10 research outputs found

    CLL derived exosomes modulate the gene expression profiles of HS-5 cells and alter the rate of their proliferation.

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    <p><b>(A)</b> HS-5 cells were treated with MEC-1 exosomes for 24hrs and a pathway array analysis of cellular RNA was performed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141429#pone.0141429.s002" target="_blank">S2 File</a>). Untreated HS-5 cells ‘spiked’ with equivalent doses of MEC-1 exosomes served as control. Validation of c-fos and ATM expression level changes determined by additional RT-qPCR is shown. Error bars represent ± S.E.M. of three independent experiments. <i>p</i> values indicating level of significance are shown. <b>(B)</b> Exosomes influence c-fos activation in recipient cells: c-fos heterodimer complex DNA binding activity was detected using an ELISA transcription factor assay kit (Active Motif). HS-5 cells were cultured alone or with exosomes as indicated. Nuclear extracts from MEC-1 and CLL exosome treated HS-5 cells show increased c-fos activation (p = 0.009 and p = 0.06 respectively). <b>(C)</b> HS-5 cells plated at a density of 3000 cells/well were incubated in the absence or presence of exosomes derived from primary CLL cell culture medium (CLL CM), CLL patient plasma or normal healthy donor plasma, (150ug/ml) and proliferation measured after 48 hrs using the CyQuant assay. Paired t-test was used to calculate p values. All data represent mean ± S.E.M of triplicate experiments.</p

    Characterization of CLL derived exosomes.

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    <p><b>A)</b> Transmission electron microscopy (TEM) image of exosomes enriched from CLL cell culture medium after 48 hrs. (a) Exosomes are visible as small 50–100 nm vesicles with bi-layered membranes (scale bar: 100 nm). (b) Exosomes were immune-isolated using magnetic beads coated with anti-HLA antibody and ultrathin sections of resultant beads processed for image analysis. The representative TEM image shows a bead coated with exosomes (scale bar: 0.2<b>μ</b>m). <b>B</b>) AFM images of exosomes from a representative CLL case immobilized on a mica surface using Peakforce Tapping mode (Multimode 8, Bruker). A) Topographic image of exosomes. <b>B</b>) DMT Modulus image C) Adhesion image: The exosomes appear as circular biconvex vesicular structures. The DMT Modulus and adhesion images of exosomes show explicit sub-structures at the centre of the vesicles (arrows). D) Schematic cross-section following the line in the indicated exosome in C). <b>C)</b> FACS analysis of B-cell antigens on CLL exosomes prepared from primary CLL cases by density ultracentrifugation and adsorbed onto 4<b>μ</b>m aldehyde-sulphate latex beads and incubated with isotype control, anti-HLA-A, B, C or CD19 antibodies followed by FITC-conjugated secondary antibody. Mean fluorescence intensities are plotted (mean ± standard error of mean (S.E.M.) (n = 9)). <b>D)</b> Representative analysis showing surface expression of CD81, HLA-DR, CD37, and integrin α4 (ITGA4) in CLL derived exosomes coupled to aldehyde-sulphate latex beads. Binding of FITC-conjugated isotype controls are included for comparison. <b>E)</b> Immunoblot analysis of CLL derived exosomes: Lysates from CLL exosome were probed for abundance of HLA-DR, MHC, IgM, Gp96, Lyn kinase, TSG 101 and Calnexin. Images are representative of analyses of 3 cases. <i>WCE-Whole cell extract</i>.</p

    MicroRNA profiles and RT-qPCR of purified exosomes and parental CLL cells.

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    <p><b>A)</b> A volcano plot of LNA array miRNA profiles of CLL cells vs exosomes samples is depicted. The x-axis shows the Log2 fold-change in miRNA expression between cellular cases and exosomes. The y-axis shows the -Log10 of the p value for each miRNA. Expressed miRs that are statistically significant between the two groups appear above the line (p < 0.05). <b>B)</b> The miRs let-7g, miR-21, miR-29a, miR-29b, and miR-26a were selected for validation of LNA array by RT-qPCR analysis (n = 5). Data represent mean ± S.E.M of triplicate experiments. <b>C)</b> miR-202-3p is enriched in exosomes compared to parental cells in the LNA array data (mean ± S.E.M). <b>D)</b> Fold change of miR expression in exosomes vs cellular CLL samples showing the enrichment of miR-202 by RT-qPCR in two representative cases.</p

    Expression of miRs 202-3p, 29a, and 21 in exosomes derived from plasma of CLL patients versus normal donors.

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    <p><b>(A)</b> Exosomes sourced from plasma obtained from CLL patients or healthy donors (n = 3 each) were subjected to RT-qPCR analysis for absolute levels of miR-202-3p using a standard curve method. The levels of miR-202-3p are significantly higher in exosomes purified from CLL plasma (p = 0.03). <b>(B)</b> Absolute levels of miR-29a and miR-21 in exosomes harvested from CLL patient and healthy donor plasma (n = 3) were determined by RT-qPCR analysis. Standard curves for miR-29a and miR-21 were generated using the miRVana<sup>TM</sup> miRNA reference panel v9.1. Statistical analysis was performed using an unpaired t-test and yielded <i>p values</i> of 0.01 and 0.2 for miR-29a and miR-21 respectively.</p

    CLL-derived exosomes are internalised by stromal cells and localise to late endosomes.

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    <p>Exosomes purified from CLL cell culture supernatants were labelled with the lipophilic dye PKH67. HS-5 stromal cells were cultured in the absence (control) or presence of fluorescent-labelled exosomes for 24hrs. Paraformaldehyde fixed HS-5 cells were permeabilised and stained for Lamp-1 or TSG-101, followed by FITC-conjugated secondary antibody and visualised by fluorescence microscopy (original magnification, ×100). Nuclei were stained using DAPI. Internalised exosomes co-localise with Lamp-1, a marker for late endosomes but not with TSG-101. A) Exosomes were internalised by HS-5 Cells and visualized for PKH67 (green), Lamp-1 (red) and DAPI (blue). Co-localisation of Lamp-1 and PKH67 appear in yellow. B) HS-5 Cells were visualized for PKH67 (green), TSG-101 (red) and DAPI (blue) and do not show co-localisation C) As controls. HS-5 cells were stained with free PKH67 dye without exosomes or antibody isotype control to exclude non-specific binding. (Scale bars: 10<b>μ</b>m).</p

    Airway leukocyte number following LESB65 infection.

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    <p>Average B lymphocyte, CD4+ve T cell, CD8+ve T cell and neutrophil count shown as mean and standard deviation per mg of tissue at 24, 48, and 72 hours post infection [N = 5 each time point]. * = p<0.05, ** = p<0.01.</p

    Expression of BAFF, CXCL13, CCL19 and CCL21 in mouse lung tissue.

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    <p>Each cytokine was measured at days 1, 2, 3 and 7 days after infection with 10<sup>6</sup> CFU of <i>P. aeruginosa</i> LESB65 by ELISA. [n = 5]. BAFF was significantly expressed at days 1 [p<0.001], 2 [p<0.01], 3 [p<0.001], and 7 [p<0.05] in comparison to day 0 control. CXCL13 at days 1, 2, 3, [all p<0.001] and 7 [p<0.01]. CCL19 at days 1 [p<0.01], 2 [p<0.001], and 7 [p<0.001]. CCL21 at days 2 [p<0.001], 3 [p<0.01] and 7 [p<0.001]. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

    BAFF expression in BAL fluid.

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    <p>Expression was measured by ELISA in BAL fluid from <i>P. aeruginosa</i> positive [n = 6] and negative [n = 14] CF patients and healthy controls [n = 7]. BAFF expression was significantly elevated [p<0.01] in both the PA infected group and non PA infected group in comparison to the control group.</p

    FACS analysis of lung inflammatory cells following <i>P. aeruginosa</i> LESB65 infection of mice.

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    <p>Representative FACS plots showing immune cell staining of airway cells. Each column shows plots from an individual mouse at each time point. All plots are lung CD45+ cells and gates were drawn relative to isotype control antibody staining for each marker. Top row shows B cells [CD45+, CD19+], middle row shows CD4 T cells [CD45+, CD3+, CD4+] and CD8 T cells [CD45+, CD3+, CD8+], bottom row shows neutrophils [CD4+, Gr-1hi, F4/80-] and monocytes/macrophages [CD45+, F4/80+, Gr-1int/low]. Numbers on gates are percentages of total CD45+ cells.</p

    Kinetics of airway infection by <i>P. aeruginosa</i> LESB65.

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    <p>Mice were challenged [intranasal] with 10<sup>6</sup> CFU. Values shown as an average Log CFU/mg of lung tissue homogenised, n = 5 mice at each time point. Time zero samples were taken at 15 minutes post infection to confirm entry into the lung and determine levels immediately post infection.</p
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