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  1. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    PubMed Central

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  2. 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

  3. 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.

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

    PubMed Central

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

    1989-01-01

    We 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 cyclosporin 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. Images PMID:2498643

  5. Plasminogen-Dependent Matriptase Activation Accelerates Plasmin Generation by Differentiating Primary Human Keratinocytes.

    PubMed

    Chen, Ya-Wen; Yin, Shi; Lai, Ying-Jung J; Johnson, Michael D; Lin, Chen-Yong

    2016-06-01

    Pericellular plasmin generation, an important pathophysiological process, can be initiated and accelerated by the autoactivation of the type 2 transmembrane serine protease matriptase and subsequent activation of urokinase plasminogen activator. The link between matriptase and plasminogen was initially thought to be one-directional: from matriptase, through plasminogen activator, to plasminogen. However, in the current study, we now show that primary human keratinocytes that are undergoing calcium-induced differentiation can rapidly activate matriptase in response to serum treatment via a mechanism dependent on intracellular calcium, protein kinase C, and phosphatidylinositol 3-kinases-based signaling. The serum factor, responsible for the induction of matriptase zymogen activation, was shown to be plasminogen. A sub-pM concentration of plasminogen (but not plasmin) acting at the cell surface is sufficient to induce matriptase activation, suggesting high potency and specificity of the induction. After matriptase zymogen activation, a proportion of active matriptase is shed into extracellular milieu and returns to the cell surface to accelerate plasmin generation. The ability of plasminogen to induce matriptase zymogen activation and the subsequent acceleration of plasmin generation by active matriptase reveals a feed-forward mechanism that allows differentiating human keratinocytes to rapidly and robustly activate pericellular proteolysis. PMID:26872599

  6. 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

  7. 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

  8. Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells☆

    PubMed Central

    Haudek-Prinz, Verena J.; Klepeisz, Philip; Slany, Astrid; Griss, Johannes; Meshcheryakova, Anastasia; Paulitschke, Verena; Mitulovic, Goran; Stöckl, Johannes; Gerner, Christopher

    2012-01-01

    Proteome profiling is the method of choice to identify marker proteins whose expression may be characteristic for certain diseases. The formation of such marker proteins results from disease-related pathophysiologic processes. In healthy individuals, peripheral blood mononuclear cells (PBMCs) circulate in a quiescent cell state monitoring potential immune-relevant events, but have the competence to respond quickly and efficiently in an inflammatory manner to any invasion of potential pathogens. Activation of these cells is most plausibly accompanied by characteristic proteome alterations. Therefore we investigated untreated and inflammatory activated primary human PBMCs by proteome profiling using a ‘top down’ 2D-PAGE approach in addition to a ‘bottom up’ LC–MS/MS-based shotgun approach. Furthermore, we purified primary human T-cells and monocytes and activated them separately. Comparative analysis allowed us to characterize a robust proteome signature including NAMPT and PAI2 which indicates the activation of PBMCs. The T-cell specific inflammation signature included IRF-4, GBP1and the previously uncharacterized translation product of GBP5; the corresponding monocyte signature included PDCD5, IL1RN and IL1B. The involvement of inflammatory activated PBMCs in certain diseases as well as the responsiveness of these cells to anti-inflammatory drugs may be evaluated by quantification of these marker proteins. This article is part of a Special Issue entitled: Integrated omics. PMID:22813876

  9. 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

  10. 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

  11. 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

  12. A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

    PubMed Central

    2013-01-01

    Background Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides. PMID:23937268

  13. Drug Metabolism Enzyme Expression and Activity in Primary Cultures of Human Proximal Tubular Cells

    PubMed Central

    Lash, Lawrence H.; Putt, David A.; Cai, Hongliang

    2008-01-01

    We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Toxicology 228, 200–218 (2006)]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism. PMID:18055091

  14. 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

  15. 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

  16. 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

  17. Molecular cloning of an activated human oncogene, homologous to v-raf, from primary stomach cancer.

    PubMed Central

    Shimizu, K; Nakatsu, Y; Sekiguchi, M; Hokamura, K; Tanaka, K; Terada, M; Sugimura, T

    1985-01-01

    Transfection with high molecular weight DNA from a primary stomach cancer induced foci of transformed NIH 3T3 cells, and the transformed cells were tumorigenic in nude mice. By screening with a human Alu-family probe, we isolated the human DNA sequence from the secondary transformant cells. This transforming sequence encompasses about 60 kilobase pairs and is unrelated to known human transforming genes. Examination of homologies between this sequence and retroviral oncogenes revealed that the human transforming sequence is closely related to the v-raf oncogene of murine transforming retrovirus 3611-MSV. Images PMID:3862088

  18. The influence of ligand-activated LXR on primary human trophoblasts

    PubMed Central

    Larkin, Jacob C.; Sears, Sarah B.; Sadovsky, Yoel

    2014-01-01

    Introduction The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. Methods We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. Results Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. Conclusion These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands. PMID:25255963

  19. Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts

    PubMed Central

    Lisse, Thomas S.; Chun, Rene F.; Rieger, Sandra; Adams, John S.; Hewison, Martin

    2013-01-01

    When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D ) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on post-transcriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOB) identified human miRNAs that were differentially regulated by > 1.5-fold following treatment with 1,25D (10nM, 6 hrs), which included miRNAs 637 and 1228. RT-PCR analyses showed that the host gene for miR-1228, low density lipoprotein receptor-related protein 1 (LRP1), was co-induced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1 – 10nM, 6hrs). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K) respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, siRNA inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated down regulation of BMP2K protein expression. This was associated with suppression of pro-differentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed post-transcriptional mechanisms. PMID:23362149

  20. The impact of Nucleofection® on the activation state of primary human CD4 T cells

    PubMed Central

    Zhang, Mingce; Ma, Zhengyu; Selliah, Nithianandan; Weiss, Greta; Genin, Anna; Finkel, Terri H.; Cron, Randy Q.

    2014-01-01

    Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1 hour later. The intracellular calcium level also increases after Nucleofection®, peaking after 1 hour before recovering 8 hours post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24 hours, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24 hours after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above. PMID:24910411

  1. The impact of Nucleofection® on the activation state of primary human CD4 T cells.

    PubMed

    Zhang, Mingce; Ma, Zhengyu; Selliah, Nithianandan; Weiss, Greta; Genin, Anna; Finkel, Terri H; Cron, Randy Q

    2014-06-01

    Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above. PMID:24910411

  2. [Primary prevention: physical activity].

    PubMed

    Schuler, G

    2004-01-01

    Traditional risk factors such as smoking, hypertension and being overweight have received considerable attention in recent years, whereas physical activity as a preventive strategy does not enjoy the same public attention. In recent years the level of physical activity has decreased dramatically in children and adolescents in favor of time spent on the internet and in front of the TV. If this trend is allowed to develop along the same direction, a sharp increase in cardiovascular disease can be anticipated. The protective action of physical activity on the cardiovascular system has been well documented in large numbers of patients, and the basic physiological mechanisms have been elucidated. Metabolic changes comprise loss of weight, reduction in triglyceride and LDL levels, as well as an increase in HDL. Insulin sensitivity is enhanced in all tissues postponing the manifestation of diabetes mellitus. Shear forces created by physical activity induce ecNOS within the endothelial lining of the arteries. This enzyme is responsible for controlling vasomotion through the elaboration of NO which causes vasodilation in the smooth muscle within the vessel wall. Utilization of preformed collateral vessels has been postulated repeatedly; so far, however, it only could be documented in animals, not in humans. Nearly all studies concerned with primary prevention have shown a significant negative correlation between energy expenditure during exercise and cardiovascular mortality, even light and moderate exercise will result in a lower incidence. In order to eliminate a sedentary life style in children and adolescents, adequate programs should be initiated in all schools; they should aim for 60 min of physical activity on a daily basis. PMID:15021990

  3. Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes.

    PubMed

    Klatt, Andreas R; Klinger, Gabriele; Paul-Klausch, Brigitte; Kühn, Gertrud; Renno, Joerg H; Wagener, Raimund; Paulsson, Mats; Schmidt, Joachim; Malchau, Gebhart; Wielckens, Klaus

    2009-11-19

    Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1beta, TNFalpha, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1beta. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage. PMID:19840795

  4. Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.

    PubMed Central

    Moon, A M; Ley, T J

    1990-01-01

    DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse beta-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR. Images PMID:2217202

  5. Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

    PubMed Central

    Pinnix, Chelsea C.; Lee, John T.; Liu, Zhao-Jun; McDaid, Ronan; Balint, Klara; Beverly, Levi J.; Brafford, Patricia A.; Xiao, Min; Himes, Benjamin; Zabierowski, Susan E.; Yashiro-Ohtani, Yumi; Nathanson, Katherine L.; Bengston, Ana; Pollock, Pamela M.; Weeraratna, Ashani T.; Nickoloff, Brian J.; Pear, Warren S.; Capobianco, Anthony J.; Herlyn, Meenhard

    2009-01-01

    The importance of MAPK signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf; yet, clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions demonstrated that Notch activity is significantly higher in melanomas than their non-transformed counterparts. The use of a constitutively-active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a ‘driving’ event in melanocytic transformation or instead a ‘passenger’ event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth suggesting that Notch, alone, is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene; this new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease. PMID:19549918

  6. Primary Guidance Activities.

    ERIC Educational Resources Information Center

    Hauck, Ruth, Comp.

    Designed for the primary grades, the guidance activities in this document cover thirty-three topical areas: capabilities, changes, cooperation, criticism, differences, family, feelings, free time, friends, following directions, handicaps, honesty, improving environment, kindness, patience, paying attention, problem solving, rejection,…

  7. Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages*

    PubMed Central

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

    2013-01-01

    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

  8. Interaction of nuclear protein p140 with human immunodeficiency virus type 1 TAR RNA in mitogen-activated primary human T lymphocytes.

    PubMed Central

    Rothblum, C J; Jackman, J; Mikovits, J; Shukla, R R; Kumar, A

    1995-01-01

    Several lines of evidence suggest that cellular proteins play a role during human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans activation. A recent report from this laboratory has shown that a 140-kDa HeLa nuclear protein (p140) binds specifically to the lower stem region of the Tat response element, TAR RNA. Since HIV-1 trans activation is most efficient in proliferating T cells, we investigated the binding of p140 to TAR RNA in unstimulated and mitogen-activated, G1-phase primary T lymphocytes. TAR RNA/protein-binding activity was low in resting cells but increased significantly within 2 h of activation and remained elevated for at least 48 h. Corresponding increases in p140 protein levels were observed with most but not all donors, suggesting that an additional nuclear factor(s) may be required for efficient binding of this protein to TAR RNA in activated T cells. PMID:7609087

  9. 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.

  10. Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes.

    PubMed

    Rondini, Elizabeth A; Duniec-Dmuchowski, Zofia; Kocarek, Thomas A

    2016-04-01

    Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism. PMID:26798158

  11. How does transcranial DC stimulation of the primary motor cortex alter regional neuronal activity in the human brain?

    PubMed Central

    Lang, Nicolas; Siebner, Hartwig R.; Ward, Nick S.; Lee, Lucy; Nitsche, Michael A.; Paulus, Walter; Rothwell, John C.; Lemon, Roger N.; Frackowiak, Richard S.

    2013-01-01

    Transcranial direct current stimulation (tDCS) of the primary motor hand area (M1) can produce lasting polarity-specific effects on corticospinal excitability and motor learning in humans. In 16 healthy volunteers, H215O positron emission tomography (PET) of regional cerebral blood flow (rCBF) at rest and during finger movements was used to map lasting changes in regional synaptic activity following 10 min of tDCS (± 1 mA). Bipolar tDCS was given through electrodes placed over the left M1 and right frontopolar cortex. Eight subjects received anodal or cathodal tDCS of the left M1, respectively. When compared to sham tDCS, anodal and cathodal tDCS induced widespread increases and decreases in rCBF in cortical and subcortical areas. These changes in rCBF were of the same magnitude as task-related rCBF changes during finger movements and remained stable throughout the 50-min period of PET scanning. Relative increases in rCBF after real tDCS compared to sham tDCS were found in the left M1, right frontal pole, right primary sensorimotor cortex and posterior brain regions irrespective of polarity. With the exception of some posterior and ventral areas, anodal tDCS increased rCBF in many cortical and subcortical regions compared to cathodal tDCS. Only the left dorsal premotor cortex demonstrated an increase in movement related activity after cathodal tDCS, however, modest compared with the relatively strong movement-independent effects of tDCS. Otherwise, movement related activity was unaffected by tDCS. Our results indicate that tDCS is an effective means of provoking sustained and widespread changes in regional neuronal activity. The extensive spatial and temporal effects of tDCS need to be taken into account when tDCS is used to modify brain function. PMID:16045502

  12. How does transcranial DC stimulation of the primary motor cortex alter regional neuronal activity in the human brain?

    PubMed

    Lang, Nicolas; Siebner, Hartwig R; Ward, Nick S; Lee, Lucy; Nitsche, Michael A; Paulus, Walter; Rothwell, John C; Lemon, Roger N; Frackowiak, Richard S

    2005-07-01

    Transcranial direct current stimulation (tDCS) of the primary motor hand area (M1) can produce lasting polarity-specific effects on corticospinal excitability and motor learning in humans. In 16 healthy volunteers, O positron emission tomography (PET) of regional cerebral blood flow (rCBF) at rest and during finger movements was used to map lasting changes in regional synaptic activity following 10 min of tDCS (+/-1 mA). Bipolar tDCS was given through electrodes placed over the left M1 and right frontopolar cortex. Eight subjects received anodal or cathodal tDCS of the left M1, respectively. When compared to sham tDCS, anodal and cathodal tDCS induced widespread increases and decreases in rCBF in cortical and subcortical areas. These changes in rCBF were of the same magnitude as task-related rCBF changes during finger movements and remained stable throughout the 50-min period of PET scanning. Relative increases in rCBF after real tDCS compared to sham tDCS were found in the left M1, right frontal pole, right primary sensorimotor cortex and posterior brain regions irrespective of polarity. With the exception of some posterior and ventral areas, anodal tDCS increased rCBF in many cortical and subcortical regions compared to cathodal tDCS. Only the left dorsal premotor cortex demonstrated an increase in movement related activity after cathodal tDCS, however, modest compared with the relatively strong movement-independent effects of tDCS. Otherwise, movement related activity was unaffected by tDCS. Our results indicate that tDCS is an effective means of provoking sustained and widespread changes in regional neuronal activity. The extensive spatial and temporal effects of tDCS need to be taken into account when tDCS is used to modify brain function. PMID:16045502

  13. 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...

  14. 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...

  15. BOLD Responses in Human Primary Visual Cortex are Insensitive to Substantial Changes in Neural Activity.

    PubMed

    Swettenham, J B; Muthukumaraswamy, S D; Singh, K D

    2013-01-01

    The relationship between blood oxygenation level dependent-functional magnetic resonance imaging (BOLD-fMRI) and magnetoencephalography (MEG) metrics were explored using low-level visual stimuli known to elicit a rich variety of neural responses. Stimuli were either perceptually isoluminant red/green or luminance-modulated black/yellow square-wave gratings with spatial frequencies of 0.5, 3, and 6 cycles per degree. Neural responses were measured with BOLD-fMRI (3-tesla) and whole head MEG. For all stimuli, the BOLD response showed bilateral activation of early visual cortex that was greater in the contralateral hemisphere. There was variation between individuals but weak, or no evidence, of amplitude dependence on either spatial frequency or the presence of luminance contrast. In contrast, beamformer analysis of MEG data showed activation in contralateral early visual cortex and revealed: (i) evoked responses with stimulus-dependent amplitude and latency; (ii) gamma and high-beta oscillations, with spatial frequency dependent peaks at approximately 30 and 50 Hz, but only for luminance-modulated gratings; (iii) The gamma and beta oscillations appeared to show different spatial frequency tuning profiles; (iv) much weaker gamma and beta responses, and at higher oscillation frequencies, for isoluminant compared to luminance-modulated gratings. The results provide further evidence that the relationship between the fMRI-BOLD response and cortical neural activity is complex, with BOLD-fMRI being insensitive to substantial changes in neural activity. All stimuli were clearly visible to participants and so the paucity of gamma oscillations to isoluminant stimuli is inconsistent with theories of their role in conscious visual perception. PMID:23482840

  16. On-line studies of activation events in primary human T lymphocytes.

    PubMed

    Bental, M; Deutsch, C

    1994-04-01

    In this paper, we review our NMR studies of human peripheral blood T lymphocytes. These studies focus on the physiological and biochemical alterations accompanying cell cycle progression. In particular, we have characterized phosphorus metabolism, glucose utilization and lactate production, and pH regulation using 31P, 13C, and 19F NMR, respectively. These studies required developing new methods for monitoring on-line stimulation of quiescent T cells under sterile, physiological conditions (i.e., CO2/HCO3- buffer, 37 degrees C) for prolonged periods of time. A perfusion system optimized for T lymphocytes inside agarose beads is described. In addition, custom-designed 19F NMR pH indicators were synthesized, characterized, and used to determine intracellular pH in quiescent lymphocytes, stimulated lymphocytes, and lymphocytes undergoing the G0-G1 transition. These unique molecular probes are described in detail. Finally, the physiological relevance of our findings regarding carbon metabolism and pH regulation is considered in the context of mitogenesis. PMID:8069534

  17. 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

  18. [Macrophage activation syndrome in primary human herpes virus-6 infection: a rare condition after liver transplantation in infants].

    PubMed

    Lecointe, D; Fabre, M; Habes, D; Mielot, F; Bernard, O; Nordmann, P

    2000-12-01

    Human herpes virus-6 primary infection generally occurs during the first three years of childhood and is generally asymptomatic. The virus has been identified as the causal agent of exanthemum subitum in children or mononucleosis-like disease in adults, and may also cause several disorders in immunocompromised patients. We report a clinical case of acute rejection observed 29 days after orthotopic liver transplantation in a 22-month-old child associated with acute hepatitis and a hemophagocytic syndrome on day 38. Human herpes virus-6 primary infection was identified based on several virological tests: seroconversion, detection of viral DNA in bone marrow and peripheral blood after polymerase chain reaction, and detection of viral replication in peripheral blood. Tests for Epstein-Barr virus, cytomegalovirus or Parvovirus B19 infections were negative. After treatment by ganciclovir (Cymévan(R)), clinical status improved. PMID:11173737

  19. 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

  20. 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

  1. 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

  2. The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase 9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro

    PubMed Central

    Allen, Terrence K; Feng, Liping; Nazzal, Matthew; Grotegut, Chad A; Buhimschi, Irina A; Murtha, Amy P

    2015-01-01

    Background Current treatment modalities for preventing preterm premature rupture of membranes (PPROM) are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase 9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term non-laboring patients. Methods Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated non-laboring patients following elective cesarean delivery (n=11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P) or medroxyprogesterone acetate (MPA) at 10-6 M concentration for 6 h followed by stimulation with TNFα at 10 ng/mL for an additional 24 h. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using RT-qPCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of

  3. Immunostimulatory activity of isoflavone-glycosides and ethanol extract from a fermented soybean product in human primary immune cells.

    PubMed

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

    2014-10-01

    Sasang constitutional medicine is a major branch of Korean traditional oriental medicine. Constitutions of Sasang medicine are classified into Taeyangin, Taeumin, Soyangin, and Soumin. We investigated immunostimulatory activities of fermented soybean product (FSP) extracts and their major bioactive compounds, isoflavone glycosides in primary immune cells isolated from the blood of Soyangin, Taeumin, and Soeumin volunteers. Results showed that the cell proliferation, nitrite, tumor necrosis factor (TNF)-α mRNA, interleukin (IL)-6 mRNA, inducible nitric oxide synthase (iNOS) mRNA, cyclooxygenase-2 (COX-2) mRNA, TNF-α protein, and IL-6 protein production of immune cells treated with a 70% ethanol Doenjang extract (DJ), a 70% ethanol Kochujang extract (KCJ), and a 70% ethanol Cheonggukjang extract (CGJ), respectively, were significantly increased, and its immunostimulatory activities by both DJ and CGJ was higher than that of KCJ in primary immune cells isolated from the blood of Soyangin volunteers. However, the cell proliferation, nitrite, TNF-α mRNA, IL-6 mRNA, iNOS mRNA, COX-2 mRNA, TNF-α protein, and IL-6 protein production by both KCJ and CGJ was higher than that of DJ in primary immune cells isolated from the blood of Taeumin and Soeumin volunteers. The major bioactive compounds, isoflavone-glycosides, in FSP were daidzin, glycitin, and genistin. Daidzin, glycitin, and genistin were used to treat primary immune cells in the same condition, the cell proliferation; iNOS mRNA expression and nitrite concentration of daidzin, glycitin, or genistin-treated immune cells isolated from the blood of Soyangin volunteers was higher than that of Taeumin and Soeumin volunteers. The effect of DJ and isoflavone glycosides on immunostimulatory activities showed similar trends. PMID:25230138

  4. 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. PMID:26888163

  5. 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

  6. 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

  7. Notch1 Signaling Is Activated in Cells Expressing Embryonic Stem Cell Proteins in Human Primary Nasopharyngeal Carcinoma

    PubMed Central

    Zhang, Yue; Peng, Jianhua; Zhang, Huxiang; Zhu, Yi; Wan, Li; Chen, Jianfu; Chen, Xiaoyun; Lin, Renyu; Li, He; Mao, XiaoOu; Jin, Kunlin

    2010-01-01

    Objective: To explore the expression of Notch1 signaling pathway in nasopharyngeal carcinoma (NPC). Methods: We performed immunocytochemistry on surgically resected NPC using antibodies against embryonic stem (ES) cell proteins and against Notch1 signaling components. Results: We found that ES cell protein markers SOX2 and OCT4 were expressed in a subpopulation of cells for all three subtypes of NPC but barely in the normal control. Double immunostaining shows that SOX2- and OCT4-positive cells coexpressed proliferative markers, suggesting that human NPC may contain cancer stem–like cells. In addition, we found that Notch1 signaling was activated in NPC. Confocal images show that the Notch1 signaling activated form and Hes1, a downstream target of Notch1 signaling, was predominantly found in SOX2- and OCT4-positive cells. Conclusion: Our findings suggest that the Notch1 signaling pathway might be a regulator of cancer stem–like cells in NPC. PMID:20211102

  8. Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes.

    PubMed

    Bachour-El Azzi, Pamela; Sharanek, Ahmad; Burban, Audrey; Li, Ruoya; Guével, Rémy Le; Abdel-Razzak, Ziad; Stieger, Bruno; Guguen-Guillouzo, Christiane; Guillouzo, André

    2015-05-01

    The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis. PMID:25690737

  9. Detection of primary cilia in human glioblastoma

    PubMed Central

    Sarkisian, Matthew R.; Siebzehnrubl, Dorit; Hoang-Minh, Lan; Deleyrolle, Loic; Silver, Daniel J.; Siebzehnrubl, Florian A.; Guadiana, Sarah M.; Srivinasan, Gayathri; Semple-Rowland, Susan; Harrison, Jeffrey K.; Steindler, Dennis A.; Reynolds, Brent A.

    2015-01-01

    Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies. PMID:24510433

  10. 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

  11. Quercetin protects primary human osteoblasts exposed to cigarette smoke through activation of the antioxidative enzymes HO-1 and SOD-1.

    PubMed

    Braun, Karl F; Ehnert, Sabrina; Freude, Thomas; Egaña, José T; Schenck, Thilo L; Buchholz, Arne; Schmitt, Andreas; Siebenlist, Sebastian; Schyschka, Lilianna; Neumaier, Markus; Stöckle, Ulrich; Nussler, Andreas K

    2011-01-01

    Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS). The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM) rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers. PMID:22203790

  12. 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 ( ...

  13. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  14. A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity.

    PubMed

    Nekhai, S; Shukla, R R; Kumar, A

    1997-10-01

    Tat protein mediates transactivation of human immunodeficiency virus type 1 (HIV-1), which results in more-efficient transcript elongation. Since phosphorylation of C-terminal domain (CTD) of RNA polymerase II correlates with its enhanced processivity, we studied the properties of a Tat-associated CTD kinase derived from mitogenically stimulated human primary T lymphocytes (TTK). TTK binds to full-length Tat and specifically phosphorylates CTD and CDK2. This dual kinase activity is characteristic of CDK-activating kinase (CAK). The CTD kinase activity is induced upon mitogenic stimulation of primary T lymphocytes. Fractionation of T-cell lysate demonstrates that Tat-associated CTD kinase activity elutes in two peaks. About 60% of Tat-associated CTD kinase copurifies with CDK2 kinase activity and contains the CAK components CDK7 and cyclin H. The rest of Tat-associated kinase is free of CDK2 kinase activity and the CAK components and thus may represent a novel CTD kinase. The kinase activities of TTK are blocked by the adenosine analog 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) as well as by the kinase inhibitor H8 at concentrations known to block transcript elongation. Importantly, the Tat-associated kinase markedly induced CAK. We suggest that the mechanism of Tat-mediated processive transcription of the HIV-1 promoter includes a Tat-associated CAK activator. PMID:9311822

  15. 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

  16. Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes

    PubMed Central

    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

  17. 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

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

    PubMed

    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

  19. 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

  20. Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells.

    PubMed

    Fahrig, R; Rupp, M; Steinkamp-Zucht, A; Bader, A

    1998-08-01

    Loss of cytochrome P-450 content is a common feature in conventional culture systems of primary hepatocytes. In contrast to the standard in vitro situation, in vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry has been reconstructed in vitro by culturing rat or human hepatocytes within two layers of collagen, thus forming a sandwich configuration. Activation of dimethylbenzanthracene (DMBA) or benzo[a]pyrene (BaP) was studied in rat and human hepatocytes. Genotoxic effects were studied in a three-dimensional co-culture model between sandwich hepatocytes and mammalian cells using the comet assay for detection of DNA strand breaks, and the HPRT test for detection of gene mutations. Sandwich hepatocytes generated active metabolites. The maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The number of DMBA- or BaP-induced genotoxic effects tended to be higher than in standard S-9 mix assays. While the ability to activate indirect carcinogens disappears within hours in primary hepatocytes, hepatocyte sandwich cultures enhance their ability to activate indirect carcinogens within 1 wk and retain this activity for up to 2 wk. This is the main advantage of the sandwich method over the more simple and conventional assays. While freshly isolated hepatocytes, regardless of whether in sandwich culture or in conventional assays, are injured by the isolation procedure and possess a corresponding reduced activation ability, hepatocytes in sandwich cultures recover over the course of a few days, and acquire a much higher ability to activate indirect carcinogens. Consequently, the indirect carcinogens BaP and DMBA, which were ineffective (BaP) or exhibited only weak effects (DMBA) at a concentration of 160nmol/ml in 1-2-day-old hepatocytes, were clearly effective (BaP) or showed about a threefold increase in genotoxicity (DMBA) in 8-day

  1. Contribution of MINCLE-SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB.

    PubMed

    Ostrop, Jenny; Jozefowski, Katrin; Zimmermann, Stephanie; Hofmann, Katharina; Strasser, Erwin; Lepenies, Bernd; Lang, Roland

    2015-09-01

    Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE. PMID:26202982

  2. Glucocorticoids induce senescence in primary human tenocytes by inhibition of sirtuin 1 and activation of the p53/p21 pathway: in vivo and in vitro evidence

    PubMed Central

    Poulsen, Raewyn C; Watts, Anna C; Murphy, Richard J; Snelling, Sarah J; Carr, Andrew J; Hulley, Philippa A

    2014-01-01

    Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. Objective To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. Methods Features of senescence (β-galactosidase activity at pH 6 (SA-β-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. Results Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-βgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. Conclusion As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue. PMID:23727633

  3. Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells.

    PubMed

    Fiskus, Warren; Verstovsek, Srdan; Manshouri, Taghi; Smith, Jacqueline E; Peth, Karissa; Abhyankar, Sunil; McGuirk, Joseph; Bhalla, Kapil N

    2013-05-01

    Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs. PMID:23445613

  4. 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

  5. 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…

  6. 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

  7. 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

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

    PubMed

    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

  9. 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

    SciTech Connect

    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.

  10. [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

  11. 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

  12. 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

    2015-01-01

    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. PMID:26007187

  13. Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

    PubMed

    Powers, James F; Korgaonkar, Parimal G; Fliedner, Stephanie; Giubellino, Alessio; Pacak, Karel; Sahagian, G Gary; Tischler, Arthur S

    2014-01-01

    There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their

  14. Cytocidal Activities of Topoisomerase 1 Inhibitors and 5-Azacytidine against Pheochromocytoma/Paraganglioma Cells in Primary Human Tumor Cultures and Mouse Cell Lines

    PubMed Central

    Powers, James F.; Korgaonkar, Parimal G.; Fliedner, Stephanie; Giubellino, Alessio; Sahagian, Karel Pacak, G. Gary.; Tischler, Arthur S.

    2014-01-01

    There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their

  15. Career Education Activities: Primary Grades.

    ERIC Educational Resources Information Center

    Gingras, Constance, Comp.; And Others

    Different aspects of life can be experienced by a child in a learning environment offered by a teacher. Such experiences will better prepare the student for the future in his dealings with other people and situations. The book offers detailed suggestions for various ways in which the teacher can introduce to the student in the primary grades…

  16. CR2 is the primary acceptor site for C3 during alternative pathway activation of complement on human peripheral B lymphocytes.

    PubMed

    Marquart, H V; Svehag, S E; Leslie, R G

    1994-07-01

    Human cells infected with certain viruses acquire the ability to activate the alternative pathway (AP) of complement. Complement receptor 2 on EBV-infected lymphoblastoid cell lines has been reported to act as the covalent binding site for C3b during AP activation. Using flow cytometry, we investigated the ability of normal human peripheral blood leukocytes to activate the AP in homologous serum. Deposition of C3 fragments was determined as a measurement of complement activation on each of the subpopulations of the blood cells. Incubating human peripheral blood leukocytes with homologous or autologous serum resulted in C3 deposition on B cells and, to a lesser extent, on monocytes and polymorphonuclear leukocytes. Complement activation in the presence of Mg2+ ions and EGTA revealed major involvement of the AP in the case of B cells, and to a lesser extent for other leukocyte populations examined. Preincubation of the leukocytes with polyclonal anti-complement receptor 2 Ab markedly decreased the C3 fragment deposition, as a result of in vitro AP activation, on B cells, indicating that on normal human B cells this receptor may be involved in AP activation. Freshly isolated, normal human B cells also bear low but significant amounts of C3d,g fragments on their membranes, indicating that this AP activation also occurs in vivo. AP activation was partially decreased in the presence of autologous erythrocytes (RBC) suggesting that complement regulatory proteins on RBC play a role in limiting the AP activation in vivo. PMID:7515925

  17. HUMAN EXPOSURE ACTIVITY PATTERNS

    EPA Science Inventory

    Human activity/uptake rate data are necessary to estimate potential human exposure and intake dose to environmental pollutants and to refine human exposure models. Personal exposure monitoring studies have demonstrated the critical role that activities play in explaining and pre...

  18. Gene targeting in primary human trophoblasts

    PubMed Central

    Rosario, Fredrick J; Sadovsky, Yoel; Jansson, Thomas

    2012-01-01

    Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts. PMID:22831880

  19. 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.

  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. PMID:26903518

  1. 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

  2. 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. PMID:26945151

  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. 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…

  5. 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

  6. Human alveolar epithelial type II cells in primary culture.

    PubMed

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  7. Human alveolar epithelial type II cells in primary culture

    PubMed Central

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-01-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  8. 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

  9. 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

  10. 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…

  11. Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation.

    PubMed

    Suzuki, K; Lovera, M; Schmachtenberg, O; Couve, E

    2015-10-01

    The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Büngner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages. PMID:26149320

  12. 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.

  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. PMID:24337472

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

    PubMed

    Sergeeva, A; He, H; Ruisaard, K; St John, L; Alatrash, G; Clise-Dwyer, K; Li, D; Patenia, R; Hong, R; Sukhumalchandra, P; You, M J; Gagea, M; Ma, Q; Molldrem, J J

    2016-07-01

    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, that 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, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) 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 the 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

  15. Cadherin Expression, Vectorial Active Transport, and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

    PubMed Central

    Slusser, Andrea; Bathula, Chandra S.; Sens, Donald A.; Somji, Seema; Sens, Mary Ann; Zhou, Xu Dong; Garrett, Scott H.

    2015-01-01

    Background Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells. Methods Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells. Results It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells. Conclusions The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype. PMID:25803827

  16. Burkholderia pseudomallei induces IL-23 production in primary human monocytes.

    PubMed

    Kulsantiwong, Panthong; Pudla, Matsayapan; Boondit, Jitrada; Wikraiphat, Chanthiwa; Dunachie, Susanna J; Chantratita, Narisara; Utaisincharoen, Pongsak

    2016-06-01

    Burkholderia pseudomallei, a gram-negative intracellular bacterium, is a causative agent of melioidosis. The bacterium has been shown to induce the innate immune response, particularly pro-inflammatory cytokine production in several of both mouse and human cell types. In the present study, we investigate host immune response in B. pseudomallei-infected primary human monocytes. We discover that wild-type B. pseudomallei is able to survive and multiply inside the primary human monocytes. In contrast, B. pseudomallei LPS mutant, a less virulent strain, is susceptible to host killing during bacterial infection. Moreover, microarray result showed that wild-type B. pseudomallei but not B. pseudomallei LPS mutant is able to activate gene expression of IL-23 as demonstrated by the up-regulation of p19 and p40 subunit expression. Consistent with gene expression analysis, the secretion of IL-23 analyzed by ELISA also showed that wild-type B. pseudomallei induces a significantly higher level of IL-23 secretion than that of B. pseudomallei LPS mutant. These results implied that IL-23 may be an important cytokine for the innate immune response during B. pseudomallei infection. The regulation of IL-23 production may drive the different host innate immune responses between patients and may relate to the severity of melioidosis. PMID:26563410

  17. Correlation of Organic Cation/Carnitine Transporter 1 and Multidrug Resistance-Associated Protein 1 Transport Activities With Protein Expression Levels in Primary Cultured Human Tracheal, Bronchial, and Alveolar Epithelial Cells.

    PubMed

    Sakamoto, Atsushi; Suzuki, Shinobu; Matsumaru, Takehisa; Yamamura, Norio; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

    2016-02-01

    Understanding how transporters contribute to the distribution of inhaled drugs in the lung is important for the discovery and development of such drugs. Protein expression levels may be useful to predict and understand drug distribution. As previously reported, organic cation/carnitine transporter 1 (OCTN1) and multidrug resistance-associated protein 1 (MRP1) have higher levels of protein expression among transporters in primary cultured human lung cells. Nevertheless, it is unclear to what extent transport activity correlates with transporter protein expression. The purpose is to evaluate whether differences in OCTN1 and MRP1 protein expression govern the respective transport activity in primary cultured human lung cells. The model substrates of 4-[4-(dimethylamino) styryl]-N-methylpyridinium iodide (ASP(+)) and carboxy-dichlorofluorescein (CDF) for OCTN1 and MRP1, respectively, were used in the lung cells from five donors. Significant correlation was found between the kinetic parameter Vmax for ASP(+) and OCTN1 protein expression in plasma membrane of tracheal, bronchial, and alveolar cells (r(2) = 0.965, 0.834, and 0.877, respectively), and between the efflux of CDF and MRP1 protein expression in plasma membrane of tracheal, bronchial, and alveolar cells (r(2) = 0.800, 0.904, and 0.790, respectively). These findings suggest that OCTN1 and MRP1 protein concentrations are determinants for drug distribution in the lung. PMID:26429295

  18. 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…

  19. 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

  20. Telomerase Contributes to Fludarabine Resistance in Primary Human Leukemic Lymphocytes

    PubMed Central

    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y.; Gryaznov, Sergei M.; Johnston, James B.; Lees-Miller, Susan P.; Assouline, Sarit E.; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells. PMID:23922990

  1. Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

    PubMed

    Shawi, May; Chu, Tsz Wai; Martinez-Marignac, Veronica; Yu, Y; Gryaznov, Sergei M; Johnston, James B; Lees-Miller, Susan P; Assouline, Sarit E; Autexier, Chantal; Aloyz, Raquel

    2013-01-01

    We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells. PMID:23922990

  2. 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.

  3. Isolation of highly enriched primary human microglia for functional studies.

    PubMed

    Rustenhoven, Justin; Park, Thomas I-H; Schweder, Patrick; Scotter, John; Correia, Jason; Smith, Amy M; Gibbons, Hannah M; Oldfield, Robyn L; Bergin, Peter S; Mee, Edward W; Faull, Richard L M; Curtis, Maurice A; Scott Graham, E; Dragunow, Mike

    2016-01-01

    Microglia, the resident macrophages of the central nervous system play vital roles in brain homeostasis through clearance of pathogenic material. Microglia are also implicated in neurological disorders through uncontrolled activation and inflammatory responses. To date, the vast majority of microglial studies have been performed using rodent models. Human microglia differ from rodent counterparts in several aspects including their response to pharmacological substances and their inflammatory secretions. Such differences highlight the need for studies on primary adult human brain microglia and methods to isolate them are therefore required. Our procedure generates microglial cultures of >95% purity from both biopsy and autopsy human brain tissue using a very simple media-based culture procedure that takes advantage of the adherent properties of these cells. Microglia obtained in this manner can be utilised for research within a week. Isolated microglia demonstrate phagocytic ability and respond to inflammatory stimuli and their purity makes them suitable for numerous other forms of in vitro studies, including secretome and transcriptome analysis. Furthermore, this protocol allows for the simultaneous isolation of neural precursor cells during the microglial isolation procedure. As human brain tissue is such a precious and valuable resource the simultaneous isolation of multiple cell types is highly beneficial. PMID:26778406

  4. 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. PMID:27595140

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

    PubMed Central

    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. PMID:25749095

  6. 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

  7. 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

  8. 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.

  9. Helicobacter hepaticus Induces an Inflammatory Response in Primary Human Hepatocytes

    PubMed Central

    Kleine, Moritz; Worbs, Tim; Schrem, Harald; Vondran, Florian W. R.; Kaltenborn, Alexander; Klempnauer, Jürgen; Förster, Reinhold; Josenhans, Christine; Suerbaum, Sebastian; Bektas, Hüseyin

    2014-01-01

    Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytomety. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a

  10. 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

  11. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    PubMed

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  12. Indoleamine 2,3-dioxygenase depletes tryptophan, activates general control non-derepressible 2 kinase and down-regulates key enzymes involved in fatty acid synthesis in primary human CD4+ T cells.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2015-10-01

    Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and exerts immunosuppressive effects on CD4(+) T cells. One mechanism is through the inhibition of aerobic glycolysis. Another prerequisite for T-cell proliferation and differentiation into effector cells is increased fatty acid (FA) synthesis. The effect of IDO on enzymes involved in FA synthesis was evaluated in primary human cells both in mixed lymphocyte reactions in the presence or not of the IDO inhibitor 1-dl-methyl-tryptophan, and in stimulated CD4(+) T cells in the presence or not of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP). IDO or TRP inhibited cell proliferation. By assessing the level of GCN2 kinase or mammalian target of rapamycin complex 1 substrates along with a kynurenine free system we showed that IDO exerts its effect mainly through activation of GCN2 kinase. IDO or TRP down-regulated ATP-citrate lyase and acetyl coenzyme A carboxylase 1, key enzymes involved in FA synthesis. Also, IDO or TRP altered the expression of enzymes that control the availability of carbon atoms for FA synthesis, such as lactate dehydrogenase-A, pyruvate dehydrogenase, glutaminase 1 and glutaminase 2, in a way that inhibits FA synthesis. In conclusion, IDO through GCN2 kinase activation inhibits CD4(+) T-cell proliferation and down-regulates key enzymes that directly or indirectly promote FA synthesis, a prerequisite for CD4(+) T-cell proliferation and differentiation into effector cell lineages. PMID:26147366

  13. 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

  14. 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…

  15. 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.

  16. Serum-free primary human fibroblast and keratinocyte coculture.

    PubMed

    Mujaj, Sally; Manton, Kerry; Upton, Zee; Richards, Sean

    2010-04-01

    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins. PMID:19929322

  17. 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…

  18. 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

  19. Oncogenic Activities of Human Papillomaviruses

    PubMed Central

    McLaughlin-Drubin, Margaret E.; Münger, Karl

    2009-01-01

    Infectious etiologies for certain human cancers have long been suggested by epidemiological studies and studies with animals. Important support for this concept came from the discovery by Harald zur Hausen’s group that human cervical carcinoma almost universally contains certain “high-risk” human papillomavirus (HPV) types. Over the years, much has been learned about the carcinogenic activities of high-risk HPVs. These studies have revealed that two viral proteins, E6 and E7, that are consistently expressed in HPV-associated carcinomas, are necessary for induction and maintenance of the transformed phenotype. Hence, HPV-associated tumors are unique amongst human solid tumors in that they are universally caused by exposure to the same, molecularly defined oncogenic agents, and the molecular signal transduction pathways subverted by these viral transforming agents are frequently disrupted in other, non-virus associated human cancers. PMID:19540281

  20. 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

  1. On the pulpal nerve supply in primary human teeth: evidence for the innervation of primary dentine.

    PubMed

    Egan, C A; Hector, M P; Bishop, M A

    1999-03-01

    The presence of nerves in human tooth pulp has been recognized for over a hundred years, and the innervation of dentine for about 40 years. These observations have been made in permanent teeth. Very few studies have reported on the innervation of the primary pulp and dentine. The purpose of this study was to describe the innervation of the primary tooth pulp-dentine complex. Ten mature primary teeth (one incisor, six canines and three molars) were used. Immediately following extraction they were divided into three sections using a diamond disc and saline coolant. They were then immersion fixed in a solution of formaldehyde and picric acid dissolved in a phosphate buffer pH 7.4). The teeth were then demineralized for 1-3 weeks in formic acid. Following complete demineralization, 30 microns sections were cut on a freezing microtome. Neural tissue was stained using a specific antibody to calcitonin gene related peptide (CGRP). Sections were mounted on glass slides and examined using light microscopy. No individual nerve fibres were seen in the control sections, suggesting that the method used was specific for CGRP-containing nerve fibres. The primary teeth appeared to be well innervated. Myelinated and unmyelinated nerves were seen. There was a dense but variable subodontoblastic plexus of nerves (plexus of Raschkow) and nerve fibres were seen to leave this to travel towards the odontoblast layer. Most terminated here, but a few penetrated the odontoblast layer to enter predentine and the dentine tubules. The maximum penetration was 125 microns but most terminated within 30 microns of the dentinopulpal junction. The coronal region was more densely innervated than the root. Within the crown the cervical third was the most densely innervated region, followed by the pulp horn and the middle third. In conclusion, this study has demonstrated that mature primary tooth contains a pulp which is well innervated and has many nerve endings terminating in or near the odontoblast

  2. 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

  3. 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

  4. 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.

  5. Productive Lytic Replication of a Recombinant Kaposi's Sarcoma-Associated Herpesvirus in Efficient Primary Infection of Primary Human Endothelial Cells

    PubMed Central

    Gao, Shou-Jiang; Deng, Jian-Hong; Zhou, Fu-Chun

    2003-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS. PMID:12941882

  6. An adenoviral vector for probing promoter activity in primary immune cells

    PubMed Central

    Tripathi, Pulak; Madan, Rajat; Chougnet, Claire; Divanovic, Senad; Ma, Xiaojing; Wahl, Larry M.; Gajewski, Thomas; Karp, Christopher L.; Hildeman, David A.

    2010-01-01

    Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter–reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells. PMID:16563424

  7. 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

  8. Oregonin reduces lipid accumulation and proinflammatory responses in primary human macrophages.

    PubMed

    Lundqvist, Annika; Magnusson, Lisa U; Ullström, Christina; Krasilnikova, Jelena; Telysheva, Galina; Dizhbite, Tatjana; Hultén, Lillemor Mattsson

    2015-03-13

    Inflammation in the vascular wall is important for the development of atherosclerosis. We have previously shown that inflammatory macrophages are more abundant in human atherosclerotic lesions than in healthy arteries. Activated macrophages produce reactive oxygen species (ROS) that promote local inflammation in atherosclerotic lesions. Here, we investigated the role of oregonin, a diarylheptanoid, on proinflammatory responses in primary human macrophages and found that oregonin decreased cellular lipid accumulation and proinflammatory cytokine secretion. We also found that oregonin decreased ROS production in macrophages. Additionally, we observed that treatment of lipopolysaccharide-exposed macrophages with oregonin significantly induced the expression of antioxidant-related genes, including Heme oxygenase-1 and NADPH dehydrogenase quinone 1. In summary, we have shown that oregonin reduces lipid accumulation, inflammation and ROS production in primary human macrophages, indicating that oregonin has anti-inflammatory bioactivities. PMID:25686497

  9. Ferritin-mediated siRNA delivery and gene silencing in human tumor and primary cells.

    PubMed

    Li, Le; Muñoz-Culla, Maider; Carmona, Unai; Lopez, Maria Paz; Yang, Fan; Trigueros, Cesar; Otaegui, David; Zhang, Lianbing; Knez, Mato

    2016-08-01

    We demonstrate a straightforward method to encapsulate siRNA into naturally available and unmodified human apoferritin. The encapsulation into apoferritin is independent of the sequence of the siRNA and provides superior protection for those sensitive molecules. High efficiency in transfection can be achieved in human tumorigenic cells, human primary mesenchymal stem cells (hMSC) and peripheral blood mononuclear cells (PBMCs). In contrast to Lipofectamine, highly effective gene silencing can be achieved with ferritin as the delivery agent in both tumor cells and PBMCs at low siRNA concentrations (10 nM). As an endogenous delivery agent, apoferritin does not induce immune activation of T- and B-cells in human PBMCs. Apoferritin shows intrinsic anti-inflammatory effects and apoferritin-mediated delivery shows a preference for immune-activated T- and B-cells, a natural selectivity which may turn useful for drug delivery in case of infections or inflammatory diseases. PMID:27187278

  10. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    PubMed Central

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

  11. 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

  12. Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts.

    PubMed

    Le Bihan, Marie-Catherine; Barrio-Hernandez, Inigo; Mortensen, Tenna Pavia; Henningsen, Jeanette; Jensen, Søren Skov; Bigot, Anne; Blagoev, Blagoy; Butler-Browne, Gillian; Kratchmarova, Irina

    2015-08-01

    Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating

  13. 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.

  14. 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, ...

  15. Testing gene therapy vectors in human primary nasal epithelial cultures

    PubMed Central

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  16. Face representation in the human primary somatosensory cortex.

    PubMed

    Nguyen, Binh T; Tran, Tuan D; Hoshiyama, Minoru; Inui, Koji; Kakigi, Ryusuke

    2004-10-01

    To investigate the representation of facial skin areas in the primary somatosensory cortex (SI), we recorded magnetic fields evoked by air pressure-induced tactile stimulation applied to six points on the face, lower lip and thumb. The thumb area in the SI was located more medial and superior to the lip area, which was consistent with Penfield's homunculus. However, the representations of all skin-covered areas including forehead, cheek, nose and chin in the SI were located between the thumb and lower lip area. There was no significant difference in location among the six facial points. Our results imply that lips occupy a large area of the face representation in the SI, whereas only a small area located between the thumb and lip areas is devoted to skin-covered surfaces. This is the first study showing that the facial skin areas in the human SI are located between the thumb and lower lip areas and close together. PMID:15380330

  17. HIV binding, penetration, and primary infection in human cervicovaginal tissue

    PubMed Central

    Maher, Diane; Wu, Xiaoyun; Schacker, Timothy; Horbul, Julie; Southern, Peter

    2005-01-01

    We have developed human cervicovaginal organ culture systems to examine the initiating events in HIV transmission after exposure to various sources of HIV infectivity, including semen. Newly infected cells were detected in the cervical submucosa 3–4 days after exposure to a primary HIV isolate. At earlier times, extensive and stable binding occurred when cervical surfaces were exposed to virions or seminal cells. Cervical mucus provided some protection for the endocervical surface, by physically trapping virions and seminal cells. Confocal microscopy combined with 3D surface reconstruction revealed that virions could both bind to the external surface of the cervical epithelium and actually penetrate beneath the epithelial surface. In quantitative assays, pretreatment with a blocking antibody directed against β1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract. PMID:16061810

  18. Testing gene therapy vectors in human primary nasal epithelial cultures.

    PubMed

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  19. 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

  20. Quantitative Subcellular Proteome and Secretome Profiling of Influenza A Virus-Infected Human Primary Macrophages

    PubMed Central

    Lietzén, Niina; Julkunen, Ilkka; Aittokallio, Tero; Matikainen, Sampsa; Nyman, Tuula A.

    2011-01-01

    Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X7 receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages. PMID:21589892

  1. 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…

  2. Primary cilia and Gli3 activity regulate cerebral cortical size

    PubMed Central

    Wilson, Sandra L.; Wilson, John P.; Wang, Chengbing; Wang, Baolin; McConnell, Susan K.

    2012-01-01

    During neural development, patterning, neurogenesis and overall growth are highly regulated and coordinated between different brain regions. Here, we show that primary cilia and the regulation of Gli activity, are necessary for the normal expansion of the cerebral cortex. We show that loss of Kif3a, an important functional component of primary cilia, leads to the degeneration of primary cilia, marked overgrowth of the cortex, and altered cell cycle kinetics within cortical progenitors. The G1 phase of the cell cycle is shortened through a mechanism likely involving reduced Gli3 activity and a resulting increase in expression of cyclin D1 and Fgf15. The defects in Gli3 activity alone are sufficient to accelerate cell cycle kinetics and cause the molecular changes seen in brains that lack cilia. Finally, we show that levels of full-length and repressor Gli3 proteins are tightly regulated during normal development and correlate with changes in expression of two known Shh-target genes, CyclinD1 and Fgf15, and with the normal lengthening of the cell cycle during corticogenesis. These data suggest that Gli3 activity is regulated through the primary cilium to control cell cycle length in the cortex and thus determine cortical size. PMID:21976438

  3. 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…

  4. 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…

  5. Talking Points: Discussion Activities in the Primary Classroom

    ERIC Educational Resources Information Center

    Dawes, Lyn

    2011-01-01

    "Talking Points: Discussion Activities in the Primary Classroom" encourages and supports classroom discussion on a range of topics, enabling children to develop the important life-skill of effective group communication. Children who can explain their own ideas and take account of the points of view and reasons of others are in the process of…

  6. 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

  7. Effect of solid freeform fabrication-based polycaprolactone/poly(lactic-co-glycolic acid)/collagen scaffolds on cellular activities of human adipose-derived stem cells and rat primary hepatocytes.

    PubMed

    Shim, Jin-Hyung; Kim, Arthur Joon; Park, Ju Young; Yi, Namwoo; Kang, Inhye; Park, Jaesung; Rhie, Jong-Won; Cho, Dong-Woo

    2013-04-01

    Highly biocompatible polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA)/collagen scaffolds in which the PCL/PLGA collagen solution was selectively dispensed into every other space between the struts were fabricated using solid freeform fabrication (SFF) technology, as we described previously. The objective of this study was to evaluate and compare the PCL/PLGA/collagen scaffolds (group 3) with PCL/PLGA-only scaffolds (group 1) and PCL/PLGA scaffolds with collagen by the dip-coating method (group 2) using human adipose-derived stem cells (hASCs) and rat primary hepatocytes. The selectively dispensed collagen formed a three-dimensional (3D) network of nanofibers in group 3, as observed by scanning electron microscopy. The compressive strength and modulus of group 3 were approximately 140 and 510 times higher, respectively, than those of a sponge-type collagen scaffold whose weak mechanical properties were regarded as a critical drawback. Proliferation and osteogenic differentiation of hASCs were promoted significantly in group 3 compared to groups 1 and 2. In addition, we found that the viability and albumin secretion ability of rat primary hepatocytes were highly retained for 10 days in group 3 but not group 1. Interestingly, hepatocyte aggregation, which enhances hepatic function through cell-cell interactions, was observed particularly in group 3. In conclusion, group 3, in which the collagen was selectively dispensed in the 3D space of the porous PCL/PLGA framework, will be a promising 3D scaffold for culturing various cell types. PMID:23430333

  8. Histopathology of the pulp of primary molars with active and arrested dentinal caries.

    PubMed

    Di Nicolo, R; Guedes-Pinto, A C; Carvalho, Y R

    2000-01-01

    The purpose of this study was to compare the histological appearance of the pulp of human primary molars with active and arrested lesions. The sample consisted of 36 primary molars (18 with active lesions and 18 with arrested lesions) extracted from 35 children between 5 to 9 years of age. The histological diagnosis was classified in normal pulp, transitional stage, partial pulpitis, total pulpitis and total necrosis, and then subdivided in three subgroups: treatable, untreatable and questionable. Results showed that normal pulp or transitional stage (treatable category) was diagnosed in 50% of teeth with arrested lesions, compared to 11.1% of teeth with active lesions. Partial pulpitis (questionable category) was present in 38.8% with arrested lesions compared to 22.2% with active lesions. Total pulpitis and total necrosis (untreatable category) was diagnosed in 11.2% with arrested lesions compared to 66.7% with active lesions. The observed frequencies of histological categories between both groups were statistically significant (P < 0.05). Histologically, pulp reaction under active and arrested lesions in primary molars revealed the formation of a basophilic calcio-traumatic line at the junction of the primary and reparative dentin, formation of reparative dentin and a regular odontoblastic layer in 60% of the cases. Results indicated that the type of lesion (active or arrested) is a good indicator of the histological status of the pulp. PMID:11314352

  9. 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.

  10. Long non-coding RNA expression in primary human monocytes.

    PubMed

    Mirsafian, Hoda; Manda, Srinivas Srikanth; Mitchell, Christopher J; Sreenivasamurthy, Sreelakshmi; Ripen, Adiratna Mat; Mohamad, Saharuddin Bin; Merican, Amir Feisal; Pandey, Akhilesh

    2016-07-01

    Long non-coding RNAs (lncRNAs) have been shown to possess a wide range of functions in both cellular and developmental processes including cancers. Although some of the lncRNAs have been implicated in the regulation of the immune response, the exact function of the large majority of lncRNAs still remains unknown. In this study, we characterized the lncRNAs in human primary monocytes, an essential component of the innate immune system. We performed RNA sequencing of monocytes from four individuals and combined our data with eleven other publicly available datasets. Our analysis led to identification of ~8000 lncRNAs of which >1000 have not been previously reported in monocytes. PCR-based validation of a subset of the identified novel long intergenic noncoding RNAs (lincRNAs) revealed distinct expression patterns. Our study provides a landscape of lncRNAs in monocytes, which could facilitate future experimental studies to characterize the functions of these molecules in the innate immune system. PMID:26778813

  11. 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

  12. 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

  13. 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.

  14. The primary inhibitor of plasmin in human plasma.

    PubMed Central

    Müllertz, S; Clemmensen, I

    1976-01-01

    A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PMID:137718

  15. 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.

  16. 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

  17. 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. PMID:27003841

  18. Hypoxia promotes liver-stage malaria infection in primary human hepatocytes in vitro.

    PubMed

    Ng, Shengyong; March, Sandra; Galstian, Ani; Hanson, Kirsten; Carvalho, Tânia; Mota, Maria M; Bhatia, Sangeeta N

    2014-02-01

    Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver-stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that could influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells to model liver-stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver-stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver-stage malaria infection efficiencies might improve under hypoxia. Using the infection of micropatterned co-cultures with Plasmodium berghei, Plasmodium yoelii or Plasmodium falciparum as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters such as hepatocyte density and height of the medium, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments revealed that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt(II) chloride, as well as a HIF-1

  19. Endothelial Interleukin-6 defines the tumorigenic potential of primary human cancer stem cells

    PubMed Central

    Krishnamurthy, Sudha; Warner, Kristy A.; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B.; Helman, Joseph I.; Taichman, Russell S.; Bellile, Emily L.; McCauley, Laurie K.; Polverini, Peter J.; Prince, Mark E.; Wicha, Max S.; Nör, Jacques E.

    2014-01-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small sub-population of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of IL-6, we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  20. Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

    PubMed

    Krishnamurthy, Sudha; Warner, Kristy A; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B; Helman, Joseph I; Taichman, Russell S; Bellile, Emily L; McCauley, Laurie K; Polverini, Peter J; Prince, Mark E; Wicha, Max S; Nör, Jacques E

    2014-11-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  1. Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

    PubMed Central

    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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. Color and surface temperature variation during bleaching in human devitalized primary teeth: an in vitro study.

    PubMed

    Gontijo, Isa T; Navarro, Ricardo S; Ciamponi, Ana Lídia; Miyakawa, Walter; Zezell, Denise Maria

    2008-01-01

    This study's purpose was to make an in vitro assessment of 2 whitening techniques in primary teeth, regarding color and temperature surface variation, during dental bleaching using different catalytic sources. Twenty-one extracted human upper central deciduous incisors were used in this in vitro study. The teeth were darkened with human blood for a period of 21 days. After preparing the teeth, they were randomly distributed into 2 groups, according to bleaching source of activation: (1) a diode laser (DL) group; and (2) a halogen lamp (HL) group. The bleaching process was performed, according to the manufacturer's guidelines, using Whiteness HP (FGM, Joinville, Brazil). The color was assessed by spectrophotometer (CIELab) and the VITA scale (3M) before and immediately after tooth whitening. The temperature increase in the radicular surface during the bleaching was registered with a thermographic camera ThermaCAM SC 3000 (Flir Systems, Danderyd, Sweden) at the Nuclear and Energy Research Institute, IPEN-CNEN (São Paulo, Brazil). There was no significant difference between the groups in terms of color changes, but there was a statistically significant difference for temperature variation. The use of a diode laser and halogen lamp both promoted whitening in devitalized primary teeth in vitro. As a catalytic source of energy, the diode laser--with the applied parameters--promoted a smaller temperature increase compared to the halogen lamp during the bleaching procedure on nonvital primary teeth. PMID:19040807

  8. 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…

  9. 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

  10. 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

  11. CD133 marks a stem cell population that drives human primary myelofibrosis

    PubMed Central

    Triviai, Ioanna; Stübig, Thomas; Niebuhr, Birte; Hussein, Kais; Tsiftsoglou, Asterios; Fehse, Boris; Stocking, Carol; Kröger, Nicolaus

    2015-01-01

    Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133+ stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133+ but not CD34+CD133− cells, with a variable burden for JAK2V617F mutation, had multipotent cloning capacity in vitro. CD133+ cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F+ myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F+ megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133+ cells harboring EZH2D265H but lacking a secondary JAK2V617F mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms. PMID:25724578

  12. Primary human hepatocytes versus hepatic cell line: assessing their suitability for in vitro nanotoxicology.

    PubMed

    Kermanizadeh, Ali; Gaiser, Birgit K; Ward, Michael B; Stone, Vicki

    2013-11-01

    The use of hepatocyte cell lines as a replacement for animal models have been heavily criticised mainly due to low expression of metabolism enzymes. This study compares primary human hepatocytes with the C3A cell line and with respect to their response to a panel of nanomaterials (NMs; two ZnO, two MWCNTs, one Ag and one positively functionalised TiO₂). The cell line was very comparable with the primary hepatocytes with regards to their cytotoxic response to the NMs (Ag > uncoated ZnO > coated ZnO). The LC₅₀ was not attained in the presence of the MWCNTs and the TiO₂ NMs. All NMs significantly increased IL-8 production, with no change in levels of TNF-α and IL-6. Albumin production was measured as an indicator of hepatic function. The authors found no change in levels of albumin with the exception of the coated ZnO NM at the LC₅₀ concentration. NM uptake was similar for both the primary hepatocytes and C3A cells as investigated by TEM. Meanwhile, the authors confirmed greater levels of CYP450 activity in untreated primary cells. This study demonstrates that the C3A cell line is a good model for investigating NM-induced hepatocyte responses with respect to uptake, cytotoxicity, pro-inflammatory cytokine production and albumin production. PMID:23009365

  13. 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

  14. Identification and Validation of Novel Contraction-Regulated Myokines Released from Primary Human Skeletal Muscle Cells

    PubMed Central

    Raschke, Silja; Eckardt, Kristin; Bjørklund Holven, Kirsten; Jensen, Jørgen; Eckel, Jürgen

    2013-01-01

    Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines. PMID:23637948

  15. Upgrading physical activity counselling in primary care in the Netherlands.

    PubMed

    Verwey, Renée; van der Weegen, Sanne; Spreeuwenberg, Marieke; Tange, Huibert; van der Weijden, Trudy; de Witte, Luc

    2016-06-01

    The systematic development of a counselling protocol in primary care combined with a monitoring and feedback tool to support chronically ill patients to achieve a more active lifestyle. An iterative user-centred design method was used to develop a counselling protocol: the Self-management Support Programme (SSP). The needs and preferences of future users of this protocol were identified by analysing the literature, through qualitative research, and by consulting an expert panel. The counselling protocol is based on the Five A's model. Practice nurses apply motivational interviewing, risk communication and goal setting to support self-management of patients in planning how to achieve a more active lifestyle. The protocol consists of a limited number of behaviour change consultations intertwined with interaction with and responses from the It's LiFe! monitoring and feedback tool. This tool provides feedback on patients' physical activity levels via an app on their smartphone. A summary of these levels is automatically sent to the general practice so that practice nurses can respond to this information. A SSP to stimulate physical activity was defined based on user requirements of care providers and patients, followed by a review by a panel of experts. By following this user-centred approach, the organization of care was carefully taken into account, which has led to a practical and affordable protocol for physical activity counselling combined with mobile technology. PMID:25539787

  16. Regulation of follistatin-like protein 1 expression and secretion in primary human skeletal muscle cells.

    PubMed

    Görgens, Sven W; Raschke, Silja; Holven, Kirsten Bjørklund; Jensen, Jørgen; Eckardt, Kristin; Eckel, Jürgen

    2013-05-01

    Follistatin-like protein 1 (Fstl1) is a secreted glycoprotein of the follistatin family. Fstl1 is secreted by C2C12 cells, and Akt1 over-expression in skeletal muscle leads to its induction in muscle and increased circulating levels. So far, secretion of Fstl1 by human myotubes and the effect of exercise on its circulating levels have not been investigated. Here, we examined both the regulation of Fstl1 expression and secretion in primary human skeletal muscle cells and the effect of acute exercise on Fstl1 serum concentrations in humans. We show that human myotubes express and secrete Fstl1 in a differentiation-dependent manner. Furthermore, IFNγ and IL-1β significantly increase Fstl1 secretion. Electrical pulse stimulation (EPS)-induced contractile activity of myotubes did not regulate Fstl1. Interestingly, we observed that 60 min cycling increased serum Fstl1 level by 22%. In conclusion, we demonstrate that Fstl1 is expressed and secreted by human myotubes and plasma Fstl1 levels are increased after exercise. PMID:23419164

  17. Human Development Program: Level VI Activity Guide.

    ERIC Educational Resources Information Center

    Ball, Geraldine

    The curriculum guide presents the activities component of the Human Development Program for grade 6. The Human Development Program (HDP) is an affective curricular approach developed by psychologists to aid teachers in instilling responsibility and self-confidence in children. The nucleus of the Human Development Program is a circle session…

  18. 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

  19. 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

  20. [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

  1. [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

  2. 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.

  3. 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. PMID:26891694

  4. 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

  5. Neutron activation analysis: A primary method of measurement

    NASA Astrophysics Data System (ADS)

    Greenberg, Robert R.; Bode, Peter; De Nadai Fernandes, Elisabete A.

    2011-03-01

    Neutron activation analysis (NAA), based on the comparator method, has the potential to fulfill the requirements of a primary ratio method as defined in 1998 by the Comité Consultatif pour la Quantité de Matière — Métrologie en Chimie (CCQM, Consultative Committee on Amount of Substance — Metrology in Chemistry). This thesis is evidenced in this paper in three chapters by: demonstration that the method is fully physically and chemically understood; that a measurement equation can be written down in which the values of all parameters have dimensions in SI units and thus having the potential for metrological traceability to these units; that all contributions to uncertainty of measurement can be quantitatively evaluated, underpinning the metrological traceability; and that the performance of NAA in CCQM key-comparisons of trace elements in complex matrices between 2000 and 2007 is similar to the performance of Isotope Dilution Mass Spectrometry (IDMS), which had been formerly designated by the CCQM as a primary ratio method.

  6. Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis

    PubMed Central

    Penolazzi, Letizia; Lampronti, Ilaria; Borgatti, Monica; Khan, Mahmud Tareq Hassan; Zennaro, Margherita; Piva, Roberta; Gambari, Roberto

    2008-01-01

    Background Osteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of Emblica officinalis fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts. Methods The effects of extracts from Emblica officinalis on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of Emblica officinalis extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments. Results Extracts of Emblica officinalis were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. Emblica officinalis increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that Emblica officinalis extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that Emblica officinalis extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene. Conclusion Induction of apoptosis of osteoclasts could be an important strategy both in

  7. NATIONAL HUMAN ACTIVITY PATTERN SURVEY (NHAPS): USE OF NATIONWIDE ACTIVITY DATA FOR HUMAN EXPOSURE ASSESSMENT

    EPA Science Inventory

    The National Human Activity Pattern Survey (NHAPS) was initiated to fill a need for updated activity information on a nationwide scale. Several recent exposure field monitoring studies have shown that human activities play a critical role in explaining the variation in human expo...

  8. 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

  9. Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.

    PubMed

    Liu, Jia; Gaj, Thomas; Yang, Yifeng; Wang, Nan; Shui, Sailan; Kim, Sojung; Kanchiswamy, Chidananda Nagamangala; Kim, Jin-Soo; Barbas, Carlos F

    2015-11-01

    Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d. PMID:26492140

  10. Biotransformation of deramciclane in primary hepatocytes of rat, mouse, rabbit, dog, and human.

    PubMed

    Monostory, Katalin; Kohalmy, Krisztina; Ludányi, Krisztina; Czira, Gábor; Holly, Sándor; Vereczkey, László; Urmös, Iván; Klebovich, Imre; Kóbori, László

    2005-11-01

    The metabolic fate of deramciclane [(1R,2S,4R)-(-)-2-phenyl-2-(2'-dimethylamino-ethoxy)-1,7,7-trimethyl-bicyclo[2.2.1]heptane], a new anxiolytic drug candidate, has been determined in rat, mouse, rabbit, dog, and human hepatocytes. Rat and rabbit cells were the most active, whereas the rate of metabolism was quite slow in human hepatocytes. During biotransformation, deramciclane underwent side chain modification and oxidation at several positions of the molecule. The side chain modification led to the formation of N-desmethyl deramciclane and phenylborneol. The oxidation of deramciclane resulted in several hydroxy-, carboxy-, and N-oxide derivatives. The hydroxylation took place at primary or secondary carbons of the camphor ring as well as at the side chain; furthermore, dihydroxylated derivatives were also found. The side chain-modified metabolites were also oxidized to hydroxy- or carboxy-derivatives. Conjugation of phase I metabolites, as a route of elimination, was also observed in rat, rabbit, and dog hepatocytes. Although there were some species differences in biotransformation of deramciclane, it was concluded that phase I metabolism in human liver cells seemed to be similar to the metabolism in the hepatocytes isolated from rat. With careful approach, the rat model may be considered to be predictive for human metabolism of deramciclane. PMID:16118331

  11. Adherence of Helicobacter pylori to primary human gastrointestinal cells.

    PubMed Central

    Clyne, M; Drumm, B

    1993-01-01

    Helicobacter pylori adheres only to gastric cells in vivo. However, the organism adheres to a wide variety of nongastric cells in vitro. In this study, we have used flow cytometry to assess the adherence of H. pylori to primary epithelial cells isolated from gastric, duodenal, and colonic biopsy specimens by collagenase digestion. After incubation of bacteria and cells together and subsequent staining with a two-stage fluorescein isothiocyanate-labelled H. pylori antibody method, cells with adherent bacteria could be easily distinguished from cells without bacteria. Binding to Kato III cells (a gastric adenocarcinoma cell line) was saturable when bacteria and cells were mixed at a ratio of 250:1. Adherence to cells isolated from gastric biopsy specimens was significantly better than adherence to cells isolated from duodenal or colonic biopsy specimens. Almost 70% of gastric cells had bacteria bound, in contrast to 30% of duodenal cells and 32% of colonic cells (P < 0.0001). There was no correlation between expression of hemagglutinins by the bacteria and ability to bind to either Kato III cells or primary epithelial cells isolated from gastric biopsy specimens. In view of the strict tropism that the organism exhibits in vivo for gastric cells, the results of this study indicate that primary cells are ideal for assessing the factors that might play a role in the pathogenesis of disease caused by the organism. Images PMID:8406792

  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. 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

  14. 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

  15. 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

  16. Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

    PubMed

    Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Al-Eryani, Laila; Prough, Russell A; States, J Christopher; Coslo, Denise M; Omiecinski, Curtis J; Cave, Matthew C

    2014-08-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  17. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    PubMed Central

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  18. Zeranol stimulates proliferation and aromatase activation in human breast preadipocytes.

    PubMed

    Zhong, Saiyi; Liu, Shouchun; Chen, Suhua; Lin, Huajuan; Wang, Weimin; Qin, Xiaoming

    2016-07-01

    Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a non‑steroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an in vitro model. The effects of zeranol on cell proliferation were measured using the MTS assay, aromatase expression levels were determined by immunocytochemical staining and reverse transcription‑polymerase chain reaction, and aromatase enzyme activity and estrogen production were analyzed using corresponding assay kits. The results demonstrated that low dose zeranol (2‑50 nM) was able to significantly promote cell proliferation, aromatase mRNA expression, aromatase activity and estrogen production in primary cultured human breast preadipocytes, thus suggesting that zeranol may act as an aromatase activator. The findings of the present study suggest that zeranol promotes breast cancer cell growth by stimulating aromatase activation and increasing estrogen biosynthesis in adipose tissue. PMID:27220457

  19. 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

  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. 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

  4. Neuroprotective effects of rosmarinic acid on ciguatoxin in primary human neurons.

    PubMed

    Braidy, N; Matin, A; Rossi, F; Chinain, M; Laurent, D; Guillemin, G J

    2014-02-01

    Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200 nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits. PMID:24097334

  5. Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages*

    PubMed Central

    Karunakaran, Denuja; Kockx, Maaike; Owen, Dylan M.; Burnett, John R.; Jessup, Wendy; Kritharides, Leonard

    2013-01-01

    Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages. PMID:23288845

  6. 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. PMID:25954072

  7. 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

  8. Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

    PubMed

    Sunthamala, Nuchsupha; Pang, Chai Ling; Thierry, Francoise; Teissier, Sebastien; Pientong, Chamsai; Ekalaksananan, Tipaya

    2014-12-01

    The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively. PMID:26484085

  9. 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…

  10. Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

    PubMed Central

    Byrd, Daniel; Amet, Tohti; Hu, Ningjie; Lan, Jie; Hu, Sishun

    2013-01-01

    Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs. PMID:23785200

  11. Oncogenic RAS-induced senescence in human primary thyrocytes: molecular effectors and inflammatory secretome involved

    PubMed Central

    Vizioli, Maria Grazia; Santos, Joana; Pilotti, Silvana; Mazzoni, Mara; Anania, Maria Chiara; Miranda, Claudia; Pagliardini, Sonia; Pierotti, Marco A.

    2014-01-01

    Oncogene-induced senescence (OIS) is a robust and sustained antiproliferative response to oncogenic stress and constitutes an efficient barrier to tumour progression. We have recently proposed that OIS may be involved in the pathogenesis of thyroid carcinoma by restraining tumour progression as well as the transition of well differentiated to more aggressive variants. Here, an OIS inducible model was established and used for dissecting the molecular mechanisms and players regulating senescence in human primary thyrocytes. We show that oncogenic RAS induces senescence in thyrocytes as judged by changes in cell morphology, activation of p16INK4a and p53/p21CIP1 tumour suppressor pathways, senescence-associated β-galactosidase (SA-β-Gal) activity, and induction of proinflammatory components including IL-8 and its receptor CXCR2. Using RNA interference (RNAi) we demonstrate that p16INK4a is necessary for the onset of senescence in primary thyrocytes as its depletion rescues RAS-induced senescence. Furthermore, we found that IL-8/CXCR2 network reinforces the growth arrest triggered by oncogenic RAS, as its abrogation is enough to resume proliferation. Importantly, we observed that CXCR2 expression coexists with OIS markers in thyroid tumour samples, suggesting that CXCR2 contributes to senescence, thus limiting thyroid tumour progression. PMID:25268744

  12. Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta.

    PubMed Central

    Takahashi, N; Takahashi, Y; Putnam, F W

    1986-01-01

    We have determined the primary structure of human placental factor XIIIa, an enzyme [fibrinoligase, transglutaminase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine gamma-glutamyltransferase)] that forms intermolecular isopeptide bonds between fibrin molecules as the last step in blood coagulation. Placental factor XIIIa is an unglycosylated polypeptide chain of 730 amino acid residues (Mr = 83,005) that appears to be identical to the a subunit of the plasma zymogen factor XIII. Ca2+-dependent activation of factor XIIIa by thrombin removes a blocked amino-terminal peptide and unmasks a reactive thiol group at Cys-314. A second specific cleavage after Lys-513 by thrombin inactivates factor XIIIa and produces an amino-terminal 56-kDa fragment and a 24-kDa fragment. The amino acid sequence of factor XIIIa is unique and does not exhibit internal homology, but its active center is similar to that of the thiol proteases. The probable Ca2+-binding site of factor XIIIa has been identified by homology to the high-affinity sites in calmodulins. Knowledge of the primary structure of factor XIIIa will aid elucidation of the mechanism of its enzymatic action and that of the many tissue transglutaminases of which it is the prototype. This will also facilitate production of factor XIIIa by recombinant DNA technology for use in treatment of congenital factor XIII deficiencies and in the postoperative healing of wounds. Images PMID:2877456

  13. 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

  14. Touch activates human auditory cortex.

    PubMed

    Schürmann, Martin; Caetano, Gina; Hlushchuk, Yevhen; Jousmäki, Veikko; Hari, Riitta

    2006-05-01

    Vibrotactile stimuli can facilitate hearing, both in hearing-impaired and in normally hearing people. Accordingly, the sounds of hands exploring a surface contribute to the explorer's haptic percepts. As a possible brain basis of such phenomena, functional brain imaging has identified activations specific to audiotactile interaction in secondary somatosensory cortex, auditory belt area, and posterior parietal cortex, depending on the quality and relative salience of the stimuli. We studied 13 subjects with non-invasive functional magnetic resonance imaging (fMRI) to search for auditory brain areas that would be activated by touch. Vibration bursts of 200 Hz were delivered to the subjects' fingers and palm and tactile pressure pulses to their fingertips. Noise bursts served to identify auditory cortex. Vibrotactile-auditory co-activation, addressed with minimal smoothing to obtain a conservative estimate, was found in an 85-mm3 region in the posterior auditory belt area. This co-activation could be related to facilitated hearing at the behavioral level, reflecting the analysis of sound-like temporal patterns in vibration. However, even tactile pulses (without any vibration) activated parts of the posterior auditory belt area, which therefore might subserve processing of audiotactile events that arise during dynamic contact between hands and environment. PMID:16488157

  15. 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

  16. 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

  17. 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

  18. Human Development Program: Level III Activity Guide.

    ERIC Educational Resources Information Center

    Bessell, Harold

    The curriculum guide presents the activities component of the Human Development Program for the third grade. The Human Development Program (HDP) is an affective curricular approach developed by psychologists to help teachers instill responsibility and self-confidence in children. Following a brief overview of the HDP and explanation of the Magic…

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

    PubMed Central

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

    2016-01-01

    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

  20. 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

  1. 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

  2. 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

  3. Brincidofovir (CMX001) Inhibits BK Polyomavirus Replication in Primary Human Urothelial Cells

    PubMed Central

    Tylden, Garth D.; Hirsch, Hans H.

    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. PMID:25801568

  4. 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

  5. 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.

  6. 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

  7. Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation.

    PubMed

    Bufi, Nathalie; Saitakis, Michael; Dogniaux, Stéphanie; Buschinger, Oscar; Bohineust, Armelle; Richert, Alain; Maurin, Mathieu; Hivroz, Claire; Asnacios, Atef

    2015-05-01

    T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/-80 Pa), dendritic cells (DCs, 440 +110/-90 Pa), and macrophages (MPHs, 900 +110/-100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young's modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation. PMID:25954876

  8. Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation

    PubMed Central

    Bufi, Nathalie; Saitakis, Michael; Dogniaux, Stéphanie; Buschinger, Oscar; Bohineust, Armelle; Richert, Alain; Maurin, Mathieu; Hivroz, Claire; Asnacios, Atef

    2015-01-01

    T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/−80 Pa), dendritic cells (DCs, 440 +110/−90 Pa), and macrophages (MPHs, 900 +110/−100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young’s modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation. PMID:25954876

  9. Recurrent epimutations activate gene body promoters in primary glioblastoma.

    PubMed

    Nagarajan, Raman P; Zhang, Bo; Bell, Robert J A; Johnson, Brett E; Olshen, Adam B; Sundaram, Vasavi; Li, Daofeng; Graham, Ashley E; Diaz, Aaron; Fouse, Shaun D; Smirnov, Ivan; Song, Jun; Paris, Pamela L; Wang, Ting; Costello, Joseph F

    2014-05-01

    Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein. PMID:24709822

  10. Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

    PubMed Central

    Foster, John L.; Molina, Rene P.; Luo, Tianci; Arora, Vivek K.; Huang, Yaoxing; Ho, David D.; Garcia, J. Victor

    2001-01-01

    We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes. PMID:11160665

  11. 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. PMID:21637921

  12. Astaxanthin prevents and reverses the activation of mouse primary hepatic stellate cells.

    PubMed

    Yang, Yue; Bae, Minkyung; Kim, Bohkyung; Park, Young-Ki; Koo, Sung I; Lee, Ji-Young

    2016-03-01

    Activation of hepatic stellate cells (HSCs) is a critical step that leads to the development of liver fibrosis. We showed that astaxanthin (ASTX), a xanthophyll carotenoid, displays antifibrogenic effects in LX-2 cells, a human HSC cell line. In this study, we further determined the effect of ASTX on HSC activation and inactivation using primary HSCs from C57BL/6J mice. Quiescent and activated HSCs were incubated with ASTX (25μM) at different stages of activation. ASTX prevented the activation of quiescent HSCs, as evidenced by the presence of intracellular lipid droplets and reduction of α-smooth muscle actin, an HSC activation marker. Also, ASTX reverted activated HSCs to a quiescent phenotype with the reappearance of lipid droplets with a concomitant increase in lecithin retinol acyltransferase mRNA. Cellular accumulation of reactive oxygen species was significantly reduced by ASTX, which was attributable to a decrease in NADPH oxidase 2 expression. The antifibrogenic effect of ASTX was independent of nuclear erythroid 2-related factor 2 as it was observed in HSCs from wild-type and Nrf2(-/-) mice. In conclusion, ASTX inhibits HSC activation and reverts activated HSCs to a quiescent state. Further investigation is warranted to determine if ASTX effectively prevents the development of liver fibrosis. PMID:26895661

  13. Profiling bioactivity of the ToxCast chemical library using BioMAP primary human cell systems.

    PubMed

    Houck, Keith A; Dix, David J; Judson, Richard S; Kavlock, Robert J; Yang, Jian; Berg, Ellen L

    2009-10-01

    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, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency's (EPA's) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFkappaB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA. PMID:19773588

  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. Pericellular oxygen concentration of cultured primary human trophoblasts

    PubMed Central

    Chen, Baosheng; Longtine, Mark S.; Nelson, D. Michael

    2012-01-01

    Introduction Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. Methods We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. Results The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤ 0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. Conclusions We conclude that culture conditions of 21% oxygen are unable to replicate the pO2 of 40–60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions. PMID:23211472

  16. 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

  17. Molecular genetics of human primary microcephaly: an overview

    PubMed Central

    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

  18. 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

  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. 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…

  1. 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

  2. Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro: an initial characterization

    PubMed Central

    2013-01-01

    Background A good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney. Results We carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics. Conclusions On the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells. PMID:24308307

  3. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

    PubMed

    March, Sandra; Ramanan, Vyas; Trehan, Kartik; Ng, Shengyong; Galstian, Ani; Gural, Nil; Scull, Margaret A; Shlomai, Amir; Mota, Maria M; Fleming, Heather E; Khetani, Salman R; Rice, Charles M; Bhatia, Sangeeta N

    2015-12-01

    The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development. PMID:26584444

  4. Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers.

    PubMed

    Iizuka, Masayoshi; Takahashi, Yoshihisa; Mizzen, Craig A; Cook, Richard G; Fujita, Masatoshi; Allis, C David; Frierson, Henry F; Fukusato, Toshio; Smith, M Mitchell

    2009-05-01

    In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder. PMID:19393168

  5. 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

  6. HIV-1 increases TLR responses in human primary astrocytes

    PubMed Central

    Serramía, M Jesús; Muñoz-Fernández, M Ángeles; Álvarez, Susana

    2015-01-01

    Astrocytes are the major glial cell within the central nervous system and have a number of important physiological properties related to brain homeostasis. They provide trophic support to neurons and are immune cells with key roles during states-of-inflammation. The potential for production of proinflammatory cytokines and its consequences has been studied in the context of HIV-1 infection of normal human astrocytes (NHA). NHA express TLR3, TLR4, and TLR5. TLR3 ligation induced the strongest proinflammatory polarizing response, characterized by generation of high levels of TNF-α, IL-6, and IL-8. HIV-1 increased the transient production of key inflammatory mediators, and exposure to LPS of HIV-1-infected cells increased significantly the cytokine secretion. We confirmed that it is necessary viral gene expression from the moment of pretreatment with antiretrovirals inhibited totally HIV-1-induced TLR response. The higher response to LPS from HIV-1-infected cells did not correlate with TLR4 or MyD88 increased expression. LPS responsiveness of infected cells parallels MHC class II expression, but not CD14. HIV-1-infected NHA present increased sensitivity to the proinflammatory effects of LPS. If this phenomenon occurs in vivo, it will contribute to the immunopathogenesis of this disease and may ultimately offer novel targets for immunomodulatory therapy. PMID:26671458

  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. 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

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

    PubMed

    Stefanon, Bruno; Pomari, Elena; Colitti, Monica

    2015-07-01

    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. Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

    PubMed Central

    Gao, Dan; Madi, Mohamed; Ding, Cherlyn; Fok, Matthew; Steele, Thomas; Ford, Christopher; Hunter, Leif

    2014-01-01

    Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue. PMID:24918199

  11. 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

  12. 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

  13. 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

  14. 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

  15. Understanding human dynamics in microblog posting activities

    NASA Astrophysics Data System (ADS)

    Jiang, Zhihong; Zhang, Yubao; Wang, Hui; Li, Pei

    2013-02-01

    Human activity patterns are an important issue in behavior dynamics research. Empirical evidence indicates that human activity patterns can be characterized by a heavy-tailed inter-event time distribution. However, most researchers give an understanding by only modeling the power-law feature of the inter-event time distribution, and those overlooked non-power-law features are likely to be nontrivial. In this work, we propose a behavior dynamics model, called the finite memory model, in which humans adaptively change their activity rates based on a finite memory of recent activities, which is driven by inherent individual interest. Theoretical analysis shows a finite memory model can properly explain various heavy-tailed inter-event time distributions, including a regular power law and some non-power-law deviations. To validate the model, we carry out an empirical study based on microblogging activity from thousands of microbloggers in the Celebrity Hall of the Sina microblog. The results show further that the model is reasonably effective. We conclude that finite memory is an effective dynamics element to describe the heavy-tailed human activity pattern.

  16. Physical environment virtualization for human activities recognition

    NASA Astrophysics Data System (ADS)

    Poshtkar, Azin; Elangovan, Vinayak; Shirkhodaie, Amir; Chan, Alex; Hu, Shuowen

    2015-05-01

    Human activity recognition research relies heavily on extensive datasets to verify and validate performance of activity recognition algorithms. However, obtaining real datasets are expensive and highly time consuming. A physics-based virtual simulation can accelerate the development of context based human activity recognition algorithms and techniques by generating relevant training and testing videos simulating diverse operational scenarios. In this paper, we discuss in detail the requisite capabilities of a virtual environment to aid as a test bed for evaluating and enhancing activity recognition algorithms. To demonstrate the numerous advantages of virtual environment development, a newly developed virtual environment simulation modeling (VESM) environment is presented here to generate calibrated multisource imagery datasets suitable for development and testing of recognition algorithms for context-based human activities. The VESM environment serves as a versatile test bed to generate a vast amount of realistic data for training and testing of sensor processing algorithms. To demonstrate the effectiveness of VESM environment, we present various simulated scenarios and processed results to infer proper semantic annotations from the high fidelity imagery data for human-vehicle activity recognition under different operational contexts.

  17. 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

  18. The Bile Acid Chenodeoxycholic Acid Increases Human Brown Adipose Tissue Activity.

    PubMed

    Broeders, Evie P M; Nascimento, Emmani B M; Havekes, Bas; Brans, Boudewijn; Roumans, Kay H M; Tailleux, Anne; Schaart, Gert; Kouach, Mostafa; Charton, Julie; Deprez, Benoit; Bouvy, Nicole D; Mottaghy, Felix; Staels, Bart; van Marken Lichtenbelt, Wouter D; Schrauwen, Patrick

    2015-09-01

    The interest in brown adipose tissue (BAT) as a target to combat metabolic disease has recently been renewed with the discovery of functional BAT in humans. In rodents, BAT can be activated by bile acids, which activate type 2 iodothyronine deiodinase (D2) in BAT via the G-coupled protein receptor TGR5, resulting in increased oxygen consumption and energy expenditure. Here we examined the effects of oral supplementation of the bile acid chenodeoxycholic acid (CDCA) on human BAT activity. Treatment of 12 healthy female subjects with CDCA for 2 days resulted in increased BAT activity. Whole-body energy expenditure was also increased upon CDCA treatment. In vitro treatment of primary human brown adipocytes derived with CDCA or specific TGR5 agonists increased mitochondrial uncoupling and D2 expression, an effect that was absent in human primary white adipocytes. These findings identify bile acids as a target to activate BAT in humans. PMID:26235421

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

    PubMed

    Chou, Yu-Chi; Chen, Ruei-Lin; Lai, Zheng-Sheng; Song, Jen-Shin; Chao, Yu-Sheng; Shen, Che-Kun James

    2015-07-01

    Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias. PMID:25986606

  8. 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

  9. VHZ is a novel centrosomal phosphatase associated with cell growth and human primary cancers

    PubMed Central

    2010-01-01

    Background VHZ is a VH1-like (member Z) dual specific protein phosphatase encoded by DUSP23 gene. Some of the dual specific protein phosphatases (DSPs) play an important role in cell cycle control and have shown to be associated with carcinogenesis. Here, the expression of VHZ associated with cell growth and human cancers was investigated. Results We generated a mouse monoclonal antibody (mAb clone#209) and rabbit polyclonal antibodies (rAb) against VHZ. We performed cell proliferation assay to learn how VHZ is associated with cell cycle by retroviral transduction to express VHZ, VHZ(C95S), and control vector in MCF-7 cells. Overexpression of VHZ [but not VHZ(C95S)] in MCF-7 cells promoted cell proliferation compared to control cells. shRNA-mediated knockdown of VHZ in MCF-7 cells showed that reduction of VHZ resulted in increased G1 but decreased S phase cell populations. Using indirect immunofluorescence, we showed that both exogenous and endogenous VHZ protein was localized at the centrosome in addition to its cytoplasmic distribution. Furthermore, using immunohistochemistry, we revealed that VHZ protein was overexpressed either in enlarged centrosomes (VHZ-centrosomal-stain) of some invasive ductal carcinomas (IDC) Stage I (8/65 cases) or in entire cytoplasm (VHZ-cytosol-stain) of invasive epithelia of some IDC Stage II/III (11/47 cases) of breast cancers examined. More importantly, upregulation of VHZ protein is also associated with numerous types of human cancer, in particular breast cancer. VHZ mAb may be useful as a reagent in clinical diagnosis for assessing VHZ positive tumors. Conclusions We generated a VHZ-specific mAb to reveal that VHZ has a novel subcellular localization, namely the centrosome. VHZ is able to facilitate G1/S cell cycle transition in a PTP activity-dependent manner. The upregulation of its protein levels in primary human cancers supports the clinical relevance of the protein in cancers. PMID:20509867

  10. 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.

  11. LINE-1 Retrotransposition Activity in Human Genomes

    PubMed Central

    Beck, Christine R.; Collier, Pamela; Macfarlane, Catriona; Malig, Maika; Kidd, Jeffrey M.; Eichler, Evan E.; Badge, Richard M.; Moran, John V.

    2010-01-01

    Summary Long Interspersed Element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of highly active or ‘hot’ L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s which are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that ‘hot’ L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of inter-individual genetic variation. PMID:20602998

  12. 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.

  13. Children's Economic Activities and Primary School Attendance in Rural Guatemala.

    ERIC Educational Resources Information Center

    Clark, Carol A. M.

    To investigate whether low school attendance rates in Guatemala (about 35% of primary school aged children do not attend) are due primarily to the need for children in low income families to contribute to family income or child care and other housekeeping tasks, time use data were collected in 4 rural villages from mothers of 369 children, aged…

  14. Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages

    PubMed Central

    Miettinen, Juho J.; Matikainen, Sampsa

    2012-01-01

    Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense. PMID:22973042

  15. Transcriptomic Analysis of Human Primary Bronchial Epithelial Cells after Chloropicrin Treatment.

    PubMed

    Pesonen, Maija; Storvik, Markus; Kokkola, Tarja; Rysä, Jaana; Vähäkangas, Kirsi; Pasanen, Markku

    2015-10-19

    Chloropicrin is a vaporizing toxic irritant that poses a risk to human health if inhaled, but the mechanism of its toxicity in the respiratory tract is poorly understood. Here, we exposed human primary bronchial epithelial cells (HBEpC) to two concentrations of chloropicrin (10-50 μM) for 6 or 48 h and used genomic microarray, flow cytometry, and TEM-analysis to monitor cellular responses to the exposures. The overall number of differentially expressed transcripts with a fold-change > ± 2 compared to controls increased with longer exposure times. The initial response was activation of genes with a higher number of up- (512 by 10 μM and 408 by 40 μM chloropicrin) rather than down-regulated transcripts (40 by 10 μM and 215 by 40 μM chloropicrin) at 6 h seen with both exposure concentrations. The number of down-regulated transcripts, however, increased with the exposure time. The differentially regulated transcripts were further examined for enriched Gene Ontology Terms (GO) and KEGG-pathways. According to this analysis, the "ribosome" and "oxidative phosphorylation" were the KEGG-pathways predominantly affected by the exposure. The predominantly affected (GO) biological processes were "protein metabolic process" including "translation," "cellular protein complex assembly," and "response to unfolded protein." Furthermore, the top pathways, "NRF2-activated oxidative stress" and "Ah-receptor signaling," were enriched in our data sets by IPA-analysis. Real time qPCR assay of six selected genes agreed with the microarray analysis. In addition, chloropicrin exposure increased the numbers of late S and/or G2/M-phase cells as analyzed by flow cytometry and induced autophagy as revealed by electron microscopy. The targets identified are critical for vital cellular functions reflecting acute toxic responses and are potential causes for the reduced viability of epithelial cells after chloropicrin exposure. PMID:26352163

  16. 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...

  17. 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...

  18. CONSOLIDATED HUMAN ACTIVITY DATABASE (CHAD) WEBSITE

    EPA Science Inventory

    The Consolidated Human Activity Database (CHAD) has been developed by ManTech Environmental for the Environmental Protection Agency's National Exposure Research Laboratory (NERL). This database was created to support exposure/intake dose/risk assessments. The overall design incor...

  19. 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…

  20. 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

  1. 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…

  2. NET amyloidogenic backbone in human activated neutrophils.

    PubMed

    Pulze, L; Bassani, B; Gini, E; D'Antona, P; Grimaldi, A; Luini, A; Marino, F; Noonan, D M; Tettamanti, G; Valvassori, R; de Eguileor, M

    2016-03-01

    Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role. PMID:26462606

  3. Active control of a 30 m ring interferometric telescope primary mirror.

    PubMed

    Dai, Yichun; Liu, Zhong; Jin, Zhenyu; Xu, Jun; Lin, Jing

    2009-02-01

    The active control of a primary mirror for a ring aperture segmented telescope is different from that of a full aperture segmented telescope. Two active maintenance proposals for the ring telescope designed from the segmented patterns are outlined. We present a preliminary calculation of noise propagation and analysis of primary mirror mode characteristics for each proposal. The modulation transfer functions (MTFs) of the primary mirror corresponding to each maintenance method at several typical working wavelengths are also simulated. PMID:19183591

  4. 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.

  5. Physical Education Activities Book for the Primary Grades.

    ERIC Educational Resources Information Center

    Green, Arthur S.

    The guide is divided into 10 chapters, the first on general program organization, the rest on specific activities. The nine activities are as follows: posture; calisthenics; active recreational games; stunts, tumbling, and apparatus skills; track and field events; team sports; fundamental and creative or interpretive rhythms; dramatized rhythms;…

  6. 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.

  7. 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

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

    PubMed Central

    González-Gómez, P.; de la Fuente, M.; Hernández-Laín, Aurelio; Mira, H.; Sánchez-Gómez, P.; Garcia-Fuentes, M.

    2015-01-01

    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

  9. Utilisation of joint movement range in arboreal primates compared with human subjects: an evolutionary frame for primary osteoarthritis.

    PubMed Central

    Alexander, C J

    1994-01-01

    OBJECTIVE--To determine whether an arboreal lifestyle required full use of movement ranges underutilised in nine joint groups in humans, because under-utilisation of available movement range may be associated with susceptibility to primary osteoarthritis. METHODS--Utilisation of the nine joint groups was studied in two species of primate exercising in a simulated arboreal environment, using 'focal animal' observation techniques supplemented by telephoto photography and by review of archival material from other sources. Fifteen apes were observed over a total observation period of 20.2 man-hours and 152 films were analysed for utilisation of movement range. RESULTS--With one exception, all the movement ranges reported to be under-utilised in humans were fully utilised by the apes in climbing activities. The exception, metacarpophalangeal extension, was an essential component of the chimpanzee ground progression mode of knuckle walking. CONCLUSIONS--The underused movement range in several human joints is explicable as residual capacity from a semiarboreal lifestyle. If the correlation with primary osteoarthritis is confirmed, it suggests that the disease may reflect a disparity between inherited capacity and current need. The significance of the result lies in its implication that primary osteoarthritis may be preventable. Images PMID:7826133

  10. Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

    PubMed Central

    Verdon, Daniel; Chen, Jennifer; Taylor, John A.; Dunbar, P. Rod

    2013-01-01

    The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7–78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6–12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes. PMID:23646140

  11. 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

  12. 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.

  13. 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

  14. Concurrent Validity of a Self-Reported Physical Activity “Vital Sign” Questionnaire With Adult Primary Care Patients

    PubMed Central

    Joy, Elizabeth A.; Gren, Lisa H.; Shaw, Janet M.

    2016-01-01

    Introduction No tool currently used by primary health care providers to assess physical activity has been evaluated for its ability to determine whether or not patients achieve recommended levels of activity. The purpose of this study was to assess concurrent validity of physical activity self-reported to the brief (<30 sec) Physical Activity “Vital Sign” questionnaire (PAVS) compared with responses to the lengthier (3–5 min), validated Modifiable Activity Questionnaire (MAQ). Methods Agreement between activity reported to the PAVS and MAQ by primary care patients at 2 clinics in 2014 was assessed by using percentages and κ coefficients. Agreement consisted of meeting or not meeting the 2008 Aerobic Physical Activity Guidelines for Americans (PA Guidelines) of the US Department of Health and Human Services. We compared self-reported usual minutes per week of moderate-to-vigorous physical activity among patients at a primary care clinic in 2014 who reported to PAVS and to MAQ by using Pearson correlation and Bland–Altman plots of agreement. Results Among 269 consenting patients who reported physical activity, PAVS results agreed with those of MAQ 89.6% of the time and demonstrated good agreement in identifying patients who did not meet PA Guidelines recommendations (κ = 0.55, ρ = 0.57; P < .001). Usual minutes per week of moderate-to-vigorous physical activity reported to PAVS had a high positive correlation with the same reported to MAQ (r = 0.71; P < .001). Conclusion PAVS may be a valid tool for identifying primary care patients who need counseling about physical activity. PAVS should be assessed further for agreement with repeated objective measures of physical activity in the patient population. PMID:26851335

  15. Antiviral Effects of Artesunate on Polyomavirus BK Replication in Primary Human Kidney Cells

    PubMed Central

    Sharma, Biswa Nath; Marschall, Manfred; Henriksen, Stian

    2014-01-01

    Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 μM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 μM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 μM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2′-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 μM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions. PMID:24145549

  16. 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

  17. Cyclopalladated primary amines: a preliminary study of antiproliferative activity through apoptosis induction.

    PubMed

    Albert, Joan; Bosque, Ramon; Crespo, Margarita; García, Graciela; Granell, Jaume; López, Concepción; Lovelle, María Victoria; Qadir, Romana; González, Asensio; Jayaraman, Anusha; Mila, Enric; Cortés, Roldán; Quirante, Josefina; Calvis, Carme; Messeguer, Ramon; Badía, Josefa; Baldomà, Laura; Cascante, Marta

    2014-09-12

    Twelve cyclometallated palladium(II) complexes containing primary aromatic amines [benzylamine (a), (R)-1-(1-naphthyl)ethylamine (b) and 2-phenylaniline (c)] as anionic bidentate (C,N)(-) ligands have been evaluated against a panel of human adenocarcinoma cell lines (A549 lung, MDA-MB231 and MCF7 breast, and the cisplatin resistant HCT116 colon). The results revealed a remarkable antiproliferative activity of the triphenylphosphane mononuclear compounds 3-4 (series a, b, c) and the best inhibition was provided for 3c and 4c with the 2-phenylaniline ligand and a six membered chelate ring. Interestingly, 3c and 4c were 14 and 19 times more potent than cisplatin for the inhibition of the cisplatin resistant HCT116 human adenocarcinoma cell line, respectively. Cyclopalladated complexes 3c and 4c exercise their antiproliferative activity over A549 cells mainly through the induction of apoptosis (38 and 31-fold increase in early apoptotic cells, respectively). PMID:25063943

  18. 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

  19. 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

    2015-01-01

    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

  20. 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

  1. 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

  2. Blood Oxygen Level-Dependent Activation of the Primary Visual Cortex Predicts Size Adaptation Illusion

    PubMed Central

    Pooresmaeili, Arezoo; Arrighi, Roberto; Biagi, Laura; Morrone, Maria Concetta

    2016-01-01

    In natural scenes, objects rarely occur in isolation but appear within a spatiotemporal context. Here, we show that the perceived size of a stimulus is significantly affected by the context of the scene: brief previous presentation of larger or smaller adapting stimuli at the same region of space changes the perceived size of a test stimulus, with larger adapting stimuli causing the test to appear smaller than veridical and vice versa. In a human fMRI study, we measured the blood oxygen level-dependent activation (BOLD) responses of the primary visual cortex (V1) to the contours of large-diameter stimuli and found that activation closely matched the perceptual rather than the retinal stimulus size: the activated area of V1 increased or decreased, depending on the size of the preceding stimulus. A model based on local inhibitory V1 mechanisms simulated the inward or outward shifts of the stimulus contours and hence the perceptual effects. Our findings suggest that area V1 is actively involved in reshaping our perception to match the short-term statistics of the visual scene. PMID:24089504

  3. Differential susceptibility of primary cultured human skin cells to hypericin PDT in an in vitro model.

    PubMed

    Popovic, A; Wiggins, T; Davids, L M

    2015-08-01

    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well established that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. The aim of this study was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model which represented both the epidermal and dermal cellular compartments of human skin. Cell viability analysis revealed a differential cytotoxic response to a range of HYP-PDT doses in all the human skin cell types, showing that Fb (LD50=1.75μM) were the most susceptible to HYP-PDT, followed by Mc (LD50=3.5μM) and Kc (LD50>4μM HYP-PDT) These results correlated with the morphological analysis which displayed distinct morphological changes in Fb and Mc, 24h post treatment with non-lethal (1μM) and lethal (3μM) doses of HYP-PDT, but the highest HYP-PDT doses had no effect on Kc morphology. Fluorescent microscopy displayed cytoplasmic localization of HYP in all the 3 skin cell types and additionally, HYP was excluded from the nuclei in all the cell types. Intracellular ROS levels measured in Fb at 3μM HYP-PDT, displayed a significant 3.8 fold (p<0.05) increase in ROS, but no significant difference in ROS levels occurred in Mc or Kc. Furthermore, 64% (p<0.005) early apoptotic Fb and 20% (p<0.05) early apoptotic Mc were evident; using fluorescence activated cell sorting (FACS), 24

  4. 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

  5. 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

  6. Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinoline Analogues

    PubMed Central

    Shukla, Nikunj M.; Malladi, Subbalakshmi S.; Mutz, Cole A.; Balakrishna, Rajalakshmi; David, Sunil A.

    2010-01-01

    Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene sidechain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N1-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N1 and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC50 value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-α induction activities in whole human blood models. PMID:20481492

  7. 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).

  8. The ubiquitin ligase APC(Cdh1) is required to maintain genome integrity in primary human cells.

    PubMed

    Engelbert, D; Schnerch, D; Baumgarten, A; Wäsch, R

    2008-02-01

    Ensuring precise DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. Proteolytic control of cell cycle regulators by the anaphase-promoting complex, activated by Cdh1 (APC(Cdh1)), is responsible for a stable G1 phase after mitotic exit allowing accurate preparation for DNA replication in the following S phase. APC(Cdh1) target proteins are frequently upregulated in tumor cells and the inactivation of human Cdh1 might interfere with genome integrity by target stabilization. Here we show that APC(Cdh1) is required for maintaining genomic integrity in primary human cells. Lentiviral-delivered strong and stable suppression of Cdh1 by RNA interference (RNAi) causes aberrant accumulation of several APC(Cdh1) target proteins, such as cyclin A, B, Aurora A or Plk1, which control accurate and equal distribution of the genetic information to daughter cells. This induces a premature and prolonged S phase, mitotic-entry delay and defects in chromosome separation and cytokinesis. Cell cycle deregulation by stable knockdown of Cdh1 leads to activation of p53/p21 and genomic instability, which is further increased by codepletion of p53. Thus, stabilization of APC(Cdh1) targets may initiate aberrant DNA replication and chromosome separation, and trigger a p53 response by deregulating G1 in primary human cells. PMID:17700535

  9. The W. M. Keck Telescope segmented primary mirror active control system

    SciTech Connect

    Jared, R.C.; Arthur, A.A.; Andreae, S.; Biocca, A.; Cohen, R.W.; Fuertes, J.M.; Franck, J.; Gabor, G.; Llacer, J.; Mast, T.; Meng, J.; Merrick, T.; Minor, R.; Nelson, J.; Orayani, M.; Salz, P.; Schaefer, B.; Witebsky, C.

    1989-07-01

    The ten meter diameter primary mirror of the W. M. Keck Telescope is a mosaic of thirty-six hexagonal mirrors. An active control system stabilizes the primary mirror. The active control system uses 168 measurements of the relative positions of adjacent mirror segments and 3 measurements of the primary mirror position in the telescope structure to control the 108 degrees of freedom needed to stabilize the figure and position of the primary mirror. The components of the active control system are relative position sensors, electronics, computers, actuators that position the mirrors, and software. The software algorithms control the primary mirror, perform star image stacking, emulate the segments, store and fit calibration data, and locate hardware defects. We give an overview of the active control system, its functional requirements and test measurements. 12 refs.

  10. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

    PubMed

    Harvey, H A; Ketterer, M R; Preston, A; Lubaroff, D; Williams, R; Apicella, M A

    1997-06-01

    In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected. PMID:9169783

  11. 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

  12. Vascular status in human primary and permanent teeth in health and disease.

    PubMed

    Rodd, Helen D; Boissonade, Fiona M

    2005-04-01

    The present study sought to compare the vascular status of human primary teeth with that of human permanent teeth, and to determine whether caries or painful pulpitis was associated with changes in vascularity. Coronal pulps were removed from 62 primary and 62 permanent mandibular molars with a known pain history. Teeth were categorized as intact, moderately carious or grossly carious. Pulp sections were labelled with Ulex europaeus I lectin (UEIL), which is a marker of human vascular endothelium. Image analysis was then used to quantify the percentage area of UEIL-labelled tissue (vascularity) and the number of blood vessels present within three regions: the pulp horn, the subodontoblastic region, and the mid-coronal pulp. Only the mid-coronal region of the primary tooth pulp was found to be significantly more vascular than the corresponding area of the permanent tooth pulp. Both dentitions showed a significant increase in vascularity within the pulp horn region with caries progression, but this was not accompanied by an increase in vessel number. There was no correlation between vascularity and pain symptoms. These findings suggest that the primary tooth pulp is more vascular than its successor within the mid-coronal region. However, the functional and clinical significance of this finding remains speculative. PMID:15819818

  13. [Biologic and molecular genetic properties of a transplantable human primary gastric cancer in nude mice].

    PubMed

    Chen, S S

    1989-05-01

    A human primary gastric cancer tissue (adenocarcinoma II-III) was transplanted into nude mice (SWISS/DF. nu/nu). It has been transferred for 8 generations at 56 sites in 28 nude mice with transplantable rate of 100%. The transplanted tumor is designated as transplantable human primary gastric cancer-1 in nude mice (THPGC-1). The growth of THPGC-1 is rather rapid and the size of transplanted tumor reaches 1 cm2, 4-5 weeks after transfer. The morphology and histochemistry of the original tumor were retained well in the initial and serial transplanted tumors. THPGC-1 could secret carcinoembryonic antigen (CEA). After intravenous or intraperitoneal injection of 131I-antiCEA monoclonal antibody into the THPGC-1 bearing nude mice, the radiolabeled antibody was concentrated and localized in the tumor as shown by gamma-camera analysis. Similar pattern of lactate dehydrogenase isoenzyme was observed both in primary gastric cancer tissue and THPGC-1 tissue. Chromosomal examination revealed that THPGC-1 was human aneuploid ones. Southern blot analysis showed that the pattern of repetitive DNA bands and the structures of 28s, rDNA, c-H-ras and c-myc genes in THPGC-1 were identical to the original primary gastric cancer DNA. The results suggest that THPGC-1 be a reliable model for the research of the molecular biology of cancer cells and experimental gastric cancer diagnosis and treatment. PMID:2693024

  14. 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

  15. Human olfactory lateralization requires trigeminal activation.

    PubMed

    Croy, Ilona; Schulz, Max; Blumrich, Anna; Hummel, Cornelia; Gerber, Johannes; Hummel, Thomas

    2014-09-01

    Rats are able to lateralize odors. This ability involves specialized neurons in the orbitofrontal cortex which are able to process the left, right and bilateral presentation of stimuli. However, it is not clear whether this function is preserved in humans. Humans are in general not able to differentiate whether a selective olfactory stimulant has been applied to the left or right nostril; however exceptions have been reported. Following a screening of 152 individuals with an olfactory lateralization test, we identified 19 who could lateralize odors above chance level. 15 of these "lateralizers" underwent olfactory fMRI scanning in a block design and were compared to 15 controls matched for age and sex distribution. As a result, both groups showed comparable activation of olfactory eloquent brain areas. However, subjects with lateralization ability had a significantly enhanced activation of cerebral trigeminal processing areas (somatosensory cortex, intraparietal sulcus). In contrast to controls, lateralizers furthermore exhibited no suppression in the area of the trigeminal principal sensory nucleus. An exploratory study with an olfactory change detection paradigm furthermore showed that lateralizers oriented faster towards changes in the olfactory environment. Taken together, our study suggests that the trigeminal system is activated to a higher degree by the odorous stimuli in the group of "lateralizers". We conclude that humans are not able to lateralize odors based on the olfactory input alone, but vary in the degree to which the trigeminal system is recruited. PMID:24825502

  16. 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

  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. Cell wounding activates phospholipase D in primary mouse keratinocytes.

    PubMed

    Arun, Senthil N; Xie, Ding; Howard, Amber C; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L; Bollag, Wendy B

    2013-03-01

    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

  19. 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. PMID:21508549

  20. [Characterization of genetic alterations in primary human melanomas carrying BRAF or NRAS mutation].

    PubMed

    Lázár, Viktória

    2013-06-01

    Human malignant melanoma is one of the most aggressive forms of skin cancer with an exceptionally bad prognosis. Melanoma often displays constitutively activated MAPK pathway through BRAF or NRAS mutations. It is also known that these mutations are almost never simultaneously present and that they appear at early stages and preserved throughout tumor progression, although it is proved that these alterations alone are insufficient to cause tumor progression. Therefore the first aim of our study was to evaluate those distinct genetic alterations which can properly differentiate the three important molecular subtypes of primary melanomas with a) BRAF, b) NRAS mutation and c) WT (wild type for both loci). High-resolution array comparative genomic hybridization (array CGH) was used to assess genome-wide analysis of DNA copy number alterations. Primary melanomas with BRAF mutation more frequently exhibited losses on 10q23-10q26 and gains on chromosome 7 and 1q23-1q25 compared to melanomas with NRAS mutation. Loss on the 11q23-11q25 sequence was found mainly in conjunction with NRAS mutation. Based on these results, we proved the existence of marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups, which might suggest that these mutations contribute to the development of malignant melanoma in conjunction with distinct cooperating oncogenic events. In general, it is an interesting phenomenon suggesting that these mutations provide probably the "guiding force" for these tumors and it also suggests that there are alternative genetic pathways to melanoma. These additional oncogenic events which are associated with BRAF or NRAS mutations can provide rational additional targets for a combination therapy with kinase inhibitors. In this study we also investigated the specific dynamic activities among different signalling pathways highlighting the frequent alterations of genes involved in the signalling interactions between the MAPK-JAK pathways

  1. High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

    PubMed Central

    Zhao, Yangbing; Zheng, Zhili; Cohen, Cyrille J.; Gattinoni, Luca; Palmer, Douglas C.; Restifo, Nicholas P.; Rosenberg, Steven A.; Morgan, Richard A.

    2006-01-01

    The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes. PMID:16140584

  2. Establishment of a Novel Primary Human Skeletal Myoblast Cellular Model for Chikungunya Virus Infection and Pathogenesis

    PubMed Central

    Hussain, Khairunnisa’ Mohamed; Lee, Regina Ching Hua; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

    2016-01-01

    Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia and is now considered endemic in countries across Asia and Africa. The tissue tropism of CHIKV infection in humans remains, however, ill-defined. Due to the fact that myositis is commonly observed in most patients infected with CHIKV, we sought to develop a clinically relevant cellular model to better understand the pathogenesis of CHIKV infection. In this study, primary human skeletal muscle myoblasts (HSMM) were established as a novel human primary cell line that is highly permissive to CHIKV infection, with maximal amounts of infectious virions observed at 16 hours post infection. Genome-wide microarray profiling analyses were subsequently performed to identify and map genes that are differentially expressed upon CHIKV infection. Infection of HSMM cells with CHIKV resulted in altered expressions of host genes involved in skeletal- and muscular-associated disorders, innate immune responses, cellular growth and death, host metabolism and virus replication. Together, this study has shown the establishment of a clinically relevant primary human cell model that paves the way for the further analysis of host factors and their involvement in the various stages of CHIKV replication cycle and viral pathogenesis. PMID:26892458

  3. 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

  4. 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.

  5. Sucrose activates human taste pathways differently from artificial sweetener.

    PubMed

    Frank, Guido K W; Oberndorfer, Tyson A; Simmons, Alan N; Paulus, Martin P; Fudge, Julie L; Yang, Tony T; Kaye, Walter H

    2008-02-15

    Animal models suggest that sucrose activates taste afferents differently than non-caloric sweeteners. Little information exists how artificial sweeteners engage central taste pathways in the human brain. We assessed sucrose and sucralose taste pleasantness across a concentration gradient in 12 healthy control women and applied 10% sucrose and matched sucralose during functional magnet resonance imaging. The results indicate that (1) both sucrose and sucralose activate functionally connected primary taste pathways; (2) taste pleasantness predicts left insula response; (3) sucrose elicits a stronger brain response in the anterior insula, frontal operculum, striatum and anterior cingulate, compared to sucralose; (4) only sucrose, but not sucralose, stimulation engages dopaminergic midbrain areas in relation to the behavioral pleasantness response. Thus, brain response distinguishes the caloric from the non-caloric sweetener, although the conscious mind could not. This could have important implications on how effective artificial sweeteners are in their ability to substitute sugar intake. PMID:18096409

  6. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    PubMed Central

    Ham, Hyoungjun; Billadeau, Daniel D.

    2013-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synapse. Concurrently, lytic granules undergo minus-end directed movement and accumulate at the microtubule-organizing center through the interaction with microtubule motor proteins, followed by polarization of the lethal cargo toward the target cell. Ultimately, myosin-dependent movement of the lytic granules toward the NK cell plasma membrane through F-actin channels, along with soluble N-ethylmaleimide-sensitive factor attachment protein receptor-dependent fusion, promotes the release of the lytic granule contents into the cleft between the NK cell and target cell resulting in target cell killing. Herein, we will discuss several disease-causing mutations in primary immunodeficiency syndromes and how they impact NK cell-mediated killing by disrupting distinct steps of this tightly regulated process. PMID:24478771

  7. 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)

  8. 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.…

  9. Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells

    PubMed Central

    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

  10. 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...

  11. 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

  12. Impact of Some Environmental Education Outdoor Activities on Nigerian Primary School Pupils' Environmental Knowledge

    ERIC Educational Resources Information Center

    Ajiboye, Josiah O.; Olatundun, Sunday Adekojo

    2010-01-01

    This study investigated the impact of outdoor educational activities on pupils' knowledge of some environmental issues and problems. A pretest, posttest, control group, quasi-experimental design was adopted for the study. Four hundred and eighty primary 5 pupils from 12 randomly selected primary schools in Ibadan, Nigeria, constituted the subjects…

  13. Taking Active Learning into the Primary School: A Matter of New Practices?

    ERIC Educational Resources Information Center

    Stephen, Christine; Ellis, Jennifer; Martlew, Joan

    2010-01-01

    This paper examines the extension of active learning pedagogical practices familiar in preschool settings to the first class of primary school. Policy and practice guidance in the UK is advocating the benefits of experiential learning as a way of engaging young children as they move into primary school but for teachers this means a move to new…

  14. 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…

  15. 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.

  16. Expression Profiling of LPS Responsive miRNA in Primary Human Macrophages

    PubMed Central

    Naqvi, Afsar Raza; Zhong, Sheng; Dang, Hong; Fordham, Jezrom B; Nares, Salvador; Khan, Asma

    2016-01-01

    microRNAs (miRNAs) have emerged as important regulators of the innate and adaptive immune response. The purpose of the present study was to interrogate miRNA profiles of primary human macrophages challenged with bacterial lipopolysaccharide (LPS) with focus on expression kinetics. We employed Nanostring platform to precisely characterize the changes in miRNA expression following different doses and durations of LPS exposure. Differentially expressed miRNAs were identified in response to LPS challenge with convergent and divergent expression profiles. Pathway analysis of LPS-responsive miRNAs revealed regulation of biological processes linked to key cell signaling (including PIK3-Akt, MAP kinase, ErbB) and pathogen response pathways. Our data provide a comprehensive miRNA profiling of human primary macrophages treated with LPS. These results show that bacterial Toll like receptor (TLR) ligands can temporally modulate macrophage miRNA expression. PMID:27307950

  17. 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

  18. Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells

    PubMed Central

    Wang, Weiping; Brautigan, David L

    2008-01-01

    Background Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood. Results Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium. Conclusion The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium. PMID:19036150

  19. Human hippocampal theta activity during virtual navigation.

    PubMed

    Ekstrom, Arne D; Caplan, Jeremy B; Ho, Emily; Shattuck, Kirk; Fried, Itzhak; Kahana, Michael J

    2005-01-01

    This study examines whether 4-8-Hz theta oscillations can be seen in the human hippocampus, and whether these oscillations increase during virtual movement and searching, as they do in rodents. Recordings from both hippocampal and neocortical depth electrodes were analyzed while six epileptic patients played a virtual taxi-driver game. During the game, the patients alternated between searching for passengers, whose locations were random, and delivering them to stores, whose locations remained constant. In both hippocampus and neocortex, theta increased during virtual movement in all phases of the game. Hippocampal and neocortical theta activity were also significantly correlated with each other, but this correlation did not differ between neocortex and hippocampus and within disparate neocortical electrodes. Our findings demonstrate the existence of movement-related theta oscillations in human hippocampus, and suggest that both cortical and hippocampal oscillations play a role in attention and sensorimotor integration. PMID:16114040

  20. 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

  1. Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

    PubMed Central

    Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

    1998-01-01

    In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

  2. 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.

  3. 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

  4. 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

  5. Carnosinase activity of human gastrointestinal mucosa.

    PubMed Central

    Sadikali, F; Darwish, R; Watson, W C

    1975-01-01

    Carnosinase, the dipeptidase which hydrolyses carnosine and other histidine-containing dipeptides, was assayed in mucosal tissues of the human and of the rat gut. Kinetic properties of the intestinal enzyme were found to be similar to carnosinase of other animal tissues. Little or no activity was detected in human gastric or colonic mucosa, and the levels were lower in duodenal than jejunal mucosa. The distribution of carnosinase is similar to that of the disaccharidases. Mean carnosinase activity was 8-8 units/g weight in 15 patients with histologically normal mucosa compared with 5-7 units in five with villous atrophy. The enzyme levels increased with histological improvement of the mucosa in patients with coeliac disease on a gluten-free diet. Tolerance curves for carnosine and its constitutent amino acids showed malabsorption of the dipeptide in a patient with carnosinase deficiency. It is concluded that the intestinal mucosa has much less hydrolase activity for carnosine than for glycylglycine and other dipeptidases, and the relatively slow hydrolysis appears to be the rate-limiting step in the total absorptive process. PMID:1237444

  6. "Are You Human Beings?"Order and Knowledge Construction through Questioning in Primary Classroom Interaction

    ERIC Educational Resources Information Center

    Margutti, Piera

    2006-01-01

    This article examines how question-answer sequences are constructed in primary school instructional activities. The interaction between teacher and students in two 3rd-year groups is analyzed using a conversation-analytic approach. Four questioning patterns--"yes-no, alternative, wh-questions", and a "non-interrogative format" very frequently used…

  7. Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol

    PubMed Central

    Dilwali, Sonam; Patel, Pratik B.; Roberts, Daniel S.; Basinsky, Gina M.; Harris, Gordon J.; Emerick, Kevin; Stankovic, Konstantina M.

    2014-01-01

    Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. PMID:24910344

  8. 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

  9. 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…

  10. 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

  11. Poly(ethylene glycol)-containing hydrogels promote the release of primary granules from human blood-derived polymorphonuclear leukocytes

    PubMed Central

    Cohen, Hannah Caitlin; Lieberthal, Tyler Jacob; Kao, W. John

    2014-01-01

    Polymorphonuclear leukocytes (PMNs) are recruited to sites of injury and biomaterial implants. Once activated, PMNs can exocytose their granule subsets to recruit monocytes (MCs) and mediate MC/macrophage activation. We investigated the release of myeloperoxidase (MPO), a primary granule marker, and matrix metalloproteinase-9 (MMP-9), a tertiary granule marker, from human blood-derived PMNs cultured on poly(ethylene glycol) (PEG) hydrogels, polydimethylsiloxane (PDMS), tissue culture polystyrene (TCPS) and gelatin-PEG (GP) hydrogels, with and without the presence of the bacterial peptide formyl-Met-Leu-Phe. Supernatants from PMN cultures on PEG-containing hydrogels (i.e., PEG and GP hydrogels) had higher concentrations of MPO than those from PMN cultures on PDMS or TCPS at 2 hours. PMNs on all biomaterials released comparable levels of MMP-9 at 2 hours, indicating that PMNs cultured on PEG-containing hydrogels have different mechanisms of release for primary and tertiary granules. Src family kinases were involved in the release of MPO from PMNs cultured on PEG hydrogels, TCPS and GP hydrogels and in the release of MMP-9 from PMNs cultured on all four materials. The increased release of primary granules from PMNs on PEG-containing hydrogels did not significantly increase MC chemotaxis, indicating that additional co-effectors in the dynamic inflammatory milieu in vivo modulate PMN-mediated MC recruitment. PMID:24497370

  12. 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

  13. 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

    2016-01-01

    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

  14. Modulation of aflatoxin B1-mediated genotoxicity in primary cultures of human hepatocytes by diindolylmethane, curcumin, and xanthohumols.

    PubMed

    Gross-Steinmeyer, Kerstin; Stapleton, Patricia L; Tracy, Julia H; Bammler, Theo K; Strom, Stephen C; Buhler, Donald R; Eaton, David L

    2009-12-01

    This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol. PMID:19770484

  15. 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

  16. 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

  17. The altered landscape of the human skin microbiome in patients with primary immunodeficiencies

    PubMed Central

    Oh, Julia; Freeman, Alexandra F.; Park, Morgan; Sokolic, Robert; Candotti, Fabio; Holland, Steven M.; Segre, Julia A.; Kong, Heidi H.

    2013-01-01

    While landmark studies have shown that microbiota activate and educate host immunity, how immune systems shape microbiomes and contribute to disease is incompletely characterized. Primary immunodeficiency (PID) patients suffer recurrent microbial infections, providing a unique opportunity to address this issue. To investigate the potential influence of host immunity on the skin microbiome, we examined skin microbiomes in patients with rare monogenic PIDs: hyper-IgE (STAT3-deficient), Wiskott-Aldrich, and dedicator of cytokinesis 8 syndromes. While specific immunologic defects differ, a shared hallmark is atopic dermatitis (AD)–like eczema. We compared bacterial and fungal skin microbiomes (41 PID, 13 AD, 49 healthy controls) at four clinically relevant sites representing the major skin microenvironments. PID skin displayed increased ecological permissiveness with altered population structures, decreased site specificity and temporal stability, and colonization with microbial species not observed in controls, including Clostridium species and Serratia marcescens. Elevated fungal diversity and increased representation of opportunistic fungi (Candida, Aspergillus) supported increased PID skin permissiveness, suggesting that skin may serve as a reservoir for the recurrent fungal infections observed in these patients. The overarching theme of increased ecological permissiveness in PID skin was counterbalanced by the maintenance of a phylum barrier in which colonization remained restricted to typical human-associated phyla. Clinical parameters, including markers of disease severity, were positively correlated with prevalence of Staphylococcus, Corynebacterium, and other less abundant taxa. This study examines differences in microbial colonization and community stability in PID skin and informs our understanding of host–microbiome interactions, suggesting a bidirectional dialogue between skin commensals and the host organism. PMID:24170601

  18. Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

    PubMed Central

    Ghavami, S; Cunnington, R H; Gupta, S; Yeganeh, B; Filomeno, K L; Freed, D H; Chen, S; Klonisch, T; Halayko, A J; Ambrose, E; Singal, R; Dixon, I M C

    2015-01-01

    Transforming growth factor-β1 (TGF-β1) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β1 may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β1-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β1 to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β1 promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β1 in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β1-induced autophagy is required for the fibrogenic response in hATMyofbs. PMID:25789971

  19. 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

  20. 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

  1. EVI1 and MDS1/EVI1 Expression During Primary Human Hematopoietic Progenitor Cell Differentiation into Various Myeloid Lineages

    PubMed Central

    Steinleitner, Katarina; Rampetsreiter, Paulina; Köffel, Rene; Ramanathan, Gajalakshmi; Mannhalter, Christine; Strobl, Herbert; Wieser, Rotraud

    2012-01-01

    Background and Aim Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in myeloid leukemia. We therefore studied its expression and function in cluster of differentiation 34 positive (CD 34+) primary human hematopoietic progenitor cells. Materials and Methods CD34+ cells were differentiated into various myeloid lineages using appropriate cytokines. EVI1 expression was measured by quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) and intranuclear fluorescence activated cell sorting (FACS). Experimental manipulation of EVI1 levels was achieved using retroviral infection. Results EVI1 mRNA and its variant myelodysplastic syndrome 1 (MDS1)/EVI1, which gives rise to a partially antagonistic protein, were detectable in CD34+ cells, but their levels declined rapidly during differentiation into the granulocytic, monocytic, dendritic, erythroid, and megakaryocytic lineages. Similarly, EVI1 protein levels decreased during myeloid differentiation. Attempts to experimentally express EVI1 in CD34+ and U937 cells indicated that ectopic expression of EVI1 may cause growth arrest, apoptosis and/or senescence of human hematopoietic cells. Conclusion EVI1 is expressed in human hematopoietic progenitor cells, but is down-regulated during differentiation. Ectopic expression of EVI1 may activate cellular safeguards against oncogene activation. PMID:23155256

  2. NFκB2/p100 is a key factor for endotoxin tolerance in human monocytes: a demonstration using primary human monocytes from patients with sepsis.

    PubMed

    Cubillos-Zapata, Carolina; Hernández-Jiménez, Enrique; Toledano, Víctor; Esteban-Burgos, Laura; Fernández-Ruíz, Irene; Gómez-Piña, Vanesa; Del Fresno, Carlos; Siliceo, María; Prieto-Chinchiña, Patricia; Pérez de Diego, Rebeca; Boscá, Lisardo; Fresno, Manuel; Arnalich, Francisco; López-Collazo, Eduardo

    2014-10-15

    Endotoxin tolerance (ET) is a state of reduced responsiveness to endotoxin stimulation after a primary bacterial insult. This phenomenon has been described in several pathologies, including sepsis, in which an endotoxin challenge results in reduced cytokine production. In this study, we show that the NFκ L chain enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed and accumulated in a well-established in vitro human monocyte model of ET. The p100 accumulation in these cells inversely correlated with the inflammatory response after LPS stimulation. Knocking down NFκB2/p100 using small interfering RNA in human monocytes further indicated that p100 expression is a crucial factor in the progression of ET. The monocytes derived from patients with sepsis had high levels of p100, and a downregulation of NFκB2/p100 in these septic monocytes reversed their ET status. PMID:25225662

  3. The transcriptional activity of human Chromosome 22

    PubMed Central

    Rinn, John L.; Euskirchen, Ghia; Bertone, Paul; Martone, Rebecca; Luscombe, Nicholas M.; Hartman, Stephen; Harrison, Paul M.; Nelson, F. Kenneth; Miller, Perry; Gerstein, Mark; Weissman, Sherman; Snyder, Michael

    2003-01-01

    A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A)+ RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22. PMID:12600945

  4. 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

  5. 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

  6. New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells.

    PubMed

    Lavery, Karen; Hawley, Sara; Swain, Pamela; Rooney, Robert; Falb, Dean; Alaoui-Ismaili, Moulay Hicham

    2009-07-01

    Bone Morphogenetic Proteins (BMPs) are members of the TGF-beta superfamily of growth factors. Several BMPs exhibit osteoinductive bioactivities, and are critical for bone formation in both developing and mature skeletal systems. BMP-7 (OP-1) is currently used clinically in revision of posterolateral spine fusions and long bone non-unions. The current study characterizes BMP-7 induced gene expression during early osteoblastic differentiation of human mesenchymal stem cells (hMSC). Primary hMSC were treated with BMP-7 for 24 or 120 h and gene expression across the entire human genome was evaluated using Affymetrix HG-U133 Plus 2.0 Arrays. 955 probe sets representing 655 genes and 95 ESTs were identified as differentially expressed and were organized into three major expression profiles (Profiles A, B and C) by hierarchical clustering. Genes from each profile were classified according to biochemical pathway analyses. Profile A, representing genes upregulated by BMP-7, revealed strong enrichment for established osteogenic marker genes, as well as several genes with undefined roles in osteoblast function, including MFI2, HAS3, ADAMTS9, HEY1, DIO2 and FGFR3. A functional screen using siRNA suggested roles for MFI2, HEY1 and DIO2 in osteoblastic differentiation of hMSC. Profile B contained genes transiently downregulated by BMP-7, including numerous genes associated with cell cycle regulation. Follow-up studies confirmed that BMP-7 attenuates cell cycle progression and cell proliferation during early osteoblastic differentiation. Profile C, comprised of genes continuously downregulated by BMP-7, exhibited strong enrichment for genes associated with chemokine/cytokine activity. Inhibitory effects of BMP-7 on cytokine secretion were verified by analysis of enriched culture media. Potent downregulation of CHI3L1, a potential biomarker for numerous joint diseases, was also observed in Profile C. A focused evaluation of BMP, GDF and BMP inhibitor expression elucidated feedback

  7. 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

  8. Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.

    PubMed

    Broeders, Jessica J W; Parmentier, Céline; Truisi, Germaine L; Jossé, Rozenn; Alexandre, Eliane; Savary, Camille C; Hewitt, Philip G; Mueller, Stefan O; Guillouzo, André; Richert, Lysiane; van Eijkeren, Jan C H; Hermens, Joop L M; Blaauboer, Bas J

    2015-12-25

    Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 μM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data. PMID:25458484

  9. Astrocytes As the Main Players in Primary Degenerative Disorders of the Human Central Nervous System

    PubMed Central

    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

  10. 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

  11. 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

  12. 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

  13. Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors.

    PubMed

    Bowers, Alex J; Boylan, John F

    2004-03-17

    The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression. PMID:15019993

  14. 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. PMID:26216948

  15. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins

    PubMed Central

    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

    2015-01-01

    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. PMID:26216948

  16. Global human appropriation of net primary production doubled in the 20th century.

    PubMed

    Krausmann, Fridolin; Erb, Karl-Heinz; Gingrich, Simone; Haberl, Helmut; Bondeau, Alberte; Gaube, Veronika; Lauk, Christian; Plutzar, Christoph; Searchinger, Timothy D

    2013-06-18

    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

    DOE PAGESBeta

    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

  18. 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.

  19. Naïve adult stem cells isolation from primary human fibroblast cultures.

    PubMed

    Wenzel, Vera; Roedl, Daniela; Ring, Johannes; Djabali, Karima

    2013-01-01

    Over the last decade, several adult stem cell populations have been identified in human skin (1-4). The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory (5, 6). These early studies described a multipotent precursor cell population from adult mammalian dermis (5). These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons (5). Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers (6). Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies. Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies (7). The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable (7). These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures (7). We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are

  20. Nef-Mediated Downregulation of CD4 Enhances Human Immunodeficiency Virus Type 1 Replication in Primary T Lymphocytes

    PubMed Central

    Lundquist, Christopher A.; Tobiume, Minoru; Zhou, Jing; Unutmaz, Derya; Aiken, Christopher

    2002-01-01

    The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4+ T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef. PMID:11932428

  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. Human papillomavirus (HPV) DNA in penile carcinomas in Argentina: analysis of primary tumors and lymph nodes.

    PubMed

    Picconi, M A; Eiján, A M; Distéfano, A L; Pueyo, S; Alonio, L V; Gorostidi, S; Teyssié, A R; Casabé, A

    2000-05-01

    Among sexually transmitted diseases, infection by human papillomavirus (HPV) has become one of the most important. On the other hand, though epidemiological data show that some HPV types are closely associated with cervical cancer, few reports have been found with reference to penile carcinoma because of its rare occurrence. The aim of this study was to investigate the relationship between HPV infection and penile cancer in Argentina. A retrospective study was carried out on 38 white men with penile squamous-cell carcinoma. Sixty-five archival fixed biopsies taken from 34 primary penile tumors, 25 nodal metastases, 1 skin "satellite" metastasis and 5 histologically normal lymph nodes were used as specimens. HPV detection and typing were carried out by the polymerase chain reaction (PCR) using generic primers, combined with single-stranded conformational polymorphism (SSCP) analysis. HPV DNA was found in 71% patients, corresponding 81% of them to "high risk" types, with predominance of HPV 18. Both primary tumors and metastases showed concordance of HPV occurrence and type in both lesions. In 3 patients, HPV 16 was detected not only in primary tumors and metastases, but also in histologically normal lymph nodes. Our data indicate that most penile carcinomas in Argentine patients are etiologically related to HPV, especially to "high risk" genital types. The agreement in HPV detection between primary tumors and metastases suggests a potential viral role in tumor progression. HPV detection in otherwise histologically normal lymph nodes might be useful as early marker of a metastatic process. PMID:10745234

  3. Spread of epileptic activity in human brain

    NASA Astrophysics Data System (ADS)

    Milton, John

    1997-03-01

    For many patients with medically refractory epilepsy surgical resection of the site of seizure onset (epileptic focus) offers the best hope for cure. Determination of the nature of seizure propagation should lead to improved methods for locating the epileptic focus (and hence reduce patient morbidity) and possibly to new treatment modalities directed at blocking seizure spread. Theoretical studies of neural networks emphasize the role of traveling waves for the propagation of activity. However, the nature of seizure propagation in human brain remains poorly characterized. The spread of epileptic activity in patients undergoing presurgical evaluation for epilepsy surgery was measured by placing subdural grids of electrodes (interelectrode spacings of 3-10 mm) over the frontal and temporal lobes. The exact location of each electrode relative to the surface of the brain was determined using 3--D MRI imaging techniques. Thus it is possible to monitor the spread of epileptic activity in both space and time. The observations are discussed in light of models for seizure propagation.

  4. Human activity and rest in situ.

    PubMed

    Roenneberg, Till; Keller, Lena K; Fischer, Dorothee; Matera, Joana L; Vetter, Céline; Winnebeck, Eva C

    2015-01-01

    Our lives are structured by the daily alternation of activity and rest, of wake and sleep. Despite significant advances in circadian and sleep research, we still lack answers to many of the most fundamental questions about this conspicuous behavioral pattern. We strongly believe that investigating this pattern in entrained conditions, real-life and daily contexts-in situ-will help the field to elucidate some of these central questions. Here, we present two common approaches for in situ investigation of human activity and rest: the Munich ChronoType Questionnaire (MCTQ) and actimetry. In the first half of this chapter, we provide detailed instructions on how to use and interpret the MCTQ. In addition, we give an overview of the main insights gained with this instrument over the past 10 years, including some new findings on the interaction of light and age on sleep timing. In the second half of this chapter, we introduce the reader to the method of actimetry and share our experience in basic analysis techniques, including visualization, smoothing, and cosine model fitting of in situ recorded data. Additionally, we describe our new approach to automatically detect sleep from activity recordings. Our vision is that the broad use of such easy techniques in real-life settings combined with automated analyses will lead to the creation of large databases. The resulting power of big numbers will promote our understanding of such fundamental biological phenomena as sleep. PMID:25707281

  5. Linguistic threat activates the human amygdala

    PubMed Central

    Isenberg, N.; Silbersweig, D.; Engelien, A.; Emmerich, S.; Malavade, K.; Beattie, B.; Leon, A. C.; Stern, E.

    1999-01-01

    Studies in animals demonstrate a crucial role for the amygdala in emotional and social behavior, especially as related to fear and aggression. Whereas lesion and functional-imaging studies in humans indicate the amygdala’s participation in assessing the significance of nonverbal as well as paralinguistic cues, direct evidence for its role in the emotional processing of linguistic cues is lacking. In this study, we use a modified Stroop task along with a high-sensitivity neuroimaging technique to target the neural substrate engaged specifically when processing linguistic threat. Healthy volunteer subjects were instructed to name the color of words of either threat or neutral valence, presented in different color fonts, while neural activity was measured by using H215O positron-emission tomography. Bilateral amygdalar activation was significantly greater during color naming of threat words than during color naming of neutral words. Associated activations were also noted in sensory-evaluative and motor-planning areas of the brain. Thus, our results demonstrate the amygdala’s role in the processing of danger elicited by language. In addition, the results reinforce the amygdala’s role in the modulation of the perception of, and response to, emotionally salient stimuli. The current study further suggests conservation of phylogenetically older mechanisms of emotional evaluation in the context of more recently evolved linguistic function. PMID:10468630

  6. Physical Activity Patterns and Psychological Correlates of Physical Activity among Singaporean Primary, Secondary, and Junior College Students

    ERIC Educational Resources Information Center

    Wang, C. K. John; Koh, K. T.; Biddle, Stuart J. H.; Liu, W. C.; Chye, Stefanie

    2011-01-01

    The purpose of this research was to examine physical activity patterns and psychological correlates of physical activity among primary, secondary, and junior college students in Singapore. A sample of 3,333 school students aged 10 to 18 years took part in the study. Results showed that the younger students had significantly higher physical…

  7. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

    PubMed Central

    Funk, C D; Furci, L; FitzGerald, G A

    1990-01-01

    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, we 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 Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) 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. Images PMID:2377602

  8. 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. PMID:25534347

  9. Experimental study on the behavior of primary human osteoblasts on laser-cused pure titanium surfaces.

    PubMed

    Markwardt, Jutta; Friedrichs, Jens; Werner, Carsten; Davids, Andreas; Weise, Hartmut; Lesche, Raoul; Weber, Anke; Range, Ursula; Meißner, Heike; Lauer, Günther; Reitemeier, Bernd

    2014-05-01

    Mandibular tumor resection can lead to a mandibular segmental defect. LaserCUSING® is used to produce a mandibular implant, designed to be identical to the shape of the mandibular defect. Novel microrough surfaces result from this generative technology. In the current study, the behavior of human osteoblasts on untreated laser-cused titanium specimens or on specimens conditioned with different blasting agents was analyzed. The conditioning of these specimens resulted in surfaces with graded roughness. White light confocal microscopy and single-cell force spectroscopy were used to characterize the surface of the specimens and to quantify the initial adhesion of primary human osteoblasts to the specimens, respectively. Furthermore, cell growth, viability, apoptosis as well as mineralization of the specimens were analyzed over a time-period of 2 months. Compared to specimens that were treated with blasting agents, untreated specimens had the highest surface roughness. Quantitative SCFS measurements demonstrated that the adhesion of human primary osteoblasts was the highest on these specimens. Additionally, the untreated specimens allowed the highest number of osteoblasts to colonize. Mineralization studies showed increasing calcium and phosphor elemental composition for all specimen series. It can be concluded that untreated laser-cused titanium specimens are superior to promote the initial adhesion and subsequent colonization by osteoblast cells. PMID:23775939

  10. 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

  11. 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

  12. 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

  13. Human Papillomavirus and Cystic Node Metastasis in Oropharyngeal Cancer and Cancer of Unknown Primary Origin

    PubMed Central

    Yasui, Toshimichi; Morii, Eiichi; Yamamoto, Yoshifumi; Yoshii, Tadashi; Takenaka, Yukinori; Nakahara, Susumu; Todo, Takeshi; Inohara, Hidenori

    2014-01-01

    The clinical significance of human papillomavirus (HPV) in neck node metastasis from cancer of unknown primary (CUP) is not well established. We aimed to address the relationship of HPV status between node metastasis and the primary tumor, and also the relevance of HPV status regarding radiographically detected cystic node metastasis in head and neck squamous cell carcinoma (HNSCC) and CUP. HPV DNA was examined in 68 matched pairs of node metastasis and primary tumor, and in node metastasis from 27 CUPs. In surgically treated CUPs, p16 was examined immunohistochemically. When tonsillectomy proved occult tonsillar cancer in CUP, HPV DNA and p16 were also examined in the occult primary. Cystic node metastasis on contrast-enhanced computed tomography scans was correlated with the primary site and HPV status in another series of 255 HNSCCs and CUPs with known HPV status. Node metastasis was HPV-positive in 19/37 (51%) oropharyngeal SCCs (OPSCCs) and 10/27 (37%) CUPs, but not in non-OPSCCs. Fluid was collected from cystic node metastasis using fine needle aspiration in two OPSCCs and one CUP, and all fluid collections were HPV-positive. HPV status, including the presence of HPV DNA, genotype, and physical status, as well as the expression pattern of p16 were consistent between node metastasis and primary or occult primary tumor. Occult tonsillar cancer was found more frequently in p16-positive CUP than in p16-negative CUP (odds ratio (OR), 39.0; 95% confidence interval (CI), 1.4–377.8; P = 0.02). Radiographically, cystic node metastasis was specific to OPSCC and CUP, and was associated with HPV positivity relative to necrotic or solid node metastasis (OR, 6.2; 95% CI, 1.2–45.7; P = 0.03). In conclusion, HPV status remains unchanged after metastasis. The occult primary of HPV-positive CUP is most probably localized in the oropharynx. HPV status determined from fine needle aspirates facilitates the diagnosis of cystic node metastasis. PMID:24752007

  14. 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…

  15. 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…

  16. 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…

  17. Comparing Primary Student Teachers' Attitudes, Subject Knowledge and Pedagogical Content Knowledge Needs in a Physics Activity

    ERIC Educational Resources Information Center

    Johnston, Jane; Ahtee, Maija

    2006-01-01

    This research explores and compares primary student teachers' attitudes, subject knowledge and pedagogical content knowledge (PCK) in physics in two institutions in England and Finland, using a practical physics activity and questionnaire. Teaching of physics activities was rated unpopular both in Finland and England, although English students…

  18. 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…

  19. 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…

  20. 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,…

  1. 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

  2. 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

  3. 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

  4. Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization.

    PubMed

    Shah, Amita R; Wenke, Joseph C; Agrawal, Chandra Mauli

    2016-01-01

    Tissue-engineering scaffolds are often seeded with a single type of cell, but there has been more focus on cocultures to improve angiogenesis and bone formation for craniofacial applications. Investigation of bone-derived osteoblasts (OBs) is important because of the use of bone grafts and migration of OBs from native bone into constructs in vivo and therefore, their contribution to bone formation in vivo. The limitation of primary OBs has been their inability to mineralize without osteogenic factors in vitro. Through coculture of OBs and endothelial cells (ECs) and manipulation of the coculture ratio, mineralization can be achieved without osteogenic media or additional growth factors, thus enhancing their utility for tissue-engineering applications. An optimal ratio of EC/OB for vasculogenesis and mineralization has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios, namely, 10:1, 5:1, 1:1, 1:5, and 1:10 with EC and OB monocultures as controls. The number of vasculogenic networks in a collagen matrix was highest in ratios of 5:1 and 1:1. ECs lined up and formed capillary-like networks by day 10, which was not seen in the other groups. On polystyrene, cells were cocultured with ECs and OBs in direct contact (direct coculture) or separated by a transwell membrane (indirect coculture). At day 21, Alizarin Red staining showed mineralization on the 1:5 and 1:10 direct coculture ratios, with 1:5 having more mineralization nodules present than 1:10. No mineralization was seen in other direct coculture ratios or in any of the indirect coculture ratios. Alkaline phosphatase secretion was highest in the 1:5 direct coculture group. Vascular endothelial growth factor secretion from OBs was present in the 1:5 and 1:10 direct coculture ratios at all time points and inhibited after day 1 in other coculture groups. To improve vasculogenesis, cocultures of primary human ECs and OBs in ratios

  5. Functional 3D human primary hepatocyte spheroids made by co-culturing hepatocytes from partial hepatectomy specimens and human adipose-derived stem cells.

    PubMed

    No, Da Yoon; Lee, Seung-A; Choi, Yoon Young; Park, DoYeun; Jang, Ju Yun; Kim, Dong-Sik; Lee, Sang-Hoon

    2012-01-01

    We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes. PMID:23236387

  6. 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

  7. 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

  8. Thiostrepton is an Inducer of Oxidative and Proteotoxic Stress that Impairs Viability of Human Melanoma Cells but not Primary Melanocytes

    PubMed Central

    Qiao, Shuxi; Lamore, Sarah D.; Cabello, Christopher M.; Lesson, Jessica L.; Muñoz-Rodriguez, José L.; Wondrak, Georg T.

    2012-01-01

    Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other

  9. 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. PMID:25288153

  10. Explanted Diseased Livers – A Possible Source of Metabolic Competent Primary Human Hepatocytes

    PubMed Central

    Krech, Till; DeTemple, Daphne; Jäger, Mark D.; Lehner, Frank; Manns, Michael P.; Klempnauer, Jürgen; Borlak, Jürgen; Bektas, Hueseyin; Vondran, Florian W. R.

    2014-01-01

    Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5′-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might

  11. Effect of urokinase on the proliferation of primary cultures of human prostatic cells

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Hienert, G.; Christ, G.; Heger, M.E.; Pflueger, H.B.; Binder, B.R.

    1987-11-01

    The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of /sup 3/H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of /sup 3/H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of /sup 3/H-thymidine incorporation, whereas plasmin caused a 25% decrease of /sup 3/H-thymidine incorporation in all cultures.

  12. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

    PubMed

    Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

    2016-09-01

    Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug. PMID:27363785

  13. Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

    PubMed Central

    Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

    2010-01-01

    Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

  14. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases.

    PubMed

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz I Altaba, Ariel

    2016-04-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/- cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2(high) and AXIN2(low), which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  15. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases

    PubMed Central

    Singovski, Grigori; Bernal, Carolina; Kuciak, Monika; Siegl-Cachedenier, Irene; Conod, Arwen; Ruiz i Altaba, Ariel

    2016-01-01

    How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/− cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2high and AXIN2low, which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors. PMID:26031752

  16. 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

  17. 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

  18. 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

  19. 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

  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. 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

  2. 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

  3. Regulation of CYP27B1 mRNA Expression in Primary Human Osteoblasts.

    PubMed

    van der Meijden, K; van Essen, H W; Bloemers, F W; Schulten, E A J M; Lips, P; Bravenboer, N

    2016-08-01

    The enzyme 1α-hydroxylase (gene CYP27B1) catalyzes the synthesis of 1,25(OH)2D in both renal and bone cells. While renal 1α-hydroxylase is tightly regulated by hormones and 1,25(OH)2D itself, the regulation of 1α-hydroxylase in bone cells is poorly understood. The aim of this study was to investigate in a primary human osteoblast culture whether parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, calcium, phosphate, or MEPE affect mRNA levels of CYP27B1. Our results show that primary human osteoblasts in the presence of high calcium concentrations increase their CYP27B1 mRNA levels by 1.3-fold. CYP27B1 mRNA levels were not affected by PTH1-34, rhFGF23, calcitonin, phosphate, and rhMEPE. Our results suggest that the regulation of bone 1α-hydroxylase is different from renal 1α-hydroxylase. High calcium concentrations in bone may result in an increased local synthesis of 1,25(OH)2D leading to an enhanced matrix mineralization. In this way, the local synthesis of 1,25(OH)2D may contribute to the stimulatory effect of calcium on matrix mineralization. PMID:27016371

  4. 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. PMID:25609254

  5. 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

  6. Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines

    PubMed Central

    Adalsteinsson, Viktor; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B.; Huang, Cindy; Bowman, Brittany; Williamson, Christina; Kwon, Douglas S.; Wittrup, K. Dane; Love, J. Christopher

    2014-01-01

    Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via either or both of the CXCR1 and CXCR2 receptors, exerting profound impacts on tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors. PMID:23995780

  7. Dual allosteric activation mechanisms in monomeric human glucokinase

    PubMed Central

    Whittington, A. Carl; Larion, Mioara; Bowler, Joseph M.; Ramsey, Kristen M.; Brüschweiler, Rafael; Miller, Brian G.

    2015-01-01

    Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems. PMID:26283387

  8. 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.

  9. 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

  10. First pathological study of canine primary breast lymphoma and the description of its clinicopathological characteristics as an animal model for human primary breast lymphoma.

    PubMed

    Rismanchi, Sanaz; Muhammadnejad, Samad; Amanpour, Saeid; Muhammadnejad, Ahad

    2015-01-01

    Canine breast cancer (BC) and human BC are the most prevalent tumors in female dogs and humans, respectively. Several studies have indicated that canine BC is a good model for human BC. Unlike breast carcinomas, human primary breast lymphoma (PBL) is a rare tumor, but no case of canine PBL has been reported thus far. The current study presents a case of canine MC of the primary non-Hodgkin lymphoma (NHL) type for the first time and subsequently questions the theory of considering it as a model for human PBL. A 2-cm tumor was surgically removed from the left caudal abdominal mammary gland of a 6-year-old female dog of the terrier breed. Microscopic examination did not show any sign for the epithelial origin of the tumor. By contrast, histomorphological view and molecular pathological evaluation by immunohistochemistry showed that the tumor was of the diffuse large B-cell lymphoma (DLBCL) type [cluster of differentiation 19(+) (CD19(+)), CD20(+), CD10(+), B-cell lymphoma 6(+), CD3(-), CD15(-)]. According to the World Health Organization classification, DLBCL is considered to be an NHL. Canine NHL is common in dogs and certain investigators believe that the biological behavior and clinical course is extremely similar to human NHL, and therefore, consider it as a model of human NHL. To the best of our knowledge, the current study is the first report of canine PBL. As the most significantly reported human PBL histotype is the DLBCL type, the histomorphological and immunophenotyping characteristics of canine PBL in the study considerably match with human PBL and raise the hypothesis that it can be a model for human PBL. PMID:25469251

  11. Quantification of primary versus secondary C-H bond cleavage in alkane activation: Propane on Pt

    SciTech Connect

    Weinberg, W.H.; Sun, Yongkui )

    1991-08-02

    The trapping-mediated dissociative chemisorption of three isotopes of propane (C{sub 3}H{sub 8}, CH{sub 3}, CD{sub 2}CH{sub 3}, and C{sub 3}D{sub 8}) has been investigated on the Pt(110)-(1 {times} 2) surface, and both the apparent activation energies and the preexponential factors of the surface reaction rate coefficients have been measured. In addition, the probabilities of primary and secondary C-H bond cleavage for alkane activation on a surface were evaluated. The activation energy for primary C-H bond cleavage was 425 calories per mole greater than that of secondary C-H bond cleavage, and the two true activation energies that embody the single measured activation energy were determined for each of the three isotopes. Secondary C-H bond cleavage is also preferred on entropic grounds, and the magnitude of the effect was quantified.

  12. 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

  13. 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

  14. Excitotoxic potential of the cyanotoxin β-methyl-amino-L-alanine (BMAA) in primary human neurons.

    PubMed

    Chiu, Alexander S; Gehringer, Michelle M; Braidy, Nady; Guillemin, Gilles J; Welch, Jeffrey H; Neilan, Brett A

    2012-11-01

    The toxicity of the cyanobacterial modified amino acid, BMAA, has been described in rat, mouse and leech neurons. Particular emphasis has been placed on the potential ability of BMAA to induce neuronal damage via excitotoxic mechanisms. Here we present data indicating that the effects observed on lower organisms are also evident in a human model. Our data indicates that BMAA induces increased intracellular Ca²⁺ influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of reactive oxygen species (ROS). The amelioration of LDH release in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Additionally, we have shown that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors. PMID:22885173

  15. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  16. Gas1 inhibits cell proliferation and induces apoptosis of human primary gliomas in the absence of Shh.

    PubMed

    Domínguez-Monzón, Gabriela; Benítez, Jorge A; Vergara, Paula; Lorenzana, Rodrigo; Segovia, José

    2009-06-01

    Growth arrest specific1 (Gas1) is a protein expressed during development and when cells arrest their growth. The potential of Gas1 as an adjuvant in the treatment of cancer, and its role as a tumor suppressor have also been proposed. In this work we are addressing the molecular mechanisms by which Gas1 induces cell arrest and apoptosis of cancer cells, using primary cultures of human gliomas as a model. We had previously demonstrated the structural relationship between Gas1 and the alpha receptors for the Glial-cell line-Derived Neurotrophic Factor (GDNF) family of ligands, and showed that Gas1 acts by inhibiting the intracellular signaling induced by GDNF. There are also reports indicating that Gas1 positively cooperates with Sonic Hedgehog (Shh) during embryonic development and in this paper we analyzed the potential interactions between Gas1 and Shh. We show that human gliomas do not express Shh, whereas GDNF and the molecular components necessary to transduce its signaling are present in human gliomas. Furthermore, the over-expression of Gas1 induces cell arrest, apoptosis and prevents the activation of Akt, a crucial mediator of survival and cellular proliferation pathways. In the present work, we present evidence demonstrating that Gas1 exerts its effects inhibiting cell growth and inducing apoptosis of glioma cells in the absence of Shh. PMID:19460624

  17. 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.

  18. 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. PMID:25957688

  19. 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

  20. Redox activity of urban quasi-ultrafine particles from primary and secondary sources

    NASA Astrophysics Data System (ADS)

    Verma, Vishal; Ning, Zhi; Cho, Arthur K.; Schauer, James J.; Shafer, Martin M.; Sioutas, Constantinos

    2009-12-01

    To characterize the redox activity profiles of atmospheric aerosols from primary (traffic) and secondary photochemical sources, ambient quasi-ultrafine particles were collected near downtown Los Angeles in two different time periods - morning (6:00-9:00 PDT) and afternoon (11:00-14:00 PDT) in the summer of 2008. Detailed chemical analysis of the collected samples, including water-soluble elements, inorganic ions, organic species and water soluble organic carbon (WSOC) was conducted and redox activity of the samples was measured by two different assays: the dithiothreitol (DTT) and the macrophage reactive oxygen species (ROS) assays. Tracers of secondary photochemical reactions, such as sulfate and organic acids were higher (2.1 ± 0.6 times for sulfate, and up to 3 times for the organic acids) in the afternoon period. WSOC was also elevated by 2.5 ± 0.9 times in the afternoon period due to photo-oxidation of primary particles during atmospheric aging. Redox activity measured by the DTT assay was considerably higher for the samples collected during the afternoon; on the other hand, diurnal trends in the ROS-based activity were not consistent between the morning and afternoon periods. A linear regression between redox activity and various PM chemical constituents showed that the DTT assay was highly correlated with WSOC ( R2 = 0.80), while ROS activity was associated mostly with water soluble transition metals (Vanadium, Nickel and Cadmium; R2 > 0.70). The DTT and ROS assays, which are based on the generation of different oxidizing species by chemical PM constituents, provide important information for elucidating the health risks related to PM exposure from different sources. Thus, both primary and secondary particles possess high redox activity; however, photochemical transformations of primary emissions with atmospheric aging enhance the toxicological potency of primary particles in terms of generating oxidative stress and leading to subsequent damage in cells.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Panax ginseng induces human Type I collagen synthesis through activation of Smad signaling.

    PubMed

    Lee, Jongsung; Jung, Eunsun; Lee, Jiyoung; Huh, Sungran; Kim, Jieun; Park, Mijung; So, Jungwoon; Ham, Younggeun; Jung, Kwangseon; Hyun, Chang-Gu; Kim, Yeong Shik; Park, Deokhoon

    2007-01-01

    Skin aging appears to be principally related to a decrease in levels of Type I collagen, the primary component of the dermal layer of skin. It is important to introduce an efficient agent for effective management of skin aging; this agent should have the fewest possible side effects and the greatest wrinkle-reducing effect. In the course of screening collagen production-promoting agents, we obtained Panax ginseng C.A. Meyer. This study was designed to investigate the possible collagen production-promoting activities of Panax ginseng C.A. Meyer root extract (PGRE) in human dermal fibroblast cells. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. In this assay, PGRE activated human COL1A2 promoter activity in a concentration-dependent manner. Human Type I procollagen synthesis was also induced by PGRE. These results suggest that PGRE promotes collagen production in human dermal fibroblast cells. Additionally, we have attempted to characterize the mechanism of action of PGRE in Type I procollagen synthesis. PGRE was found to induce the phosphorylation of Smad2, an important transcription factor in the production of Type I procollagen. When applied topically in a human skin primary irritation test, PGRE did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that PGRE may be considered as an attractive, wrinkle-reducing candidate for topical application. PMID:16890388

  6. AN OVERVIEW OF HUMAN EXPOSURE MODELING ACTIVITIES AT THE U.S. EPA'S NATIONAL EXPOSURE RESEARCH LABORATORY

    EPA Science Inventory

    The computational modeling of human exposure to environmental pollutants is one of the primary activities of the US Environmental Protection Agency (USEPA)'s National Exposure Research Laboratory (NERL). Assessment of human exposures is a critical part of the overall risk assessm...

  7. Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

    SciTech Connect

    Bumpus, Namandje N.

    2011-12-15

    Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibition of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and 8

  8. 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

  9. Focus-of-attention for human activity recognition from UAVs

    NASA Astrophysics Data System (ADS)

    Burghouts, G. J.; van Eekeren, A. W. M.; Dijk, J.

    2014-10-01

    This paper presents a system to extract metadata about human activities from full-motion video recorded from a UAV. The pipeline consists of these components: tracking, motion features, representation of the tracks in terms of their motion features, and classification of each track as one of the human activities of interest. We consider these activities: walk, run, throw, dig, wave. Our contribution is that we show how a robust system can be constructed for human activity recognition from UAVs, and that focus-of-attention is needed. We find that tracking and human detection are essential for robust human activity recognition from UAVs. Without tracking, the human activity recognition deteriorates. The combination of tracking and human detection is needed to focus the attention on the relevant tracks. The best performing system includes tracking, human detection and a per-track analysis of the five human activities. This system achieves an average accuracy of 93%. A graphical user interface is proposed to aid the operator or analyst during the task of retrieving the relevant parts of video that contain particular human activities. Our demo is available on YouTube.

  10. Development of Functional Microfold (M) Cells from Intestinal Stem Cells in Primary Human Enteroids

    PubMed Central

    Rouch, Joshua D.; Scott, Andrew; Lei, Nan Ye; Solorzano-Vargas, R. Sergio; Wang, Jiafang; Hanson, Elaine M.; Kobayashi, Masae; Lewis, Michael; Stelzner, Matthias G.; Dunn, James C. Y.; Eckmann, Lars; Martín, Martín G.

    2016-01-01

    Background & Aims Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer’s patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. Methods Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. Results Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. Conclusions Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium

  11. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  12. Observation of Children's Physical Activity Levels in Primary School: Is the School an Ideal Setting for Meeting Government Activity Targets?

    ERIC Educational Resources Information Center

    Waring, Michael; Warburton, Peter; Coy, Martin

    2007-01-01

    Given the commitment (and funding) by the British government to promote physical activity among all ages, and despite the inevitable political manipulation of physical education (PE) and school sport, there is now an ideal opportunity to focus on primary schools as a key target group for the future. This study determined the physical activity…

  13. RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

    PubMed Central

    Kheradmand Kia, Sima; Verbeek, Elly; Engelen, Erik; Schot, Rachel; Poot, Raymond A.; de Coo, Irenaeus F.M.; Lequin, Maarten H.; Poulton, Cathryn J.; Pourfarzad, Farzin; Grosveld, Frank G.; Brehm, António; de Wit, Marie Claire Y.; Oegema, Renske; Dobyns, William B.; Verheijen, Frans W.; Mancini, Grazia M.S.

    2012-01-01

    Polymicrogyria is a malformation of the developing cerebr