold. The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression, significant dopamine uptake and production capacities, and enables symptomatic relief in a rat Parkinson's disease model. Taken together, our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons. PMID:25129808

  18. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

    PubMed Central

    Su, Shu; Hu, Bian; Shao, Jie; Shen, Bin; Du, Juan; Du, Yinan; Zhou, Jiankui; Yu, Lixia; Zhang, Lianru; Chen, Fangjun; Sha, Huizi; Cheng, Lei; Meng, Fanyan; Zou, Zhengyun; Huang, Xingxu; Liu, Baorui

    2016-01-01

    Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies. PMID:26818188

  19. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.

    PubMed

    Su, Shu; Hu, Bian; Shao, Jie; Shen, Bin; Du, Juan; Du, Yinan; Zhou, Jiankui; Yu, Lixia; Zhang, Lianru; Chen, Fangjun; Sha, Huizi; Cheng, Lei; Meng, Fanyan; Zou, Zhengyun; Huang, Xingxu; Liu, Baorui

    2016-01-28

    Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

  20. Early suppression effect in human primary visual cortex during Kanizsa illusion processing: A magnetoencephalographic evidence.

    PubMed

    Chernyshev, Boris V; Pronko, Platon K; Stroganova, Tatiana A

    2016-01-01

    Detection of illusory contours (ICs) such as Kanizsa figures is known to depend primarily upon the lateral occipital complex. Yet there is no universal agreement on the role of the primary visual cortex in this process; some existing evidence hints that an early stage of the visual response in V1 may involve relative suppression to Kanizsa figures compared with controls. Iso-oriented luminance borders, which are responsible for Kanizsa illusion, may evoke surround suppression in V1 and adjacent areas leading to the reduction in the initial response to Kanizsa figures. We attempted to test the existence, as well as to find localization and timing of the early suppression effect produced by Kanizsa figures in adult nonclinical human participants. We used two sizes of visual stimuli (4.5 and 9.0°) in order to probe the effect at two different levels of eccentricity; the stimuli were presented centrally in passive viewing conditions. We recorded magnetoencephalogram, which is more sensitive than electroencephalogram to activity originating from V1 and V2 areas. We restricted our analysis to the medial occipital area and the occipital pole, and to a 40-120 ms time window after the stimulus onset. By applying threshold-free cluster enhancement technique in combination with permutation statistics, we were able to detect the inverted IC effect-a relative suppression of the response to the Kanizsa figures compared with the control stimuli. The current finding is highly compatible with the explanation involving surround suppression evoked by iso-oriented collinear borders. The effect may be related to the principle of sparse coding, according to which V1 suppresses representations of inner parts of collinear assemblies as being informationally redundant. Such a mechanism is likely to be an important preliminary step preceding object contour detection. PMID:27485162

  1. Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi

    PubMed Central

    Dykan, Andrey; Rachakonda, Girish; Villalta, Fernando; Mandape, Sammed N.; Lima, Maria F.; Pratap, Siddharth; Nde, Pius N.

    2016-01-01

    The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy

  2. Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi.

    PubMed

    Udoko, Aniekanabassi N; Johnson, Candice A; Dykan, Andrey; Rachakonda, Girish; Villalta, Fernando; Mandape, Sammed N; Lima, Maria F; Pratap, Siddharth; Nde, Pius N

    2016-01-01

    The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy

  3. Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi.

    PubMed

    Udoko, Aniekanabassi N; Johnson, Candice A; Dykan, Andrey; Rachakonda, Girish; Villalta, Fernando; Mandape, Sammed N; Lima, Maria F; Pratap, Siddharth; Nde, Pius N

    2016-01-01

    The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy

  4. Bile Acid-Induced Necrosis in Primary Human Hepatocytes and in Patients with Obstructive Cholestasis

    PubMed Central

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-01-01

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. PMID:25636263

  5. CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

    PubMed Central

    Pathak, Janak L.; Bakker, Astrid D.; Verschueren, Patrick; Lems, Willem F.; Luyten, Frank P.; Klein-Nulend, Jenneke; Bravenboer, Nathalie

    2015-01-01

    Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication. PMID:26103626

  6. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    PubMed

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  7. Decoding patterns of human brain activity.

    PubMed

    Tong, Frank; Pratte, Michael S

    2012-01-01

    Considerable information about mental states can be decoded from noninvasive measures of human brain activity. Analyses of brain activity patterns can reveal what a person is seeing, perceiving, attending to, or remembering. Moreover, multidimensional models can be used to investigate how the brain encodes complex visual scenes or abstract semantic information. Such feats of "brain reading" or "mind reading," though impressive, raise important conceptual, methodological, and ethical issues. What does successful decoding reveal about the cognitive functions performed by a brain region? How should brain signals be spatially selected and mathematically combined to ensure that decoding reflects inherent computations of the brain rather than those performed by the decoder? We highlight recent advances and describe how multivoxel pattern analysis can provide a window into mind-brain relationships with unprecedented specificity, when carefully applied. However, as brain-reading technology advances, issues of neuroethics and mental privacy will be important to consider.

  8. Trehalose-Mediated Autophagy Impairs the Anti-Viral Function of Human Primary Airway Epithelial Cells

    PubMed Central

    Wu, Qun; Jiang, Di; Huang, Chunjian; van Dyk, Linda F.; Li, Liwu; Chu, Hong Wei

    2015-01-01

    Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations. PMID:25879848

  9. A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

    PubMed Central

    Parikh, Mukti R.; Belch, Andrew R.; Pilarski, Linda M; Kirshner, Julia

    2014-01-01

    Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions

  10. Preparation of gaseous CRMs from the primary system for (222)Rn activity measurement.

    PubMed

    Kim, B J; Kim, B C; Lee, K B; Lee, J M; Park, T S

    2016-03-01

    For disseminating the gaseous radon standard traceable to the KRISS primary system based on the defined solid angle counting method, two kinds of radon CRM (a glass ampule type and a stainless steel cylinder type) were developed. The activity of the CRM was certified by subtracting a residual activity from the measured activity by the primary system. After certification, the ampule CRM was used to calibrate a radon-monitoring instrument and the cylinder CRM to calibrate an HPGe system. We also improved the measurement procedure of the radon primary system. In a typical radon energy spectrum, the radon peak overlaps with the polonium peak. For more reliable and accurate measurement of radon activity, a fitting method was adopted for the evaluation of radon area in the alpha energy spectrum. The result of radon activity evaluated by using the fitting method is in good agreement with that by the previous integration method. PMID:26778761

  11. The spatial structure of transnational human activity.

    PubMed

    Deutschmann, Emanuel

    2016-09-01

    Starting from conflictive predictions of hitherto disconnected debates in the natural and social sciences, this article examines the spatial structure of transnational human activity (THA) worldwide (a) across eight types of mobility and communication and (b) in its development over time. It is shown that the spatial structure of THA is similar to that of animal displacements and local-scale human motion in that it can be approximated by Lévy flights with heavy tails that obey power laws. Scaling exponent and power-law fit differ by type of THA, being highest in refuge-seeking and tourism and lowest in student exchange. Variance in the availability of resources and opportunities for satisfying associated needs appears to explain these differences. Over time (1960-2010), the Lévy-flight pattern remains intact and remarkably stable, contradicting the popular notion that socio-technological trends lead to a "death of distance." Humans have not become more "global" over time, they rather became more mobile in general, i.e. they move and communicate more at all distances. Hence, it would be more adequate to speak of "mobilization" than of "globalization." Longitudinal change occurs only in some types of THA and predominantly at short distances, indicating regional rather than global shifts. PMID:27480376

  12. Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth.

    PubMed

    Winship, Amy; Cuman, Carly; Rainczuk, Katarzyna; Dimitriadis, Evdokia

    2015-12-01

    Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts. We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women. PMID:26506560

  13. Astrocytes As the Main Players in Primary Degenerative Disorders of the Human Central Nervous System.

    PubMed

    Capani, Francisco; Quarracino, Cecilia; Caccuri, Roberto; Sica, Roberto E P

    2016-01-01

    Along the last years it has been demonstrated that non-neural cells play a major role in the pathogenesis of the primary degenerative disorders (PDDs) of the human central nervous system. Among them, astrocytes coordinate and participate in many different and complex metabolic processes, in close interaction with neurons. Moreover, increasing experimental evidence hints an early astrocytic dysfunction in these diseases. In this mini review we summarize the astrocytic behavior in PDDs, with special consideration to the experimental observations where astrocytic pathology precedes the development of neuronal dysfunction. We also suggest a different approach that could be consider in human investigations in Alzheimer's and Parkinson's disease. We believe that the study of PDDs with human brain samples may hold the key of a paradigmatic physiopathological process in which astrocytes might be the main players. PMID:26973519

  14. Genotoxicity test of self-renovated ceramics in primary human peripheral lymphocytes.

    PubMed

    Hua, Nan; Zhu, Huifang; Zhuang, Jing; Chen, Liping

    2014-12-01

    Zirconia-based ceramics is widely used in dentistry. Different compositions of ceramics have different features. Our self-renovated ceramics become more machinable without scarifying its dental restoration properties after adjusting ratio of lanthanum phosphate (LaPO4)/yttrium oxide (Y2O3). In order to evaluate its safety, here, we tested its genotoxicity in primary human peripheral lymphocytes. The human lymphocytes cultured on three groups of different ratios of LaPO4/Y2O3 diphase ceramics for 6 days showed little effect of growth inhibition and similar effect of growth trend to the negative control. Furthermore, single-cell gel electrophoresis (comet assay) indicated that there was no significant difference of the value of tail moment between the tested ceramics and negative control, the IPS Empress II (P > 0.05). Our findings implicate that our self-renovated ceramics do not induce DNA damages in human peripheral lymphocytes and support their future clinic application.

  15. Characterizing primary human microglia: A comparative study with myeloid subsets and culture models.

    PubMed

    Melief, J; Sneeboer, M A M; Litjens, M; Ormel, P R; Palmen, S J M C; Huitinga, I; Kahn, R S; Hol, E M; de Witte, L D

    2016-11-01

    The biology of microglia has become subject to intense study, as they are widely recognized as crucial determinants of normal and pathologic brain functioning. While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro- and anti-inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro- (LPS or poly I:C) and anti-inflammatory (IL-4 or dexamethasone) stimuli. Interestingly, pro-inflammatory responses differed between microglia and monocyte-derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. GLIA 2016;64:1857-1868.

  16. Global human appropriation of net primary production doubled in the 20th century

    PubMed Central

    Krausmann, Fridolin; Erb, Karl-Heinz; Gingrich, Simone; Haberl, Helmut; Bondeau, Alberte; Gaube, Veronika; Lauk, Christian; Plutzar, Christoph; Searchinger, Timothy D.

    2013-01-01

    Global increases in population, consumption, and gross domestic product raise concerns about the sustainability of the current and future use of natural resources. The human appropriation of net primary production (HANPP) provides a useful measure of human intervention into the biosphere. The productive capacity of land is appropriated by harvesting or burning biomass and by converting natural ecosystems to managed lands with lower productivity. This work analyzes trends in HANPP from 1910 to 2005 and finds that although human population has grown fourfold and economic output 17-fold, global HANPP has only doubled. Despite this increase in efficiency, HANPP has still risen from 6.9 Gt of carbon per y in 1910 to 14.8 GtC/y in 2005, i.e., from 13% to 25% of the net primary production of potential vegetation. Biomass harvested per capita and year has slightly declined despite growth in consumption because of a decline in reliance on bioenergy and higher conversion efficiencies of primary biomass to products. The rise in efficiency is overwhelmingly due to increased crop yields, albeit frequently associated with substantial ecological costs, such as fossil energy inputs, soil degradation, and biodiversity loss. If humans can maintain the past trend lines in efficiency gains, we estimate that HANPP might only grow to 27–29% by 2050, but providing large amounts of bioenergy could increase global HANPP to 44%. This result calls for caution in refocusing the energy economy on land-based resources and for strategies that foster the continuation of increases in land-use efficiency without excessively increasing ecological costs of intensification. PMID:23733940

  17. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    SciTech Connect

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; Marson, Alexander

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

  18. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

    PubMed

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R; Subramaniam, Meena; Gate, Rachel E; Haliburton, Genevieve E; Ye, Chun J; Bluestone, Jeffrey A; Doudna, Jennifer A; Marson, Alexander

    2015-08-18

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

  19. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    DOE PAGES

    Schumann, Kathrin; Lin, Steven; Boyer, Eric; Simeonov, Dimitre R.; Subramaniam, Meena; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Bluestone, Jeffrey A.; Doudna, Jennifer A.; et al

    2015-07-27

    T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4+ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9more » RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ~40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ~20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.« less

  20. Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling.

    PubMed

    Mitchell, Troy; Lo, Andrea; Logan, Michael R; Lacy, Paige; Eitzen, Gary

    2008-11-01

    The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

  1. Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo

    PubMed Central

    Becerra, Aniuska; Warke, Rajas V.; Martin, Katherine; Xhaja, Kris; de Bosch, Norma; Rothman, Alan L.; Bosch, Irene

    2009-01-01

    We used gene expression profiling of human primary cells infected in vitro with dengue virus (DENV) as a tool to identify secreted mediators induced in response to the acute infection. Affymetrix Genechip analysis of human primary monocytes, B cells and dendritic cells infected with DENV in vitro revealed a strong induction of monocyte chemotactic protein 2 (MCP-2/CCL8), interferon gamma-induced protein 10 (IP-10/CXCL10) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10). The expression of these genes was confirmed in dendritic cells infected with DENV in vitro at mRNA and protein levels. A prospectively enrolled cohort of DENV-infected Venezuelan patients was used to measure the levels of these proteins in serum during three different periods of the disease. Results showed significant increase of MCP-2, IP-10 and TRAIL levels in DENV-infected patients during the febrile period, when compared to healthy donors and patients with other febrile illnesses. MCP-2 and IP-10 levels were still elevated during the post-febrile period while TRAIL levels dropped close to normal after defervescense. Patients with primary infections had higher TRAIL levels than patients with secondary infections during the febrile period of the disease. Increased levels of IP-10, TRAIL and MCP-2 in acute DENV infections suggest a role for these mediators in the immune response to the infection. PMID:19551822

  2. Correlates of perceptual awareness in human primary auditory cortex revealed by an informational masking experiment.

    PubMed

    Wiegand, Katrin; Gutschalk, Alexander

    2012-05-15

    The presence of an auditory event may remain undetected in crowded environments, even when it is well above the sensory threshold. This effect, commonly known as informational masking, allows for isolating neural activity related to perceptual awareness, by comparing repetitions of the same physical stimulus where the target is either detected or not. Evidence from magnetoencephalography (MEG) suggests that auditory-cortex activity in the latency range 50-250 ms is closely coupled with perceptual awareness. Here, BOLD fMRI and MEG were combined to investigate at which stage in the auditory cortex neural correlates of conscious auditory perception can be observed. Participants were asked to indicate the perception of a regularly repeating target tone, embedded within a random multi-tone masking background. Results revealed widespread activation within the auditory cortex for detected target tones, which was delayed but otherwise similar to the activation of an unmasked control stimulus. The contrast of detected versus undetected targets revealed activity confined to medial Heschl's gyrus, where the primary auditory cortex is located. These results suggest that activity related to conscious perception involves the primary auditory cortex and is not restricted to activity in secondary areas.

  3. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.

  4. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  5. Physical Activity as a Dimension of Family Life for Lower Primary School Children

    ERIC Educational Resources Information Center

    Macdonald, Doune; Rodger, Sylvia; Ziviani, Jenny; Jenkins, David; Batch, Jenny; Jones, Judy

    2004-01-01

    While questions of children's engagement in physical activity are being widely debated, little is known about how physical activity is valued and managed within families. This paper reports on qualitative data from a multi-method study on lower primary aged children. The focus of the broader study was to determine the relationships between young…

  6. Independent and Small Group Activities for Social Studies in the Primary Grades.

    ERIC Educational Resources Information Center

    Ball, Barbara; And Others

    A teachers' guide for social studies, this manual stresses geography curriculum and activities for the primary grades. It is suggested that a teacher work with one group while the other children work individually. Children first work independently for a team, and then progress to less structured small group activities. Positive reinforcement by…

  7. Experimental Activities in Primary School to Learn about Microbes in an Oral Health Education Context

    ERIC Educational Resources Information Center

    Mafra, Paulo; Lima, Nelson; Carvalho, Graça S.

    2015-01-01

    Experimental science activities in primary school enable important cross-curricular learning. In this study, experimental activities on microbiology were carried out by 16 pupils in a Portuguese grade-4 classroom (9-10?years old) and were focused on two problem-questions related to microbiology and health: (1) do your teeth carry microbes? (2) why…

  8. Teaching Primary Science: Emotions, Identity and the Use of Practical Activities

    ERIC Educational Resources Information Center

    Cripps Clark, John; Groves, Susie

    2012-01-01

    This paper uses cultural historical activity theory to examine the interactions between the choices primary teachers make in the use of practical activities in their teaching of science and the purposes they attribute to these; their emotions, background and beliefs; and the construction of their identities as teachers of science. It draws on four…

  9. The Art and Science Connection. Hands-On Activities for Primary Students.

    ERIC Educational Resources Information Center

    Tolley, Kimberley

    Most people think that the artist and the scientist live in two totally different worlds. However, art and science are only two different ways of understanding and knowing the world. To help primary students make a connection between art and science, a collection of hands-on activities have been developed. By engaging in these activities that…

  10. Elementary Environmental Learning Packet K-3, Third Revised Edition. [Primary CEL Blocks, Student Activity Cards].

    ERIC Educational Resources Information Center

    Brevard County School Board, Cocoa, FL.

    This environmental education program consists of two levels: primary and intermediate. The learning materials are activity based and incorporate process and subject area skills with knowledge and concern for the environment. The program is also interdisciplinary including activities and skills from art, language arts, mathematics, music, science,…

  11. Music Activities in Primary School: Students' Preferences in the Spanish Region of Murcia

    ERIC Educational Resources Information Center

    Vicente-Nicolás, Gregorio; Mac Ruairc, Gerry

    2014-01-01

    The aim of this study was to determine the preferences of primary school children in relation to the types of activities that typically take place in music classrooms. For the purposes of this study, these classroom-based music activities have been categorised into five areas: singing, playing instruments, listening, reading and writing music and…

  12. Waterworks Book. An Activity Book about Mississippi's Coastal Resources for Primary Grades.

    ERIC Educational Resources Information Center

    Howe, Kevin M.

    Coastal resources are highlighted in this activity book for primary school children. Special focus is given to Mississippi's coastal areas, but applications to other geographic areas can be made. Wetland concepts and conditions are developed through a variety of games, puzzles, matching exercises and pictorial explanations. Activities addressing…

  13. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    PubMed

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  14. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase

    SciTech Connect

    Funk, C.D.; Furci, L.; FitzGerald, G.A. )

    1990-08-01

    The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE), is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, the authors have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a M{sub r} 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-12lx) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.

  15. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses.

    PubMed

    Flipse, Jacky; Diosa-Toro, Mayra A; Hoornweg, Tabitha E; van de Pol, Denise P I; Urcuqui-Inchima, Silvio; Smit, Jolanda M

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  16. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

    PubMed Central

    Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-Inchima, Silvio; Smit, Jolanda M.

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts’ antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  17. Reversal of opiate-induced apoptosis by human recombinant growth hormone in murine foetus primary hippocampal neuronal cell cultures.

    PubMed

    Svensson, Anne-Lie; Bucht, Nora; Hallberg, Mathias; Nyberg, Fred

    2008-05-20

    Previous studies have shown that chronic opiates may inhibit cell growth and trigger apoptosis leading to impaired cognitive capabilities in both humans and other mammals. In contrast, growth hormone (GH) has been demonstrated to stimulate cell growth and counteract apoptosis. GH has also been shown to improve learning and memory in both human and rodents. In this work, we demonstrate that GH may reverse opiate-induced apoptosis in cells derived from prenatal mouse hippocampus. Primary hippocampal cell cultures derived from 16-day-old fetal mouse neurons were treated with morphine for 7 days during growth in the absence or presence of recombinant human GH (rhGH). The release of lactate dehydrogenase (LDH) into the culture media and the level of cleaved caspase-3 were measured. Results indicate that morphine (15 microM) decreased the cell content in a concentration-dependent manner and increased LDH release and caspase-3 activity. Thus, fetal mouse neurons treated with morphine showed less viability compared with controls. Interestingly, the addition of rhGH (1 microM) counteracted the morphine-induced effect on the cell density. Furthermore, the hormone attenuated the effects on LHD release and caspase-3 activity elicited by morphine. These results suggest that the hormone is capable of preventing or even repairing morphine-induced damage to hippocampal cells.

  18. Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt.

    PubMed

    Schulze-Bergkamen, Henning; Brenner, Dirk; Krueger, Andreas; Suess, Dorothee; Fas, Stefanie C; Frey, Christian R; Dax, Andreas; Zink, Dorothea; Büchler, Peter; Müller, Martina; Krammer, Peter H

    2004-03-01

    CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x(L). HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine(727)--but not tyrosine(705)--phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases. PMID:14999683

  19. Anticancer activity of glucomoringin isothiocyanate in human malignant astrocytoma cells.

    PubMed

    Rajan, Thangavelu Soundara; De Nicola, Gina Rosalinda; Iori, Renato; Rollin, Patrick; Bramanti, Placido; Mazzon, Emanuela

    2016-04-01

    Isothiocyanates (ITCs) released from their glucosinolate precursors have been shown to inhibit tumorigenesis and they have received significant attention as potential chemotherapeutic agents against cancer. Astrocytoma grade IV is the most frequent and most malignant primary brain tumor in adults without any curative treatment. New therapeutic drugs are therefore urgently required. In the present study, we investigated the in vitro antitumor activity of the glycosylated isothiocyanate moringin [4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate] produced from quantitative myrosinase-induced hydrolysis of glucomoringin (GMG) under neutral pH value. We have evaluated the potency of moringin on apoptosis induction and cell death in human astrocytoma grade IV CCF-STTG1 cells. Moringin showed to be effective in inducing apoptosis through p53 and Bax activation and Bcl-2 inhibition. In addition, oxidative stress related Nrf2 transcription factor and its upstream regulator CK2 alpha expressions were modulated at higher doses, which indicated the involvement of oxidative stress-mediated apoptosis induced by moringin. Moreover, significant reduction in 5S rRNA was noticed with moringin treatment. Our in vitro results demonstrated the antitumor efficacy of moringin derived from myrosinase-hydrolysis of GMG in human malignant astrocytoma cells. PMID:26882972

  20. Identification of CD146 as a marker enriched for tumor-propagating capacity reveals targetable pathways in primary human sarcoma

    PubMed Central

    Voisin, Veronique; Sato, Shingo; Hirata, Makoto; Whetstone, Heather; Han, Ilkyu; Ailles, Laurie; Bader, Gary D.; Wunder, Jay; Alman, Benjamin A.

    2015-01-01

    Tumor-propagating cells (TPCs) are believed to drive cancer initiation, progression and recurrence. These cells are characterized by enhanced tumorigenicity and self-renewal. The ability to identify such cells in primary human sarcomas relies on the dye exclusion ability of tumor side population (SP) cells. Here, we performed a high-throughput cell surface antigen screen and found that CD146 is enriched in the SP population. In vivo serial transplantation assays showed that CD146+ cells are highly tumorigenic, capable of self-renewal and thus enriches for the TPC population. In addition, depletion of SP cells from the CD146+ population show that CD146+ cells and SP cells are a distinct and overlapping TPC populations. Gene expression profiling of CD146+ and SP cells revealed multiple pathways commonly upregulated in both of these populations. Inhibition of one of these upregulated pathways, Notch signaling, significantly reduced tumor growth and self-renewal. Our data demonstrate that CD146 is an effective cell surface marker for enriching TPCs in primary human sarcomas. Targeting differentially activated pathways in TPCs may provide new therapeutic strategies for treating sarcoma. PMID:26517673

  1. Next-generation sequencing reveals low-dose effects of cationic dendrimers in primary human bronchial epithelial cells.

    PubMed

    Feliu, Neus; Kohonen, Pekka; Ji, Jie; Zhang, Yuning; Karlsson, Hanna L; Palmberg, Lena; Nyström, Andreas; Fadeel, Bengt

    2015-01-27

    Gene expression profiling has developed rapidly in recent years with the advent of deep sequencing technologies such as RNA sequencing (RNA Seq) and could be harnessed to predict and define mechanisms of toxicity of chemicals and nanomaterials. However, the full potential of these technologies in (nano)toxicology is yet to be realized. Here, we show that systems biology approaches can uncover mechanisms underlying cellular responses to nanomaterials. Using RNA Seq and computational approaches, we found that cationic poly(amidoamine) dendrimers (PAMAM-NH2) are capable of triggering down-regulation of cell-cycle-related genes in primary human bronchial epithelial cells at doses that do not elicit acute cytotoxicity, as demonstrated using conventional cell viability assays, while gene transcription was not affected by neutral PAMAM-OH dendrimers. The PAMAMs were internalized in an active manner by lung cells and localized mainly in lysosomes; amine-terminated dendrimers were internalized more efficiently when compared to the hydroxyl-terminated dendrimers. Upstream regulator analysis implicated NF-κB as a putative transcriptional regulator, and subsequent cell-based assays confirmed that PAMAM-NH2 caused NF-κB-dependent cell cycle arrest. However, PAMAM-NH2 did not affect cell cycle progression in the human A549 adenocarcinoma cell line. These results demonstrate the feasibility of applying systems biology approaches to predict cellular responses to nanomaterials and highlight the importance of using relevant (primary) cell models.

  2. Cortical entrainment of human hypoglossal motor unit activities

    PubMed Central

    Laine, Christopher M.; Nickerson, Laura A.

    2012-01-01

    Output from the primary motor cortex contains oscillations that can have frequency-specific effects on the firing of motoneurons (MNs). Whereas much is known about the effects of oscillatory cortical drive on the output of spinal MN pools, considerably less is known about the effects on cranial motor nuclei, which govern speech/oromotor control. Here, we investigated cortical input to one such motor pool, the hypoglossal motor nucleus (HMN), which controls muscles of the tongue. We recorded intramuscular genioglossus electromyogram (EMG) and scalp EEG from healthy adult subjects performing a tongue protrusion task. Cortical entrainment of HMN population activity was assessed by measuring coherence between EEG and multiunit EMG activity. In addition, cortical entrainment of individual MN firing activity was assessed by measuring phase locking between single motor unit (SMU) action potentials and EEG oscillations. We found that cortical entrainment of multiunit activity was detectable within the 15- to 40-Hz frequency range but was inconsistent across recordings. By comparison, cortical entrainment of SMU spike timing was reliable within the same frequency range. Furthermore, this effect was found to be intermittent over time. Our study represents an important step in understanding corticomuscular synchronization in the context of human oromotor control and is the first study to document SMU entrainment by cortical oscillations in vivo. PMID:22049332

  3. Structural snapshots of actively translating human ribosomes.

    PubMed

    Behrmann, Elmar; Loerke, Justus; Budkevich, Tatyana V; Yamamoto, Kaori; Schmidt, Andrea; Penczek, Pawel A; Vos, Matthijn R; Bürger, Jörg; Mielke, Thorsten; Scheerer, Patrick; Spahn, Christian M T

    2015-05-01

    Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

  4. Computational study of human head response to primary blast waves of five levels from three directions.

    PubMed

    Wang, Chenzhi; Pahk, Jae Bum; Balaban, Carey D; Miller, Mark C; Wood, Adam R; Vipperman, Jeffrey S

    2014-01-01

    Human exposure to blast waves without any fragment impacts can still result in primary blast-induced traumatic brain injury (bTBI). To investigate the mechanical response of human brain to primary blast waves and to identify the injury mechanisms of bTBI, a three-dimensional finite element head model consisting of the scalp, skull, cerebrospinal fluid, nasal cavity, and brain was developed from the imaging data set of a human female. The finite element head model was partially validated and was subjected to the blast waves of five blast intensities from the anterior, right lateral, and posterior directions at a stand-off distance of one meter from the detonation center. Simulation results show that the blast wave directly transmits into the head and causes a pressure wave propagating through the brain tissue. Intracranial pressure (ICP) is predicted to have the highest magnitude from a posterior blast wave in comparison with a blast wave from any of the other two directions with same blast intensity. The brain model predicts higher positive pressure at the site proximal to blast wave than that at the distal site. The intracranial pressure wave invariably travels into the posterior fossa and vertebral column, causing high pressures in these regions. The severities of cerebral contusions at different cerebral locations are estimated using an ICP based injury criterion. Von Mises stress prevails in the cortex with a much higher magnitude than in the internal parenchyma. According to an axonal injury criterion based on von Mises stress, axonal injury is not predicted to be a cause of primary brain injury from blasts. PMID:25409326

  5. A transcriptional sketch of a primary human breast cancer by 454 deep sequencing

    PubMed Central

    Guffanti, Alessandro; Iacono, Michele; Pelucchi, Paride; Kim, Namshin; Soldà, Giulia; Croft, Larry J; Taft, Ryan J; Rizzi, Ermanno; Askarian-Amiri, Marjan; Bonnal, Raoul J; Callari, Maurizio; Mignone, Flavio; Pesole, Graziano; Bertalot, Giovanni; Bernardi, Luigi Rossi; Albertini, Alberto; Lee, Christopher; Mattick, John S; Zucchi, Ileana; De Bellis, Gianluca

    2009-01-01

    Background The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown

  6. Computational study of human head response to primary blast waves of five levels from three directions.

    PubMed

    Wang, Chenzhi; Pahk, Jae Bum; Balaban, Carey D; Miller, Mark C; Wood, Adam R; Vipperman, Jeffrey S

    2014-01-01

    Human exposure to blast waves without any fragment impacts can still result in primary blast-induced traumatic brain injury (bTBI). To investigate the mechanical response of human brain to primary blast waves and to identify the injury mechanisms of bTBI, a three-dimensional finite element head model consisting of the scalp, skull, cerebrospinal fluid, nasal cavity, and brain was developed from the imaging data set of a human female. The finite element head model was partially validated and was subjected to the blast waves of five blast intensities from the anterior, right lateral, and posterior directions at a stand-off distance of one meter from the detonation center. Simulation results show that the blast wave directly transmits into the head and causes a pressure wave propagating through the brain tissue. Intracranial pressure (ICP) is predicted to have the highest magnitude from a posterior blast wave in comparison with a blast wave from any of the other two directions with same blast intensity. The brain model predicts higher positive pressure at the site proximal to blast wave than that at the distal site. The intracranial pressure wave invariably travels into the posterior fossa and vertebral column, causing high pressures in these regions. The severities of cerebral contusions at different cerebral locations are estimated using an ICP based injury criterion. Von Mises stress prevails in the cortex with a much higher magnitude than in the internal parenchyma. According to an axonal injury criterion based on von Mises stress, axonal injury is not predicted to be a cause of primary brain injury from blasts.

  7. Computational Study of Human Head Response to Primary Blast Waves of Five Levels from Three Directions

    PubMed Central

    Wang, Chenzhi; Pahk, Jae Bum; Balaban, Carey D.; Miller, Mark C.; Wood, Adam R.; Vipperman, Jeffrey S.

    2014-01-01

    Human exposure to blast waves without any fragment impacts can still result in primary blast-induced traumatic brain injury (bTBI). To investigate the mechanical response of human brain to primary blast waves and to identify the injury mechanisms of bTBI, a three-dimensional finite element head model consisting of the scalp, skull, cerebrospinal fluid, nasal cavity, and brain was developed from the imaging data set of a human female. The finite element head model was partially validated and was subjected to the blast waves of five blast intensities from the anterior, right lateral, and posterior directions at a stand-off distance of one meter from the detonation center. Simulation results show that the blast wave directly transmits into the head and causes a pressure wave propagating through the brain tissue. Intracranial pressure (ICP) is predicted to have the highest magnitude from a posterior blast wave in comparison with a blast wave from any of the other two directions with same blast intensity. The brain model predicts higher positive pressure at the site proximal to blast wave than that at the distal site. The intracranial pressure wave invariably travels into the posterior fossa and vertebral column, causing high pressures in these regions. The severities of cerebral contusions at different cerebral locations are estimated using an ICP based injury criterion. Von Mises stress prevails in the cortex with a much higher magnitude than in the internal parenchyma. According to an axonal injury criterion based on von Mises stress, axonal injury is not predicted to be a cause of primary brain injury from blasts. PMID:25409326

  8. Performance comparison between two axial active support schemes for 1-m thin meniscus primary mirror

    NASA Astrophysics Data System (ADS)

    Niu, D. S.; Wang, G. M.; Gu, B. Z.; Ye, Y.

    2013-03-01

    Active support scheme may decide the deformation of the optical surface figure of the primary mirror. Two main active axial support schemes are often adopted to the thin meniscus primary mirror, one scheme is that the axial supports normal to the mirror bottom surface, and the other is that the active forces parallel to the optical axis. In order to compare the performance of the two support schemes, 1-m thin meniscus primary mirror is conducted. Finite element analysis (FEA) is employed to analyze the optical surface figures of the primary mirror, and optimizations are carried out by using ANSYS for each support scheme to obtain the locations and active forces. The axial support force sensitivities are calculated for the two support schemes in a case that a single axial support has a force error of 0.5 N. The correction ability of the active support system for both of the support schemes are analyzed when an arbitrary axial support is failure. Several low order Zernike modes are modeled with MATLAB procedure, and active optics corrections are applied to these modes for the two active supports. The extra mirror surface error due to thermal deformation is also corrected with the two support schemes.

  9. Use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance.

    PubMed

    Huh, Warner K; Ault, Kevin A; Chelmow, David; Davey, Diane D; Goulart, Robert A; Garcia, Francisco A R; Kinney, Walter K; Massad, L Stewart; Mayeaux, Edward J; Saslow, Debbie; Schiffman, Mark; Wentzensen, Nicolas; Lawson, Herschel W; Einstein, Mark H

    2015-02-01

    In 2011, the American Cancer Society, the American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology updated screening guidelines for the early detection of cervical cancer and its precursors. Recommended screening strategies were cytology or cotesting (cytology in combination with high-risk human papillomavirus [hrHPV] testing). These guidelines also addressed the use of hrHPV testing alone as a primary screening approach, which was not recommended for use at that time. There is now a growing body of evidence for screening with primary hrHPV testing, including a prospective U.S.-based registration study. Thirteen experts, including representatives from the Society of Gynecologic Oncology, the American Society for Colposcopy and Cervical Pathology, the American College of Obstetricians and Gynecologists, the American Cancer Society, the American Society of Cytopathology, the College of American Pathologists, and the American Society for Clinical Pathology, convened to provide interim guidance for primary hrHPV screening. This guidance panel was specifically triggered by an application to the U.S. Food and Drug Administration (FDA) for a currently marketed HPV test to be labeled for the additional indication of primary cervical cancer screening. Guidance was based on literature review and review of data from the FDA registration study, supplemented by expert opinion. This document aims to provide information for health care providers who are interested in primary hrHPV testing and an overview of the potential advantages and disadvantages of this strategy for screening as well as to highlight areas in need of further investigation.

  10. Stress Conditions Increase Vimentin Cleavage by Omi/HtrA2 Protease in Human Primary Neurons and Differentiated Neuroblastoma Cells.

    PubMed

    Lucotte, Bérangère; Tajhizi, Mehdi; Alkhatib, Dareen; Samuelsson, Eva-Britt; Wiehager, Birgitta; Schedin-Weiss, Sophia; Sundström, Erik; Winblad, Bengt; Tjernberg, Lars O; Behbahani, Homira

    2015-12-01

    Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

  11. Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

    PubMed

    Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

    2014-01-01

    Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

  12. Shell Extracts from the Marine Bivalve Pecten maximus Regulate the Synthesis of Extracellular Matrix in Primary Cultured Human Skin Fibroblasts

    PubMed Central

    Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

    2014-01-01

    Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

  13. Persistent pain after spinal cord injury is maintained by primary afferent activity.

    PubMed

    Yang, Qing; Wu, Zizhen; Hadden, Julia K; Odem, Max A; Zuo, Yan; Crook, Robyn J; Frost, Jeffrey A; Walters, Edgar T

    2014-08-01

    Chronic pain caused by insults to the CNS (central neuropathic pain) is widely assumed to be maintained exclusively by central mechanisms. However, chronic hyperexcitablility occurs in primary nociceptors after spinal cord injury (SCI), suggesting that SCI pain also depends upon continuing activity of peripheral sensory neurons. The present study in rats (Rattus norvegicus) found persistent upregulation after SCI of protein, but not mRNA, for a voltage-gated Na(+) channel, Nav1.8, that is expressed almost exclusively in primary afferent neurons. Selectively knocking down Nav1.8 after SCI suppressed spontaneous activity in dissociated dorsal root ganglion neurons, reversed hypersensitivity of hindlimb withdrawal reflexes, and reduced ongoing pain assessed by a conditioned place preference test. These results show that activity in primary afferent neurons contributes to ongoing SCI pain. PMID:25100607

  14. Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy

    PubMed Central

    Rizzardi, G. Paolo; Harari, Alexandre; Capiluppi, Brunella; Tambussi, Giuseppe; Ellefsen, Kim; Ciuffreda, Donatella; Champagne, Patrick; Bart, Pierre-Alexandre; Chave, Jean-Philippe; Lazzarin, Adriano; Pantaleo, Giuseppe

    2002-01-01

    Primary HIV-1 infection causes extensive immune activation, during which CD4+ T cell activation supports massive HIV-1 production. We tested the safety and the immune-modulating effects of combining cyclosporin A (CsA) treatment with highly active antiretroviral therapy (HAART) during primary HIV-1 infection. Nine adults with primary HIV-1 infection were treated with CsA along with HAART. At week 8, all patients discontinued CsA but maintained HAART. Viral replication was suppressed to a comparable extent in the CsA + HAART cohort and in 29 control patients whose primary infection was treated with HAART alone. CsA restored normal CD4+ T cell levels, both in terms of percentage and absolute numbers. The increase in CD4+ T cells was apparent within a week and persisted throughout the study period. CsA was not detrimental to virus-specific CD8+ or CD4+ T cell responses. At week 48, the proportion of IFN-γ–secreting CD4+ and CD4+CCR7– T cells was significantly higher in the CsA + HAART cohort than in the HAART-alone cohort. In conclusion, rapid shutdown of T cell activation in the early phases of primary HIV-1 infection can have long-term beneficial effects and establish a more favorable immunologic set-point. Appropriate, immune-based therapeutic interventions may represent a valuable complement to HAART for treating HIV infection. PMID:11877476

  15. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    PubMed

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  16. Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells

    PubMed Central

    2013-01-01

    Background Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts. Methods Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing. Results Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed. Conclusions At least 3 different adventitious viruses were present in the batch of contaminated RPTEC. Whereas we are unable to determine whether the original RPTEC were pre-infected prior to their separation from other kidney cells, or had gotten contaminated with HCoV-NL63 from an ill laboratory worker during their preparation for commercial sale, our findings are a reminder that human-derived biologicals should always be considered as potential sources of infectious agents. Importantly, HCoV-NL63 replicates to high titers in some primary human kidney cells. PMID:23805916

  17. Persistence and Fitness of Multidrug-Resistant Human Immunodeficiency Virus Type 1 Acquired in Primary Infection

    PubMed Central

    Brenner, Bluma G.; Routy, Jean-Pierre; Petrella, Marco; Moisi, Daniela; Oliveira, Maureen; Detorio, Mervi; Spira, Bonnie; Essabag, Vidal; Conway, Brian; Lalonde, Richard; Sekaly, Rafick-Pierre; Wainberg, Mark A.

    2002-01-01

    This study examines the persistence and fitness of multidrug-resistant (MDR) viruses acquired during primary human immunodeficiency virus infection (PHI). In four individuals, MDR infections persisted over the entire study period, ranging from 36 weeks to 5 years, in the absence of antiretroviral therapy. In stark contrast, identified source partners in two cases showed expected outgrowth of wild-type (WT) virus within 12 weeks of treatment interruption. In the first PHI case, triple-class MDR resulted in low plasma viremia (1.6 to 3 log copies/ml) over time compared with mean values obtained for an untreated PHI group harboring WT infections (4.1 to 4.3 log copies/ml). Increasing viremia in PHI patient 1 at week 52 was associated with the de novo emergence of a protease inhibitor-resistant variant through a recombination event involving the original MDR virus. MDR infections in two other untreated PHI patients yielded viremia levels typical of the untreated WT group. A fourth patient's MDR infection yielded low viremia (<50 to 500 copies/ml) for 5 years despite his having phenotypic resistance to all antiretroviral drugs in his treatment regimen. In two of these PHI cases, a rebound to higher levels of plasma viremia only occurred when the M184V mutation in reverse transcriptase could no longer be detected and, in a third case, nondetection of M184V was associated with an inability to isolate virus. To further evaluate the fitness of MDR variants acquired in PHI, MDR and corresponding WT viruses were isolated from index and source partners, respectively. Although MDR viral infectivity (50% tissue culture infective dose) was comparable to that observed for WT viruses, MDR infections in each case demonstrated 2-fold and 13- to 23-fold reductions in p24 antigen and reverse transcriptase enzymatic activity, respectively. In dual-infection competition assays, MDR viruses consistently demonstrated a marked replicative disadvantage compared with WT virus. These results

  18. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4.

    PubMed

    Lager, Susanne; Gaccioli, Francesca; Ramirez, Vanessa I; Jones, Helen N; Jansson, Thomas; Powell, Theresa L

    2013-03-01

    Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸB, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.

  19. Microstructured zirconia surfaces modulate osteogenic marker genes in human primary osteoblasts.

    PubMed

    Bergemann, Claudia; Duske, Kathrin; Nebe, J Barbara; Schöne, André; Bulnheim, Ulrike; Seitz, Hermann; Fischer, Jens

    2015-01-01

    In dentistry, zirconia has been used since the early 1990s for endodontic posts, more recently for implant abutments and frameworks for fixed dental prostheses. Zirconia is biocompatible and mechanically strong enough to serve as implant material for oral implants. Although several zirconia implant systems are available, currently the scientific and clinical data for zirconia implants are not sufficient to recommend them for routine clinical use. Here the influence of microstructured yttria-stabilized zirconia (YZ) on human primary osteoblast (HOB) behavior was determined. YZ surfaces were treated by sandblasting (YZ-S), acid etching (YZ-SE) and additionally heat treatment (YZ-SEH). Morphological changes of HOB were determined by scanning electron microscopy. Actin cytoskeleton was investigated by laser scanning microscopy and analyzed by novel actin quantification software. Differentiation of HOB was determined by real time RT-PCR. Improved mechanical interlocking of primary HOB into the porous microstructure of the acid etched and additionally heat treated YZ-surfaces correlates with drastically increased osteocalcin (OCN) gene expression. In particular, OCN was considerably elevated in primary HOB after 3 days on YZ-SE (13-fold) as well as YZ-SEH (12-fold) surfaces. Shorter actin filaments without any favored orientation on YZ-SE and YZ-SEH surfaces are associated with higher roughness (Ra) values. Topographically modified yttria-stabilized zirconia is a likely material for dental implants with cell stimulating properties achieving or actually exceeding those of titanium. PMID:25578704

  20. Hemispheric asymmetry in cerebrovascular reactivity of the human primary motor cortex: an in vivo study at 7 T.

    PubMed

    Driver, Ian D; Andoh, Jamila; Blockley, Nicholas P; Francis, Susan T; Gowland, Penny A; Paus, Tomáš

    2015-05-01

    Current functional MRI (fMRI) approaches assess underlying neuronal activity through monitoring the related local variations in cerebral blood oxygenation, blood volume and blood flow. This vascular response is likely to vary across brain regions and across individuals, depending on the composition of the local vascular bed and on the vascular capacity to dilate. The most widely used technique uses the blood oxygen level dependent (BOLD) fMRI signal, which arises from a complex combination of all of these factors. The model of handedness provides a case where one brain region (dominant motor cortex) is known to have a stronger BOLD response over another (non-dominant motor cortex) during hand motor task performance. We predict that this is accompanied by a higher vascular reactivity in the dominant motor cortex, when compared with the non-dominant motor cortex. Precise measurement of end-tidal CO2 and a novel sinusoidal CO2 respiratory challenge were combined with the high sensitivity and finer spatial resolution available for fMRI at 7 T to measure BOLD cerebrovascular reactivity (CVR) in eight healthy male participants. BOLD CVR was compared between the left (dominant) and right (non-dominant) primary motor cortices of right-handed adults. Hemispheric asymmetry in vascular reactivity was predicted and observed in the primary motor cortex (left CVR = 0.60 ± 0.15%/mm Hg; right CVR = 0.47 ± 0.08%/mm Hg; left CVR > right CVR, P = 0.04), the first reported evidence of such a vascular difference. These findings demonstrate a cerebral vascular asymmetry between the left and right primary motor cortex. The origin of this asymmetry largely arises from the contribution of large draining veins. This work has implications for future motor laterality studies that use BOLD, and it is also suggestive of a vascular plasticity in the human primary motor cortex. PMID:25788020

  1. Temporal overlaps of feral cats with prey and competitors in primary and human-altered habitats on Bohol Island, Philippines.

    PubMed

    Bogdan, Vlastimil; Jůnek, Tomáš; Jůnková Vymyslická, Pavla

    2016-01-01

    The vertebrate fauna of the Philippines, known for its diversity and high proportion of endemic species, comprises mainly small- to medium-sized forms with a few large exceptions. As with other tropical ecosystems, the major threats to wildlife are habitat loss, hunting and invasive species, of which the feral cat (Felis catus) is considered the most damaging. Our camera-trapping study focused on a terrestrial vertebrate species inventory on Bohol Island and tempo-spatial co-occurrences of feral cats with their prey and competitors. The survey took place in the Rajah Sikatuna Protected Landscape, and we examined the primary rainforest, its border with agricultural land, and rural areas in the vicinity of villages. Altogether, over 2,885 trap days we captured 30 species of vertebrates-10 mammals (including Sus philippensis), 19 birds and one reptile, Varanus cumingi. We trapped 81.8% of expected vertebrates. Based on the number of events, the most frequent native species was the barred rail (Gallirallus torquatus). The highest overlap in diel activity between cats and potential prey was recorded with rodents in rural areas (Δ = 0.62); the lowest was in the same habitat with ground-dwelling birds (Δ = 0.40). Cat activity was not recorded inside the rainforest; in other habitats their diel activity pattern differed. The cats' activity declined in daylight in the proximity of humans, while it peaked at the transition zone between rainforest and fields. Both rodents and ground-dwelling birds exhibited a shift in activity levels between sites where cats were present or absent. Rodents tend to become active by day in cat-free habitats. No cats' temporal response to co-occurrences of civets (Paradoxurus hermaphroditus and Viverra tangalunga) was found but cats in diel activity avoided domestic dogs (Canis lupus familiaris). Our first insight into the ecology of this invasive predator in the Philippines revealed an avoidance of homogeneous primary rainforest and a

  2. Temporal overlaps of feral cats with prey and competitors in primary and human-altered habitats on Bohol Island, Philippines

    PubMed Central

    Bogdan, Vlastimil; Jůnková Vymyslická, Pavla

    2016-01-01

    The vertebrate fauna of the Philippines, known for its diversity and high proportion of endemic species, comprises mainly small- to medium-sized forms with a few large exceptions. As with other tropical ecosystems, the major threats to wildlife are habitat loss, hunting and invasive species, of which the feral cat (Felis catus) is considered the most damaging. Our camera-trapping study focused on a terrestrial vertebrate species inventory on Bohol Island and tempo-spatial co-occurrences of feral cats with their prey and competitors. The survey took place in the Rajah Sikatuna Protected Landscape, and we examined the primary rainforest, its border with agricultural land, and rural areas in the vicinity of villages. Altogether, over 2,885 trap days we captured 30 species of vertebrates–10 mammals (including Sus philippensis), 19 birds and one reptile, Varanus cumingi. We trapped 81.8% of expected vertebrates. Based on the number of events, the most frequent native species was the barred rail (Gallirallus torquatus). The highest overlap in diel activity between cats and potential prey was recorded with rodents in rural areas (Δ = 0.62); the lowest was in the same habitat with ground-dwelling birds (Δ = 0.40). Cat activity was not recorded inside the rainforest; in other habitats their diel activity pattern differed. The cats’ activity declined in daylight in the proximity of humans, while it peaked at the transition zone between rainforest and fields. Both rodents and ground-dwelling birds exhibited a shift in activity levels between sites where cats were present or absent. Rodents tend to become active by day in cat-free habitats. No cats’ temporal response to co-occurrences of civets (Paradoxurus hermaphroditus and Viverra tangalunga) was found but cats in diel activity avoided domestic dogs (Canis lupus familiaris). Our first insight into the ecology of this invasive predator in the Philippines revealed an avoidance of homogeneous primary rainforest and a

  3. Temporal overlaps of feral cats with prey and competitors in primary and human-altered habitats on Bohol Island, Philippines

    PubMed Central

    Bogdan, Vlastimil; Jůnková Vymyslická, Pavla

    2016-01-01

    The vertebrate fauna of the Philippines, known for its diversity and high proportion of endemic species, comprises mainly small- to medium-sized forms with a few large exceptions. As with other tropical ecosystems, the major threats to wildlife are habitat loss, hunting and invasive species, of which the feral cat (Felis catus) is considered the most damaging. Our camera-trapping study focused on a terrestrial vertebrate species inventory on Bohol Island and tempo-spatial co-occurrences of feral cats with their prey and competitors. The survey took place in the Rajah Sikatuna Protected Landscape, and we examined the primary rainforest, its border with agricultural land, and rural areas in the vicinity of villages. Altogether, over 2,885 trap days we captured 30 species of vertebrates–10 mammals (including Sus philippensis), 19 birds and one reptile, Varanus cumingi. We trapped 81.8% of expected vertebrates. Based on the number of events, the most frequent native species was the barred rail (Gallirallus torquatus). The highest overlap in diel activity between cats and potential prey was recorded with rodents in rural areas (Δ = 0.62); the lowest was in the same habitat with ground-dwelling birds (Δ = 0.40). Cat activity was not recorded inside the rainforest; in other habitats their diel activity pattern differed. The cats’ activity declined in daylight in the proximity of humans, while it peaked at the transition zone between rainforest and fields. Both rodents and ground-dwelling birds exhibited a shift in activity levels between sites where cats were present or absent. Rodents tend to become active by day in cat-free habitats. No cats’ temporal response to co-occurrences of civets (Paradoxurus hermaphroditus and Viverra tangalunga) was found but cats in diel activity avoided domestic dogs (Canis lupus familiaris). Our first insight into the ecology of this invasive predator in the Philippines revealed an avoidance of homogeneous primary rainforest and a

  4. Temporal overlaps of feral cats with prey and competitors in primary and human-altered habitats on Bohol Island, Philippines.

    PubMed

    Bogdan, Vlastimil; Jůnek, Tomáš; Jůnková Vymyslická, Pavla

    2016-01-01

    The vertebrate fauna of the Philippines, known for its diversity and high proportion of endemic species, comprises mainly small- to medium-sized forms with a few large exceptions. As with other tropical ecosystems, the major threats to wildlife are habitat loss, hunting and invasive species, of which the feral cat (Felis catus) is considered the most damaging. Our camera-trapping study focused on a terrestrial vertebrate species inventory on Bohol Island and tempo-spatial co-occurrences of feral cats with their prey and competitors. The survey took place in the Rajah Sikatuna Protected Landscape, and we examined the primary rainforest, its border with agricultural land, and rural areas in the vicinity of villages. Altogether, over 2,885 trap days we captured 30 species of vertebrates-10 mammals (including Sus philippensis), 19 birds and one reptile, Varanus cumingi. We trapped 81.8% of expected vertebrates. Based on the number of events, the most frequent native species was the barred rail (Gallirallus torquatus). The highest overlap in diel activity between cats and potential prey was recorded with rodents in rural areas (Δ = 0.62); the lowest was in the same habitat with ground-dwelling birds (Δ = 0.40). Cat activity was not recorded inside the rainforest; in other habitats their diel activity pattern differed. The cats' activity declined in daylight in the proximity of humans, while it peaked at the transition zone between rainforest and fields. Both rodents and ground-dwelling birds exhibited a shift in activity levels between sites where cats were present or absent. Rodents tend to become active by day in cat-free habitats. No cats' temporal response to co-occurrences of civets (Paradoxurus hermaphroditus and Viverra tangalunga) was found but cats in diel activity avoided domestic dogs (Canis lupus familiaris). Our first insight into the ecology of this invasive predator in the Philippines revealed an avoidance of homogeneous primary rainforest and a

  5. Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: Clues from human phylogeography

    PubMed Central

    Mokrousov, Igor; Ly, Ho Minh; Otten, Tatiana; Lan, Nguyen Ngoc; Vyshnevskyi, Boris; Hoffner, Sven; Narvskaya, Olga

    2005-01-01

    We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors. PMID:16169923

  6. Microtubule plus end–associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells

    PubMed Central

    Jovasevic, Vladimir

    2015-01-01

    Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end–tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170–mediated capture to initiate transport in primary human cells. PMID:26504169

  7. In vivo functional and myeloarchitectonic mapping of human primary auditory areas

    PubMed Central

    Dick, Frederic; Tierney, Adam Taylor; Lutti, Antoine; Josephs, Oliver; Sereno, Martin I.; Weiskopf, Nikolaus

    2012-01-01

    In contrast to vision, where retinotopic mapping alone can define areal borders, primary auditory areas such as A1 are best delineated by combining in vivo tonotopic mapping with post mortem cyto- or myelo-architectonics from the same individual. We combined high-resolution (800 μm) quantitative T1 mapping with phase-encoded tonotopic methods to map primary auditory areas (A1 and R) within the ‘auditory core’ of human volunteers. We first quantitatively characterize the highly myelinated auditory core in terms of shape, area, cortical depth profile, and position, with our data showing considerable correspondence to post-mortem myeloarchitectonic studies, both in cross-participant averages and in individuals. The core region contains two ‘mirror-image‘ tonotopic maps oriented along the same axis as observed in macaque and owl monkey. We suggest that thee two maps within the core are the human analogues of primate auditory areas A1 and R. The core occupies a much smaller portion of tonotopically organized cortex on the superior temporal plane and gyrus than is generally supposed. The multi-modal approach to defining the auditory core will facilitate investigations of structure-function relationships, comparative neuroanatomical studies, and promises new biomarkers for diagnosis and clinical studies. PMID:23152594

  8. On the relation between surface roughness of metallic substrates and adhesion of human primary bone cells.

    PubMed

    Anselme, K; Bigerelle, M

    2014-01-01

    Surface characteristics of materials, whether their topography, chemistry, or surface energy, play an essential part in osteoblast adhesion on biomaterials. Thus, the quality of cell adhesion will influence the cell's capacity to proliferate and differentiate in contact with a biomaterial. We have developed for more than ten years numerous studies on the influence of topography and chemistry of metallic substrates on the response of primary human bone cells. The originality of our approach is that contrary to most of other authors, we quantified the adhesion of primary human bone cells on metallic substrates with perfectly characterized surface topography after some hours but also over 21 days. Moreover, we have developed original statistical approaches for characterizing the relation between surface roughness and cell-adhesion parameters. In this article, we will illustrate different studies we did these last ten years concerning the development of a new adhesion parameter, the adhesion power; the correlation between short-term adhesion, long-term adhesion, and proliferation; the influence of roughness organization on cell adhesion and the development of the order parameter; our modeling approach of cell adhesion on surface topography; the relative influence of surface chemistry and topography on cell adhesion and contact angle; the relation between surface features dimensions and cell adhesion. Further, some considerations will be given on the methods for scanning surface topography for cell-adhesion studies. Finally, perspectives will be given to elucidate these intracellular mechanotransduction mechanisms induced by the deformation of cells on model sinusoidal peaks-or-valleys surfaces.

  9. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    PubMed

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

  10. Chemokine-Targeted Mouse Models of Human Primary and Metastatic Colorectal Cancer

    PubMed Central

    Chen, Huanhuan Joyce; Sun, Jian; Huang, Zhiliang; Hou, Harry; Arcilla, Myra; Rakhilin, Nikolai; Joe, Daniel J.; Choi, Jiahn; Gadamsetty, Poornima; Milsom, Jeff; Nandakumar, Govind; Longman, Randy; Zhou, Xi Kathy; Edwards, Robert; Chen, Jonlin; Chen, Kai Yuan; Bu, Pengcheng; Wang, Lihua; Xu, Yitian; Munroe, Robert; Abratte, Christian; Miller, Andrew D.; Gümüş, Zeynep H.; Shuler, Michael; Nishimura, Nozomi; Edelmann, Winfried; Shen, Xiling; Lipkin, Steven M.

    2015-01-01

    Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired sub-cutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening. PMID:26006007

  11. Primary prevention of human papillomavirus-dependent neoplasia: no condom, no sex.

    PubMed

    Epstein, Richard J

    2005-11-01

    Cervix cancer is one of several neoplastic disorders that arise following transfer of human papillomavirus (HPV) during unprotected sexual intercourse, and like most other sexually transmitted diseases (STDs), is largely preventable by consistent condom use. This primary prevention strategy has received little support, however, when compared with massive secondary prevention initiatives involving cervical screening. The reasons for this anomalous situation are complex, and include: (i) the asymptomatic nature of most primary HPV infections; (ii) widespread ignorance concerning the venereal aetiology of HPV-related cancers; (iii) the common but incorrect belief that condom use does not reduce HPV transmission; (iv) the perceived irrelevance of safe sex campaigns based on reducing transmission of human immunodeficiency virus (HIV) in high-HPV but low-HIV countries such as the Philippines; (v) the promotion of oral contraception by the medical and pharmaceutical sectors as the sexual prophylaxis of choice; and (vi) the assumption that HPV vaccines will solve the problem. Here it is proposed that the high prevalence of non-HIV STDs, including distressing disorders such as genital warts and herpes simplex, can be exploited with greater efficacy as a public health deterrent to unsafe sex and HPV transmission. Targeting a "mutually assured infection" campaign at vulnerable subgroups such as teenagers and oral contraceptive users could help reverse the global expansion of HPV-related cancers. PMID:16223580

  12. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells.

    PubMed

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  13. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    NASA Astrophysics Data System (ADS)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  14. SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

    PubMed Central

    2013-01-01

    Background Genetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer. Methods Microarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction. Results Overall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro. Conclusions The relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence. PMID:24165198

  15. Somatotopic source arrangement of 600 Hz oscillatory magnetic fields at the human primary somatosensory hand cortex.

    PubMed

    Curio, G; Mackert, B M; Burghoff, M; Neumann, J; Nolte, G; Scherg, M; Marx, P

    1997-10-01

    Based on low-noise superconducting quantum interference devices (SQUIDs) magnetoencephalography allows the non-invasive detection of low-amplitude high-frequency brain responses evoked about 20 ms after electric hand nerve stimulation. The main spectral energy of these brief oscillatory bursts (near 600 Hz) is in the range typical for rapidly repeated action potentials. Here, the magnetic fields of median and ulnar nerve evoked 600 Hz bursts are shown to exhibit a somatotopic arrangement at the primary somatosensory hand cortex closely resembling that of the concomitant postsynaptic primary cortical response (¿N20m'). Two possible burst generators are discussed: (1) repetitive spike volleys conducted along the terminal segments of somatotopically arranged thalamocortical axons, and (2) early intracortical spike activity in nerve-specific subterritories of the 3b hand area.

  16. LymPHOS 2.0: an update of a phosphosite database of primary human T cells.

    PubMed

    Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

    2015-01-01

    LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org.

  17. LymPHOS 2.0: an update of a phosphosite database of primary human T cells

    PubMed Central

    Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

    2015-01-01

    LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org. PMID:26708986

  18. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  19. Ontario primary care reform and quality improvement activities: an environmental scan

    PubMed Central

    2013-01-01

    Background Quality improvement is attracting the attention of the primary health care system as a means by which to achieve higher quality patient care. Ontario, Canada has demonstrated leadership in terms of its improvement in healthcare, but the province lacks a structured framework by which it can consistently evaluate its quality improvement initiatives specific to the primary healthcare system. The intent of this research was to complete an environmental scan and capacity map of quality improvement activities being built in and by the primary healthcare sector (QI-PHC) in Ontario as a first step to developing a coordinated and sustainable framework of primary healthcare for the province. Methods Data were collected between January and July 2011 in collaboration with an advisory group of stakeholder representatives and quality improvement leaders in primary health care. Twenty participants were interviewed by telephone, followed by review of relevant websites and documents identified in the interviews. Data were systematically examined using Framework Analysis augmented by Prior’s approach to document analysis in an iterative process. Results The environmental scan identified many activities (n = 43) designed to strategically build QI-PHC capacity, identify promising QI-PHC practices and outcomes, scale up quality improvement-informed primary healthcare practice changes, and make quality improvement a core organizational strategy in health care delivery, which were grouped into clusters. Cluster 1 was composed of initiatives in the form of on-going programs that deliberately incorporated long-term quality improvement capacity building through province-wide reach. Cluster 2 represented activities that were time-limited (research, pilot, or demonstration projects) with the primary aim of research production. The activities of most primary health care practitioners, managers, stakeholder organizations and researchers involved in this scan demonstrated a

  20. Methods and theory in bone modeling drift: comparing spatial analyses of primary bone distributions in the human humerus.

    PubMed

    Maggiano, Corey M; Maggiano, Isabel S; Tiesler, Vera G; Chi-Keb, Julio R; Stout, Sam D

    2016-01-01

    This study compares two novel methods quantifying bone shaft tissue distributions, and relates observations on human humeral growth patterns for applications in anthropological and anatomical research. Microstructural variation in compact bone occurs due to developmental and mechanically adaptive circumstances that are 'recorded' by forming bone and are important for interpretations of growth, health, physical activity, adaptation, and identity in the past and present. Those interpretations hinge on a detailed understanding of the modeling process by which bones achieve their diametric shape, diaphyseal curvature, and general position relative to other elements. Bone modeling is a complex aspect of growth, potentially causing the shaft to drift transversely through formation and resorption on opposing cortices. Unfortunately, the specifics of modeling drift are largely unknown for most skeletal elements. Moreover, bone modeling has seen little quantitative methodological development compared with secondary bone processes, such as intracortical remodeling. The techniques proposed here, starburst point-count and 45° cross-polarization hand-drawn histomorphometry, permit the statistical and populational analysis of human primary tissue distributions and provide similar results despite being suitable for different applications. This analysis of a pooled archaeological and modern skeletal sample confirms the importance of extreme asymmetry in bone modeling as a major determinant of microstructural variation in diaphyses. Specifically, humeral drift is posteromedial in the human humerus, accompanied by a significant rotational trend. In general, results encourage the usage of endocortical primary bone distributions as an indicator and summary of bone modeling drift, enabling quantitative analysis by direction and proportion in other elements and populations.

  1. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

    PubMed Central

    Sather, Blythe D.; Romano Ibarra, Guillermo S.; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F.; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A.; Scharenberg, Andrew M.; Rawlings, David J.

    2016-01-01

    Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV) – mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP – modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. PMID:26424571

  2. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

    PubMed

    Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

    2015-09-30

    Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

  3. Response to flavone acetic acid (NSC 347512) of primary and metastatic human colorectal carcinoma xenografts.

    PubMed Central

    Giavazzi, R.; Garofalo, A.; Damia, G.; Garattini, S.; D'Incalci, M.

    1988-01-01

    The antitumour activity of flavone acetic acid (FAA) was evaluated against two human colorectal carcinoma (HCC) lines, HCC-P2988 and HCC-M1410, transplanted into nude mice. On repeated i.v. injection of 200 mg kg-1 every 4 days FAA was moderately active against the s.c. growing HCC-P2988. HCC-M1410 transplanted s.c. was almost unresponsive in the same experimental conditions. In contrast, FAA (200 mg kg-1 i.v. every 4 days, repeated three times) significantly reduced liver tumour colonies produced by the HCC-M1410 cells injected intrasplenically into nude mice. These findings suggest that FAA has potential activity against human colorectal carcinoma, particularly against liver metastases. Images Figure 3 Figure 4 PMID:3355765

  4. Treatment of active duty military with PTSD in primary care: A follow-up report.

    PubMed

    Cigrang, Jeffrey A; Rauch, Sheila A M; Mintz, Jim; Brundige, Antoinette; Avila, Laura L; Bryan, Craig J; Goodie, Jeffrey L; Peterson, Alan L

    2015-12-01

    First-line trauma-focused therapies offered in specialty mental health clinics do not reach many veterans and active duty service members with posttraumatic stress disorder (PTSD). Primary care is an ideal environment to expand access to mental health care. Several promising clinical case series reports of brief PTSD therapies adapted for primary care have shown positive results, but the long-term effectiveness with military members is unknown. The purpose of this study was to determine the long-term outcome of an open trial of a brief cognitive-behavioral primary care-delivered protocol developed specifically for deployment-related PTSD in a sample of 24 active duty military (15 men, 9 women). Measures of PTSD symptom severity showed statistically and clinically significant reductions from baseline to posttreatment that were maintained at the 6-month and 1-year follow-up assessments. Similar reductions were maintained in depressive symptoms and ratings of global mental health functioning. PMID:26519833

  5. Treatment of active duty military with PTSD in primary care: A follow-up report.

    PubMed

    Cigrang, Jeffrey A; Rauch, Sheila A M; Mintz, Jim; Brundige, Antoinette; Avila, Laura L; Bryan, Craig J; Goodie, Jeffrey L; Peterson, Alan L

    2015-12-01

    First-line trauma-focused therapies offered in specialty mental health clinics do not reach many veterans and active duty service members with posttraumatic stress disorder (PTSD). Primary care is an ideal environment to expand access to mental health care. Several promising clinical case series reports of brief PTSD therapies adapted for primary care have shown positive results, but the long-term effectiveness with military members is unknown. The purpose of this study was to determine the long-term outcome of an open trial of a brief cognitive-behavioral primary care-delivered protocol developed specifically for deployment-related PTSD in a sample of 24 active duty military (15 men, 9 women). Measures of PTSD symptom severity showed statistically and clinically significant reductions from baseline to posttreatment that were maintained at the 6-month and 1-year follow-up assessments. Similar reductions were maintained in depressive symptoms and ratings of global mental health functioning.

  6. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  7. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  8. Differential effects of antidepressants on glucocorticoid receptors in human primary blood cells and human monocytic U-937 cells.

    PubMed

    Heiske, Andreas; Jesberg, Jutta; Krieg, Jürgen-Christian; Vedder, Helmut

    2003-04-01

    A number of data support the assumption that antidepressants (ADs) normalize the altered function of the hypothalamic-pituitary-adrenocortical (HPA) system involved in the pathophysiology of depressive disorder via direct effects on glucocorticoid receptors (GRs). In the present study, we examined the tricyclic ADs desipramine (DESI) and imipramine (IMI), the noradrenaline reuptake inhibitor maprotiline (MAPRO), and the noradrenergic and specific serotonergic AD (NaSSA) mirtazapine (MIR) for their effects on GR expression in primary human leukocytes and in monocytic U-937 cells. Semiquantitative RT-PCR indicated that the ADs exert differential effects on GR-mRNA levels in both primary human leukocytes and U-937 cells: whereas MAPRO and IMI did not induce pronounced changes in GR-mRNA levels, DESI and MIR significantly decreased the amounts of GR-mRNA in both cell systems. Further characterization of the effects of MIR revealed a time dependency of the regulation with an initial increase of GR-mRNA levels above control levels after 2.5 h of treatment and a decrease after 4, 24, and 48 h of incubation. A dose-response analysis demonstrated maximal effects of MIR at a concentration of 10(-7) M. Immunohistochemical studies showed that MIR increased the GR protein levels in a time-dependent manner and that this upregulation appeared earlier by additional treatment with dexamethasone (DEX). A translocation of the GR protein from the cytoplasm to the nucleus was induced between 24 and 48 h of treatment with MIR and MIR/DEX, respectively. Taken together, our data further support the assumption that ADs influence the neuroendocrine and immune system via effects on cellular GRs. PMID:12655328

  9. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

    SciTech Connect

    Acikgoez, Ali; Karim, Najibulla; Giri, Shibashish; Schmidt-Heck, Wolfgang; Bader, Augustinus

    2009-01-15

    Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c{sub i,j} (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p{sub k} (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the

  10. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    SciTech Connect

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.

  11. Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

    PubMed

    Frieauff, W; Martus, H J; Suter, W; Elhajouji, A

    2013-01-01

    The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

  12. Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry.

    PubMed Central

    Zolla-Pazner, S; O'Leary, J; Burda, S; Gorny, M K; Kim, M; Mascola, J; McCutchan, F

    1995-01-01

    The immunologic relatedness of the various human immunodeficiency virus type 1 (HIV-1) clades was determined with 13 human anti-HIV-1 monoclonal antibodies (MAbs) to six immunogenic regions of the HIV-1 structural proteins. The immunoreactivity of the native, oligomeric viral envelope glycoproteins expressed on the surfaces of human peripheral blood mononuclear cells infected in vitro with primary isolates from clades A through E was determined by flow cytometry. Some epitopes in the immunodominant region of gp41 and the C terminus of gp120 appear to be HIV-1 group specific in that they are expressed on the surfaces of cells in cultures infected with the majority of viruses tested from clades A to E. Epitopes within the V3 region appear to be clade restricted. Surprisingly, one MAb to an epitope in the C terminus of gp120 was entirely clade B specific. Staining with anti-V2 and anti-CD4 binding domain (CD4bd) reagents was infrequently detected. Anti-CD4bd MAbs stained only CD4-negative T cells because the CD4bd of gp120 appeared to be complexed with membrane CD4. When present, the epitopes of V2 and the CD4bd appeared to be expressed on cells infected with various clades. Thus, the results suggest that MAbs to gp41, the C terminus, and the V3 loop of gp120 are most useful in serotyping primary isolates of HIV-1, providing group-specific, clade-restricted, and clade-specific reagents. The use of the immunofluorescent method with the reagents described herein distinguishes infection with clade B from that with all other HIV-1 clades. With additional MAbs, this technique will allow a broadly applicable, reproducible, and practical method for serotyping HIV-1. PMID:7745728

  13. Activating Older Adults With Serious Mental Illness for Collaborative Primary Care Visits

    PubMed Central

    Bartels, Stephen J.; Aschbrenner, Kelly A.; Rolin, Stephanie A.; Hendrick, Delia Cimpean; Naslund, John A.; Faber, Marjan J.

    2016-01-01

    Objective Persons with serious mental illness frequently receive inadequate medical care and are more likely to experience difficulty navigating the health care system compared with the general population. To address this gap in quality, we developed a program of peer co-led collaborative activation training for primary care (CAT-PC) designed to improve “patient activation” and person-centered care in primary care visits for middle-aged and older adults with serious mental illness and cardiovascular risk. This report presents pilot study feasibility and participant outcomes for CAT-PC. Method A pre-post pilot evaluation of CAT-PC included N = 17 adults (age ≥ 50) with serious mental illness and cardiovascular health risk conditions, and N = 6 primary care providers. CAT-PC consists of 9 weekly peer co-led patient education and skills training sessions and a 45-min video-based training for primary care providers. Pre-post measures included the Patient Activation Measure (PAM), Perceived Efficacy in Patient-Physician Interactions (PEPPI), Autonomy Preference Index (API) for preferred role in primary care encounters, and Social Skills Performance Assessment (SSPA) role-play test for medical visits. Results All 17 participants attended 5 or more sessions. Post-intervention improvement was found for patient activation and simulated performance of medical visit communication skills. Trends were observed for improved self-efficacy in provider interactions and greater preference for a more collaborative role in decision-making. Conclusions and Implications CAT-PC is a brief, peer co-led education and skills training intervention potentially improving patient activation in primary care encounters and providing an important missing component in emerging models of “patient-centered behavioral health homes” for this high-risk group. PMID:24219769

  14. Structural snapshots of actively translating human ribosomes

    PubMed Central

    Behrmann, Elmar; Loerke, Justus; Budkevich, Tatyana V.; Yamamoto, Kaori; Schmidt, Andrea; Penczek, Pawel A.; Vos, Matthijn R.; Bürger, Jörg; Mielke, Thorsten; Scheerer, Patrick; Spahn, Christian M.T.

    2015-01-01

    Summary Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. While their mode of action is often compared to that of mechanical machines, a crucial difference is that at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and three-dimensional variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements. PMID:25957688

  15. New activity pattern in human interactive dynamics

    NASA Astrophysics Data System (ADS)

    Formentin, Marco; Lovison, Alberto; Maritan, Amos; Zanzotto, Giovanni

    2015-09-01

    We investigate the response function of human agents as demonstrated by written correspondence, uncovering a new pattern for how the reactive dynamics of individuals is distributed across the set of each agent’s contacts. In long-term empirical data on email, we find that the set of response times considered separately for the messages to each different correspondent of a given writer, generate a family of heavy-tailed distributions, which have largely the same features for all agents, and whose characteristic times grow exponentially with the rank of each correspondent. We furthermore show that this new behavioral pattern emerges robustly by considering weighted moving averages of the priority-conditioned response-time probabilities generated by a basic prioritization model. Our findings clarify how the range of priorities in the inputs from one’s environment underpin and shape the dynamics of agents embedded in a net of reactive relations. These newly revealed activity patterns might be universal, being present in other general interactive environments, and constrain future models of communication and interaction networks, affecting their architecture and evolution.

  16. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary