text:
Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2
Aled Clayton, J. Paul Mitchell, Jacquelyn Court, Malcolm D. Mason and Zsuzsanna Tabi
Aled Clayton
J. Paul Mitchell
Jacquelyn Court
Malcolm D. Mason
Zsuzsanna Tabi
DOI: 10.1158/0008-5472.CAN-06-3456 Published August 2007
Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4+ T cells, CD8+ T cells, and NK cells revealed that CD8+ T-cell proliferation was not inhibited in the absence of CD4+ T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2–mediated CD25 up-regulation, affecting all but the CD3+CD8− T-cell subset. IL-2–induced Foxp3 expression by CD4+CD25+ cells was not inhibited by tumor exosomes, and the suppressive function of CD4+CD25+ T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell–independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor β1 (TGFβ1), which contributed to the antiproliferative effects, shown by using neutralizing TGFβ1-specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated "double hit" to cellular immunity strongly implicates the role of exosomes in tumor immune evasion. [Cancer Res 2007;67(15):7458–66]
Tumor resistance to immune response
Exosomes are a population of nanometer-sized vesicles, actively secreted by a diverse range of living cells, with physiologic functions that include immune modulation ( 1). Several reports document the immune-activating capacity of exosomes isolated from dendritic cells ( 2– 5) and other cell types ( 6, 7), yet cells such as intestinal epithelia may produce tolerogenic exosomes ( 8).
The immunology of tumor exosomes is poorly understood, with reported activatory ( 9) and inhibitory effects ( 10); differences are probably dependent on the exosome phenotype. Exosomes are produced in advanced human malignancies, shown by the analysis of malignant effusions ( 11, 12), and likely enter the circulating exosome pool ( 13). However, patients with malignant tumor effusions do not spontaneously mount a protective antitumor immune response, suggesting that the immune system is either insufficiently activated or nonresponsive to tumor exosomes in vivo. Another possibility is that tumor exosomes actively impair immune responses, and this is the subject of the current report.
Interleukin-2 (IL-2) is of central importance in the homeostasis of lymphoid cells and in promoting the survival, proliferation, and functional differentiation of several lymphocyte subsets, including natural killer (NK) cells, CD8+ T cells, and CD4+CD25+ regulatory T cells (Treg; refs. 14– 16). Here, the responses of peripheral blood lymphocytes (PBL), obtained from healthy donors, to IL-2 in the absence or presence of tumor exosomes was compared. We show impaired lymphocyte proliferation in response to IL-2 in the presence of tumor exosomes. This inhibition requires the CD4+ T-cell subset. We highlight a novel mechanism in which IL-2–mediated up-regulation of CD25 is selectively inhibited by tumor exosomes in NK cells and CD8+ CTL but not CD8− T cells. The cytotoxic function of NK cells was impaired following tumor exosome treatment, but CD4+CD25+ Treg cells remained IL-2 responsive through induction of Foxp3 expression, and their inhibitory function was enhanced by tumor exosomes. Our data show that tumor exosomes express membrane-associated transforming growth factor β1 (TGFβ1), which contributes to the antiproliferative effects. Exosomes may therefore be physiologically important for skewing immune responses away from cytotoxic effector mechanisms toward Treg cell responses, contributing to tumor immune escape.
Tumor cells and exosome purification. Data presented were obtained with tumor cell line–derived exosomes (mesothelioma cell line established within the department from pleural fluid; obtained from a patient with advanced pleural malignant mesothelioma). Additional tumor cell lines were used in some experiments, as stated, including prostate cancer cells (DU145 and PC3), the myelogenous leukemia line K562, the T-cell lymphoma Jurkat (from EACC), and the natural killer cell line NKL (ref. 17; a gift from Dr. E. Wang, Medical Biochemistry and Immunology, Cardiff University, Cardiff, United Kingdom). EBV-immortalized B cells were prepared as described ( 18). Cultures were confirmed free of Mycoplasma by monthly screening by PCR (purchased from Minerva Biolabs). Cells were maintained in RPMI 1640 supplemented with 10% exosome-depleted fetal bovine serum and antibiotics. Medium from confluent cultures (5–7 days) was depleted of cells and debris by centrifugation before ultracentrifugation (LE80K, Beckman Coulter) on a 30% sucrose/D2O cushion ( 19). The cushion was collected, diluted in excess PBS, and exosomes were pelleted by ultracentrifugation. The protein content of each preparation was quantified using the bicinchoninic acid protein assay (Pierce).
Electron microscopy. A method similar to that previously reported ( 6) was used to visualize exosome preparations. Briefly, frozen exosomes were thawed on ice and resuspended in 1% glutaraldehyde in PBS (pH 7.4). A 5-μL drop of the suspension was pipetted onto a pioloform-coated copper grid and allowed to stand for 5 min at room temperature. The grid was transferred to a 50-μL drop of double distilled water for 2 min and this process was repeated seven times for a total of eight water washes. The sample was stained on ice for 10 min with a 5-μL drop of 2% methyl cellulose containing 2% uranyl acetate. Excess fluid was removed with a piece of Whatman filter paper and allowed to air dry for 10 min before viewing by transmission electron microscopy (Philips EM 208, FEI).
Western blotting. Ten micrograms of whole-cell lysates were prepared as previously described ( 20), or 10 μg of purified exosomes were made to 40-μL final volume in sample buffer (6 mol/L urea, 2% SDS, 30% glycerol, 50 mmol/L Tris-HCl, and freshly added 5% v/v β2-mercaptoethanol). Samples were subjected to electrophoresis through 10% bis-acrylamide gels and transferred to polyvinylidene difluoride membranes. Following overnight blocking in 3% nonfat milk in 0.5% Tween 20 in PBS (PBS-T), primary antibody (added at 0.2–2 μg/mL) was added for 1 h, followed by three washes in PBS-T. Molecular weight markers (Cruz Marker, Santa Cruz Biotechnology, Inc.) and primary antibodies were detected using Cruz Marker–compatible goat anti-mouse immunoglobulin G-horseradish peroxidase (IgG-HRP) conjugated at 1:12,000 dilution in PBS-T for 30-min incubation followed by five washes in PBS-T. Bands were visualized using the ECL+ system (GE Healthcare). The antibodies used included TGS101, lysosomal integral membrane protein 1 (LAMP-1), intercellular adhesion molecule (ICAM)-1, β1-integrin, heat shock protein (hsp)-70, hsp90, Her-2/neu, histone H1, and calnexin-specific monoclonal antibodies (mAb; Santa Cruz Biotechnology). Anti-CD81 and anti-CD9 mAbs (Santa Cruz Biotechnology) were used on samples prepared with nonreducing conditions. Anti–MIC-A and anti–MIC-B were from R&D Systems. Anti-mesothelin and anti-acetylcholinesterase mAbs were from Abcam Plc. Anti–class I (clone HC10) was a gift from the Medical Research Council Co-operative, Cardiff University. Rat anti-grp94 (gp96; Stressgen) was detected using antirat IgG-HRP conjugated antibody (Dako) at 1:15,000 dilution in PBS-T. In this case, molecular weight markers (high-range rainbow markers) were from Bio-Rad.
Isolation and analysis of lymphocytes. PBL from healthy donors, purified by Histopaque (Sigma), were subjected to 2-h adhesion to plastic to remove adherent monocytes. The nonadherent cells were used in subsequent experiments. Multicolor flow cytometry (FACSCanto, Becton Dickinson) running FACSDiva software was done with antibodies from Becton Dickinson: CD3-allophycocyanin (APC), CD8-phycoerythrin (PE)-Cy7, CD4-APC-Cy7, CD56-PE-Cy5, and CD16-PE-Cy5 (used together) and CD25-FITC. Foxp3 analysis was done using eBioscience kit following the manufacturer's instructions. NK cells were defined by the lack of CD3 expression and as CD56+CD16−, CD56+CD16+, or CD56−CD16+. Purification of lymphocyte subsets was done using CD4+ or CD8+ T-cell negative isolation kits (Miltenyi Biotec) following the manufacturer's instructions. CD56+ NK cells were purified first by depleting CD3+ T cells, followed by positive isolation of CD56+ cells (Miltenyi Biotec).
Lymphocyte proliferation and cytotoxicity assay. Lymphocytes were treated with recombinant human IL-2 (0–500 units/mL; Proleukin) or phytohemagglutinin (PHA; Sigma) to trigger proliferation. In some experiments, TGFβ1 (from Peprotech) was added as an inhibitor of proliferation/cytotoxicity. Proliferating cells in 96-well plates were pulsed with 1 μCi/well of [3H]thymidine for the final 18 h of cultures before harvesting and measuring [3H]thymidine incorporation (Microbeta-3, Perkin-Elmer). For NK cytotoxicity assays, NK cells were enriched (using anti-CD3 antibody–coated Dynal beads), and the remaining population (confirmed as >79% CD3− by flow cytometry) was used in standard 4-h 51Cr release assays with K562 cells as targets.
Treg cell isolation and in vitro suppression assay. Healthy donor PBL (50 × 106) were subjected to CD4+CD25+ isolation using a Treg isolation kit (Miltenyi Biotec) following the manufacturer's instructions. Fractions before and after isolation were compared by flow cytometry, staining with anti–CD4-APCCy7 (Becton Dickinson) and anti–CD25-PE (Miltenyi Biotec), to assess purity. The CD4+CD25+ fraction was left untreated for 24 h or treated with IL-2 (250 units/mL) and/or exosomes (10 μg/105 cells). The cells were washed in 10× volume (in culture medium) to remove IL-2 and exosomes and added back (at 2 × 103, 10 × 103, or 50 × 103 per well) to unfractionated PBL from the same donor (100 × 103 per well). Proliferation was then initiated with 1 μg/mL PHA (Sigma) and, after 3 days, [3H]thymidine incorporation was measured.
ELISA for IL-10 and TGFβ1. ELISA development reagents (duo-set kit) for human IL-10 and human TGFβ1 were purchased from R&D Systems, with subsequent assays done as recommended by the manufacturer. Blocking, substrate, and stopping solutions were purchased from R&D Systems. High-binding microtitre ELISA strips were from Greiner. In TGFβ1 colocalization experiments, antihuman CD81 mAb (carrier protein free; Becton Dickinson) was substituted for the TGFβ1 capture antibody, with the remainder of the assay done exactly as for the TGFβ1 ELISA. Absorbance was measured with wavelength correction (A450–A540 nm) on a 3550 Microplate Reader (Bio-Rad).
TGFβ1 neutralization. Lymphocytes were treated with IL-2 (250 units/mL) to trigger proliferation in the presence or absence of recombinant human TGFβ1 (5 ng/mL) or tumor exosomes (10 μg/105 cells). In some wells, increasing concentrations (up to 10 μg/mL) of a well-characterized TGFβ1 neutralizing antibody (clone 141322, R&D Systems; refs. 21, 22) were added. After 3 days, the degree of proliferation was assessed by measuring [3H]thymidine incorporation.
Statistical analysis. All data are represented as mean ± SE. Comparisons between exosome-treated and nontreated groups were done using paired t tests, calculated using graphing and statistical software Prism 4 (version 4.03) from GraphPad.
Tumor exosome characterization. Purification of exosomes derived from cultured cancer cells (of various types) revealed small (30–100 nm in diameter) vesicular structures, typical of exosomes in general ( 2, 6, 23), by electron microscopy (Supplementary Fig. S1A). Preparations were free of contaminating membranous fragments or electron dense apoptotic bodies (as previously described; ref. 24). The molecular phenotype of the exosomes was also typical of exosomes from other sources. Comparing whole-cell lysates with exosome preparations revealed an absence of endoplasmic reticulum–resident proteins (calnexin and grp94) or nuclear proteins (histone H1) from the exosomes. Yet, exosomes were relatively enriched in the multivesicular body marker TSG101 and the GPI-anchored protein acetyl-cholinesterase (Supplementary Fig. S1B). Exosomes also stained positively for CD9, CD81, LAMP-1, glyceraldehyde-3-phosphate dehydrogenase, hsp70, hsp90, β1-integrin, and ICAM-1. Tumor-associated antigens, such as Her-2/neu and mesothelin, and MHC molecules including members of the MICA-family were also exosomally expressed (Supplementary Fig. S1C).
The effect of tumor exosomes on IL-2–mediated lymphocyte proliferation. We first investigated the effect of IL-2 on lymphocyte proliferation in the presence or absence of tumor exosomes. In the presence of exosomes, IL-2–mediated proliferation was severely impaired, measured by a marked inhibition of [3H]thymidine incorporation, after only 48 h following IL-2 treatment (Supplementary Fig. S2A). CFSE labeling and multicolor flow cytometry revealed that the antiproliferative effect of tumor exosomes affected CD3+CD8+, CD3+CD8−, and CD3− lymphocytes (not shown). Flow cytometry also revealed an exosome-driven impairment in morphologic changes (assessed by increased forward scatter/side scatter) associated with IL-2–driven lymphocyte blast formation (Supplementary Fig. S2B and C). We conclude that tumor exosomes strongly impair proliferative responses to IL-2 in all lymphocyte populations.
The antiproliferative exosome effects are mediated principally by the CD4+ T-cell subset. We next studied whether the antiproliferative effect of exosomes was retained on fractionating peripheral blood lymphocytes into various subpopulations. Using immunomagnetic beads, CD4+ T cells, CD8+ T cells, and CD56+ NK cells were purified and stimulated with IL-2 in the presence or absence of tumor exosomes. The proliferative response was assessed after 3 days ( Fig. 1 ). Tumor exosomes strongly inhibited IL-2–mediated lymphocyte proliferation in unfractionated cells (>67% inhibition at 250 units/mL IL-2 in the experiment shown; Fig. 1A). The antiproliferative effect remained evident in the purified CD4+ T-cell population (50% inhibition; Fig. 1B). Whereas purified CD8+ T cells responded strongly to IL-2, there was no exosome-mediated inhibition of this proliferative response ( Fig. 1C), and the proliferation of CD56+ NK cells was only weakly inhibited by tumor exosomes (24% inhibition; Fig. 1D). The CD56+ NK cell fraction, however, contained ∼10% non-CD56+ cells, which may account for the partial antiproliferative exosome effect. We therefore repeated the experiments using the NK cell line NKL ( Fig. 1E). Although tumor exosomes exerted an antiproliferative effect on NKL cells, this was weak and was undetectable at IL-2 doses of ≥200 units/mL. In contrast, a CD4+ T-cell line, Jurkat, showed high sensitivity to the antiproliferative effects of tumor exosomes ( Fig. 1F). In none of the studies was there evidence of apoptotic death mediated by exosomes or by IL-2 (assessed by Annexin V binding; data not shown). We conclude, therefore, that it is principally through the CD4+ T-cell population that the antiproliferative effect of exosomes is mediated, as CD56+ cells are only weekly inhibited, and the IL-2–driven proliferation of CD8+ T cells is not impaired by direct interactions with tumor exosomes.
The antiproliferative exosome effects are mediated principally by CD4+ T cells. Healthy donor PBL were left unfractionated (A) or purified into CD4+ T-cell (B), CD8+ T-cell (C), or CD3−CD56+ NK cell subsets. Cells (105 per well) were treated with IL-2 [0, 50, or 250 units/mL (A–C) or, due to limiting cell numbers, 0 and 250 units/mL (D)] in the presence or absence of tumor exosomes. Proliferation was assessed by measuring [3H]thymidine incorporation at day 3. Columns, mean of triplicates; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Lymphocyte subset purity, assessed by flow cytometry (histogram insets), was 59.3% CD4+ T cells (A), 95.2% CD4+ T cells (B), 95.4% CD8+ T cells (C), and 89.8% NK Cells (D). Representative of two experiments done with PBL from different donors. E and F, two cell lines (NKL and Jurkat, respectively) were treated in a similar manner.
The effect of exosomes on CD25 expression. We hypothesized that inhibition of IL-2–driven proliferation may be due to exosome-mediated impairment of the IL-2 receptor. We examined possible changes in the expression of the IL-2Rα chain (CD25), which is required for the formation of the high-affinity receptor complex. In unfractionated PBL, IL-2 stimulated a dose-dependent elevation in the proportion of CD25-positive CD8+ T cells, CD8− T cells, and NK cells. In the presence of tumor exosomes, however, this induction was significantly inhibited, but in a cell type–specific manner as it was not seen in CD3+CD8− T cells ( Fig. 2 ). This observation shows that the expression of the high-affinity IL-2 receptor is selectively impaired on cytotoxic effector cells, remaining readily inducible on the Treg cell–containing population, suggesting that Treg cells may selectively remain functional in the presence of tumor exosomes.
Tumor exosomes selectively impair up-regulation of CD25. Lymphocytes were examined for the expression of CD25 6 d after treatment with IL-2 in the presence or absence of tumor exosomes. A, dot plot representation of CD25 expression of CD3+ gated cells. B to D, quantification of changes in the proportion of CD25-positive CD8+ (B), CD8− (C), and NK cells (D), and CD25 expression levels [mean fluorescence intensity (MFI)]. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01. †, in the absence of IL-2, there were <100 events in the NK cell gate. Representative of three experiments done with different donors.
Tumor exosomes do not impair the function of Treg cells. To address whether IL-2–dependent responses, other than proliferation, were retained by the CD3+CD8− population, we examined the effect of IL-2 on the expression of the forkhead transcription factor Foxp3 ( 14), exclusively expressed by Treg cells ( 25). Following 18-h stimulation with IL-2, there was a 4-fold increase in the proportion of Foxp3-positive CD3+CD4+ cells. Importantly, however, there was no exosome-mediated impairment of Foxp3 induction ( Fig. 3A and C ), indicating that this cell population remained sensitive to proliferation-independent IL-2 effects. Yet, in the same samples, IL-2–mediated elevation of CD25 expression was inhibited by exosomes, but again only in the CD4− population ( Fig. 3B and D). Interestingly, addition of exosomes alone (in the absence of IL-2) was sufficient to significantly increase the proportion of Foxp3+ (but not CD25+) CD4+ cells, showing tumor exosomes as a stimulus in their own right for inducing a Treg cell phenotype. We next tested whether the ability of Treg cells to suppress immune responses was affected by exposure to tumor exosomes. CD4+CD25+ Treg cells were purified from fresh PBL ( Fig. 4A ) and were left untreated for 24 h or activated with IL-2 and/or tumor exosomes to elevate Foxp3 expression. The cells were subsequently washed, to remove IL-2 and exosomes, before adding at increasing numbers (2,000–50,000 per well) to 105 unfractionated lymphocytes. Proliferation was initiated by addition of PHA (1 μg/mL) and, 3 days later, measured by [3H]thymidine incorporation. In the absence of PHA, there was minimal background cell division (107 ± 38 cpm) on day 3, whereas PHA stimulated a strong proliferative response (10,461 ± 403 cpm). Purified Treg cells did not proliferate in response to PHA treatment (37 ± 5 cpm). Adding increasing numbers of Treg cells to PHA-stimulated autologous PBL showed their capacity to inhibit proliferation. Of the conditions used to preactivate Treg cells, it was clear that a combination of IL-2 with tumor exosomes significantly enhanced their inhibitory capacity (67 ± 4% inhibition of proliferation) at 1:10 Treg/PBL ratio ( Fig. 4B). Treatment of Treg cells with IL-2 alone (40 ± 3%) or with exosomes alone (41 ± 14%) both stimulated inhibitory functions above that of untreated Treg cells (25 ± 5%). Tumor exosomes, therefore, when combined with IL-2, strongly enhanced the inhibitory functions of Treg cells, and tumor exosomes were a stimulus in their own right, boosting the suppressive function of Treg cells.
IL-2–mediated induction of Foxp3 is not impaired by tumor exosomes. Analysis of Foxp3 (A and C) or CD25 (B and D) expression after 18-h stimulation with IL-2 (250 units/mL) in the presence or absence of tumor exosomes (10 μg/105 PBL). The IL-2–driven induction of Foxp3 expression is not inhibited by tumor exosomes. Similarly, CD25 expression is unhindered by exosomes in the CD4+ cells, but significant CD25 impairment is evident in the CD4− cells. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Representative of three experiments done with different donors.
Treg cells retain inhibitory functions after tumor exosome treatment. CD4+CD25+ Treg cells were enriched (using Miltenyi Treg isolation kit) to 68.5% (A) before 24-h incubation alone, with IL-2 (200 units/mL), combination of IL-2 and exosomes (10 μg/105 Treg), or exosomes alone. Treg cells were washed in 10× volume with RPMI 1640 to remove traces of IL-2 and exosomes before adding (at 2 × 103–50 × 103 per well) to autologous nonadherent PBL (105 per well) in 96-well plates. PHA (1 μg/mL final) was added and proliferation measured after 3 d. Graph depicts proliferation relative to that of PHA-treated cells in the absence of Treg cell addition (set to a value of 1). Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01. Representative of two experiments done with different donors (B).
Tumor exosomes impair cytotoxic cell functions. The consequence of exposure to tumor exosomes on NK cell cytolytic function was next examined. In the absence of exosomes, activation of purified (T-cell depleted) NK cells with 200 units/mL IL-2 for 3 days led to efficient cytotoxic killing of K562 cells. Adding exosomes with IL-2, however, resulted in a significant and dose-dependent reduction in killing ( Fig. 5A ), comparable to that achieved by high-dose (50 ng/mL) TGFβ1. This inhibition could not simply be explained by differences in NK cell numbers after 3-day proliferation with IL-2 because cell numbers were equalized through matched effector/target cell ratios for each group. Furthermore, the depletion of T cells suggests a direct exosome-mediated inhibition of NK cell killing capacity, which is unlikely to involve Treg cell–suppressive effects. These experiments have also been successfully reproduced using the NK cell line NKL ( Fig. 5B), further confirming a Treg cell–independent mechanism for impaired NK killing.
Tumor exosomes inhibit NK cell killing function. NK cell–enriched lymphocytes were treated with tumor exosomes (0.25–4 μg/105 PBL) or TGFβ1 in the presence of 200 units/mL IL-2, and on day 4, the capacity to kill K562 target cells was measured by standard chromium release assay. Graph depicts percent specific K562 lysis at various effector/target cell ratios, as indicated. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01. Representative of three experiments done with different donors (A). Similarly, the cytolytic function of the NKL cell line was tested (B).
Tumor exosomes express TGFβ1. To better understand the molecular mechanisms underlying the inhibitory effects of tumor exosomes, we questioned whether immunosuppressive cytokines were present in our exosome preparations. We focused on the possible expression of IL-10 and TGFβ1 by tumor exosomes because these factors are strongly implicated in immune evasion mechanisms and may be responsible for the antiproliferative (or anticytotoxic) effects observed in our study. IL-10 and TGFβ1 ELISAs were done in parallel on the same samples of exosomes derived from tumor cells. Whereas IL-10 was consistently absent, TGFβ1 was measurable in mesothelioma exosomes (Supplementary Fig. S3A) and in exosomes isolated from other cancer cell types (Supplementary Fig. S3B). The average TGFβ1 content for mesothelioma-derived exosomes was 7.12 ± 1.14 pg TGFβ1/μg exosomes (mean ± SE of five preparations). To ascertain whether TGFβ1 was present as a soluble contaminant or associated with the exosome membrane, we took two approaches: First, freshly prepared exosomes were split into three aliquots. One aliquot (labeled original) was used to assess starting TGFβ1 quantity. An equal aliquot was resuspended in >300× volume of PBS and pelleted at 100,000 × g to wash away any soluble TGFβ1. The final aliquot was incubated at room temperature for 2 h with 2,000 pg/mL recombinant human TGFβ1 before washing. Supplementary Fig. S3C reveals that washing exosomes in vast excess volume of PBS had little effect on TGFβ1 levels. Similarly, exogenously added soluble TGFβ1 did not increase exosomal TGFβ1 levels, suggesting that TGFβ1 was unlikely present as a soluble contaminant in the exosome preparations. In addition, from the TGF spiking experiment, it seems that either TGFβ1 is unable to passively adsorb onto the exosome membrane under these conditions or available binding sites on the exosome surface were already occupied with TGFβ1. We also did a variation of the ELISA technique, where microtitre plates were coated with either TGFβ1 capture antibody or anti-CD81 mAb (to bind exosomes to the plate based on their expression of CD81). Following sample addition and subsequent washing steps, TGFβ1 content was assessed exactly as done for the TGFβ1 ELISA (Supplementary Fig. S3D). Results revealed that the CD81 antibody–coated wells were unable to bind soluble TGFβ1, yet a signal was measured in wells containing exosomes, showing colocalization of CD81 with TGFβ1. We conclude that mesothelioma and other tumor cell exosomes express membrane-associated TGFβ1.
Exosomal TGFβ1 contributes to antiproliferative effects and is more potent than soluble TGFβ1. We investigated whether exosomally expressed TGFβ1 was functional in mediating the antiproliferative effects of tumor exosomes. Lymphocytes were treated with IL-2 (250 units/mL) in the presence of soluble TGFβ1 (5 ng/mL) or tumor exosomes (10 μg/105 PBL). Both conditions resulted in a significant impairment of IL-2–stimulated proliferation. Addition of TGFβ neutralizing antibody (but not irrelevant antibody, not shown) inhibited the antiproliferative effect of soluble TGFβ1 and exosomes in a dose-dependent manner ( Fig. 6A ), showing that TGFβ1 expressed by exosomes was indeed involved in the antiproliferative effect. When comparing the relative potency of soluble TGFβ1 with exosomally delivered TGFβ1 (where 1 μg exosomes = 7 pg TGFβ1 measured by ELISA), it was evident that exosomal TGFβ1 achieved a potent inhibitory effect at significantly lower doses than soluble TGFβ1 ( Fig. 6B).
Exosomal-TGFβ1 is functional and potent. A, lymphocyte proliferation, triggered with 250 units/mL IL-2, was inhibited by ∼50% after the addition of soluble TGFβ1 (5 ng/mL) or tumor exosomes (10 μg/105 PBL). Adding TGFβ neutralizing antibody (0.1–10 μg/mL) restored the proliferative response in a dose-dependent manner. Results of 3-d [3H]thymidine incorporation assay, presented as percent inhibition of the IL-2 response. Columns, mean (n = 3); bars, SE. B, exosomes (1 or 10 μg/105 PBL) or TGFβ1 (0.5–10 ng/mL) was added to IL-2–treated (50 units/mL) lymphocytes and proliferation assessed 3 d later. Percent inhibition of the IL-2–mediated proliferation response against TGFβ1 dose, assuming 1 μg exosomes = 7 pg TGFβ1. Points, mean (n = 3); bars, SE.
The priming of cytotoxic NK and CD8+ T cells in readiness for killing function is driven by IL-2 but, paradoxically, Treg cells and their immune suppressive functions are also supported by IL-2 ( 15). Evidence from the clinic has shown that, in certain cancers, administering IL-2 as therapy can boost Treg cells and contribute to tumor immune escape ( 16). Our study investigated whether exosomes produced by cancer cells were involved in modulating T-cell and NK cell responses to IL-2. Tumor exosomes had the capacity to strongly inhibit IL-2–driven lymphocyte proliferation, preventing blast formation, in all subpopulations. On isolation of lymphocyte subsets, however, it became clear that although NK cell proliferation was weekly impaired following direct interactions with exosomes, the principal antiproliferative effect is mediated through the CD4+ T-cell subset, as CD8+ T cells were impervious to the antiproliferative effects of tumor exosomes in the absence of CD4+ T cells.
When we investigated more subtle aspects of this phenomenon, we discovered a unique selectivity in this exosome effect. IL-2 induces the expression of the high-affinity IL-2Rα chain (CD25), and this occurs in CD3+CD8+ T cells, CD3+CD8− T cells, and CD3− CD56+/16+ NK cell subpopulations. However, in the presence of tumor exosomes, this CD25 induction is markedly inhibited in all but the CD3+CD8− T cells. Further phenotypic analysis revealed that in the presence of tumor exosomes, the Treg cell–containing population remained capable of responding to IL-2, exemplified by induced Foxp3 expression. The Treg cell–mediated inhibition of proliferation was enhanced by tumor exosomes and further boosted when added in conjunction with IL-2. In contrast, freshly isolated NK cells activated with IL-2 exhibited strong cytotoxicity against K562 targets, but this function was significantly impaired following tumor exosome treatment through a mechanism independent of Treg cells. Our data therefore identify a novel mechanism by which tumors may drive immune responses away from cytotoxic effector mechanisms while not impairing, but supporting, Treg cell activities.
The exosomes in our study were from cultured mesothelioma cell lines, isolated from tissue samples or pleural fluid taken from patients with advanced malignant pleural mesothelioma. These exosomes may therefore represent an extreme tumor exosome type, being derived from a highly aggressive cancer type, with patient's survival after diagnosis typically ∼9 to 12 months ( 26). Yet the exosome morphology was similar to those from other (tumor and nontumor) sources, and they expressed numerous markers typical of exosomes in general ( 27) as well as tumor-associated proteins such as Her-2/neu ( 11, 23), mesothelin ( 28), and MICA/B ( 29). In this sense, therefore, these are unremarkable tumor exosomes, and it may be that our observation is equally applicable across multiple tumor cell types. We have indeed observed the inhibition of lymphocyte proliferation (stimulated by various nonphysiologic mitogens such as PHA and CD3/CD28 antibody–coated beads) using exosomes isolated from mesothelioma and other tumor types: breast carcinoma (T47D), prostate cancer (DU145 and PC3), and an EBV-immortalized B cells. 3 Whether such diverse exosome types can selectively impair IL-2 responses, as we have shown here, remains to be determined, and this is the subject of ongoing investigations in our laboratory. Other researchers have also previously described the inhibition of lymphocyte proliferation by exosomes. For example, EBV-encoded latent membrane protein-1 is expressed by exosomes secreted by EBV-immortalized B lymphocytes. These exosomes were able to impair proliferative responses to PHA and CD3/CD28 beads in a manner that may have been latent membrane protein-1 dependent ( 30). This molecule is not expressed by non-EBV malignancies and is therefore not sufficient to explain what seems to be an antiproliferative tumor exosome effect in general.
Suppressive factors such as IL-10 or TGFβ are produced by many tumor types and may mediate antiproliferative and anticytotoxic effects similar to those we observed for tumor exosomes. When we examined the possible presence of these cytokines in our exosome preparations, we found that IL-10 was not present at detectable levels but TGFβ1 was consistently found in tumor exosomes (from multiple tumor types). Further studies showed that exosomes express a membrane-associated form of TGFβ1 that could not be removed by ultracentrifugation washing steps. This raised the possibility that tumor exosomes were mediating at least some of their effects through delivery of TGFβ1 to lymphocytes. Administering a well-characterized TGFβ1 neutralizing antibody to IL-2– and exosome-treated lymphocytes partially restored proliferative capacity, revealing that exosomal TGFβ1 was indeed functional and thus contributes to the inhibitory effect.
The amount of TGFβ1 present in mesothelioma-derived exosomes was very low, at 7 pg/μg of exosomes. In our experiments, therefore, the effective dose of exosomal TGFβ1 with which lymphocytes were treated was 350 pg/mL. When we compared the effects of soluble TGFβ1 on lymphocyte proliferation, it was clear that this dose was not sufficient to suppress IL-2–mediated proliferative responses. In fact, 0.35 pg/mL exosomal TGFβ1 administered to lymphocytes stimulated with 50 units/mL IL-2 mediated an antiproliferative effect comparable to 5,000 pg/mL soluble TGFβ1. This suggested that TGFβ1 presented in the form of exosomes was ∼1,400 times more potent than the soluble form under these conditions. Differences in the cellular response to soluble TGFβ1 were also evident, such as a general impairment of CD25 (IL-2Rα) expression (not shown) rather than the cell type–selective effect obtained with exosomes. Enhanced potency of membrane versus soluble TGFβ1 in other systems has previously been described ( 31). It may arise from sustained signaling by membrane TGFβ resulting in distinctive intracellular signaling events (such as Notch1 activation), which do not occur with soluble TGFβ. It is possible that exosomal TGFβ1 achieves similar sustained signaling effects that explain their relative functional potency. The role of other exosomal molecules in assisting the delivery of TGFβ1 to lymphocytes and in directing the cell type selectivity, which we report here, clearly warrants further investigations. Furthermore, the nature of TGFβ1 anchorage to the exosome membrane remains unknown, and the possible involvement of latent associated peptide ( 32, 33) or β-glycan ( 34, 35), as in other systems, requires clarification.
Previous reports have also shown that exosome-NK cell ( 9) and - T-cell ( 36, 37) interactions are possible, with outcomes dependent on the exosome phenotype. Although antigen presenting cell–derived exosomes may directly activate T-cell responses ( 36), direct interactions between T cells and tumor exosomes seem to be inhibitory. For example, Andreola et al. ( 37) have suggested that Fas ligand–positive exosomes may directly deliver death signals to activated (Fas-positive) T cells. They suggest this as a possible mechanism by which tumors delete tumor-specific T cells in vivo, and similar studies have also been published more recently ( 38). Other reports suggest that tumor exosomes are responsible for defective signaling responses in T cells by down-modulating CD3-ζ and Janus-activated kinase 3, resulting in apoptosis ( 39), although many of these studies relied on immortalized T-cell lines (Jurkat cells), rather than freshly isolated T cells, and very high doses of exosomes (400 μg/mL). In none of the experiments presented in this report, or in allied investigations within our laboratory, was there evidence of exosome-mediated loss in CD4+ or CD8+ T cells, indicating that Fas ligand–driven T-cell death is not a universal consequence of tumor exosome interaction with T cells.
A recent report by Gastapar et al. ( 9) highlighted a role for hsp70 expression by tumor exosome in their interactions with NK cells. Their experimental system involved colorectal cancer cell lines, producing either hsp70 surface positive exosomes or a subline (otherwise identical) producing hsp70 surface negative exosomes. The former exosomes were potent at activating the migratory and cytolytic functions of NK cells, whereas the hsp70 negative exosomes remained nonactivating. Thus, even with such a closely controlled experimental system, differences in the phenotype of the exosomes were absolutely key to their immunologic effects. Although the mesothelioma-derived exosomes used in our study were hsp70 positive (by Western blot), it does not necessarily follow that hsp70 was present at the exosome surface, as we showed for other exosome types, even after robust cell stress ( 20). The fact that mesothelioma-derived exosomes conspicuously impaired the killing function of fresh NK cells and an NK cell line suggests that either the exosomes are hsp70 surface negative or this potential activatory mechanism is overwhelmed by inhibitory processes occurring in parallel.
Several studies have shown activation of immune responses by tumor exosomes, as prophylactic agents ( 40) or as therapeutic vaccines in murine cancer models ( 23) and in humans ( 11, 41), usually by loading them onto enriched dendritic cell populations. By binding and endocytosing exosomes, dendritic cells may cross-present exosome-delivered antigens to T cells and stimulate antitumor immunity ( 11, 23). Much of the literature relating to tumor exosome vaccines, therefore, are in reality dendritic cell–based vaccines with exosomes acting as an antigen source. Liu et al. ( 10) recently showed that pretreating mice with tumor exosomes before vaccination with tumor exosome–pulsed dendritic cells was counterproductive, resulting in accelerated growth of explanted tumors through a mechanism involving exosome-mediated impairment of NK cell functions ( 10). Their report showed that murine tumor exosomes were capable of inhibiting IL-2–induced proliferation of NK cells and of inhibiting NK cell cytolytic function ( 10). Our study supports such findings and confirms that these effects are also observed with human tumor exosomes in the absence of dendritic cells as carriers for exosomes.
To date, however, Treg cells as potential exosome-reactive cells have not been described, and the capacity of tumor exosomes to inhibit cytotoxic effector cells while in parallel supporting Treg cell phenotype and function was hitherto unknown. These exosome effects, which are at least in part due to membrane TGFβ1 expression, provide a coordinated "double hit" to cellular immunity. Tumor exosomes are likely to represent an important mechanism contributing to immune evasion in cancer.
Grant support: Cancer Research Wales.
We thank Dr. A. Hann (Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom) for electron microscopic analysis of exosome preparations, and Dr. Robert Steadman (Institute of Nephrology, Cardiff University, Cardiff, United Kingdom) for discussions about TGFβ.
↵3 Our unpublished observations.
Received September 19, 2006.
Revision received May 18, 2007.
Accepted May 24, 2007.
Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002; 2: 569–79.
Zitvogel L, Regnault A, Lozier A, et al. Eradication of established murine tumours using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998; 4: 594–600.
Chaput N, Schartz NEC, Andre F, et al. Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection. J Immunol 2004; 172: 2137–46.
Andre F, Chaput N, Schartz NEC, et al. Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J Immunol 2004; 172: 2126–36.
Segura E, Nicco C, Lombard B, et al. ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood 2005; 106: 216–23.
Raposo G, Nijman HW, Stoorvogel W, et al. B Lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996; 183: 1161–72.
Skokos D, Le_Panse S, Villa I, et al. Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. J Immunol 2001; 166: 868–76.
Karlsson M, Lundin S, Dahlgren U, et al. "Tolerosomes" are produced by intestinal epithelial cells. Eur J Immunol 2001; 31: 2892–900.
Gastpar R, Gehrmann M, Bausero M, et al. Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 2005; 65: 5238–47.
Liu C, Yu S, Zinn K, et al. Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function. J Immunol 2006; 176: 1375–85.
Andre F, Schartz NE, Movassagh M, et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet 2002; 360: 295–305.
Bard MP, Hegmans JP, Hemmes A, et al. Proteomic analysis of exosomes isolated from human malignant pleural effusions. Am J Respir Cell Mol Biol 2004; 31: 114–21.
Caby M, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005; 17: 879–87.
Zhou J, Zhang J, Lichtenheld MG, Meadows GG. A role for NF-κB activation in perforin expression of NK cells upon IL-2 receptor signaling. J Immunol 2002; 169: 1319–25.
Zorn E, Nelson EA, Mohseni M, et al. IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT dependent mechanism and induces the expansion of these cells in vivo. Blood 2006; 108: 1571–9.
Ahmadzadeh M, Rosenberg SA. IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients. Blood 2006; 107: 2409–14.
Robertson M, Cochran K, Cameron C, et al. Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukaemia. Exp Hematol 1996; 24: 406–15.
Doyle A, Griffiths JB, Newel DG. Establishment of human B lymphoblastoid cell lines from peripheral blood mononuclear cells. In: Cell and tissue culture laboratory procedures. Porton Down (Salisbury, UK): Wiley; 1994. p. 21–6.
Lamparski H, Metha-Damani A, Yao J, et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 2002; 270: 211–26.
Clayton A, Turkes A, Hossein N, Mason MD, Tabi Z. Inducible heat shock protein expression in B cell exosomes. J Cell Sci 2005; 118: 3631–8.
Lewkowicz P, Lewkowicz N, Sasiak A, Tchorzewski H. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol 2006; 177: 7155–63.
Manigold T, Shin E-C, Mizukoshi E, et al. Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C. Blood 2006; 107: 4424–32.
Wolfers J, Lozier A, Raposo G, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001; 7: 297–303.
Thery C, Boussac M, Veron P, et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 2001; 166: 7309–18.
Fontenot JD, Rasmussen JP, Williams LM, et al. Regulatory T Cell Lineage Specification by the Forkhead Transcription Factor Foxp3. Immunity 2005; 22: 329–41.
Byrne MJ, Nowak AK. Modified RECIST criteria for assessment of response in malignant pleural mesothelioma. Ann Oncol 2004; 15: 257–60.
Théry C, Clayton A, Amigorena S, Raposo G. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protocol Cell Biol 2006: Unit 3.22.
Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 1996; 93: 136–40.
Clayton A, Tabi Z. Exosomes and the MICA-NKG2D system in cancer. Blood Cells Mol Dis 2005; 34: 206–13.
Dukers DF, Meij P, Vervoort MB, et al. Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol 2000; 165: 663–70.
Ostroukhova M, Qi Z, Oriss TB, et al. Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-β. J Clin Invest 2006; 116: 996–1004.
Gandhi R, Anderson DE, Weiner HL. Cutting Edge: immature human dendritic cells express latency-associated peptide and inhibit T cell activation in a TGF-β-dependent manner. J Immunol 2007; 178: 4017–21.
Nakamura K, Kitani A, Fuss I, et al. TGF-β1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol 2004; 172: 834–42.
Blobe GC, Liu X, Fang SJ, How T, Lodish HF. A novel mechanism for regulating transforming growth factor β (TGF-β) signaling. Functional modulation of type-III TGF-β receptor expression theough interaction with PDZ domain protein, GIPC. J Biol Chem 2001; 276: 39608–17.
Cheifetz S, Massague J. Transforming growth factor-β (TGF-β) receptor proteoglycan. Cell surface expression and ligand binding in the absence of glycosaminoglycan chains. J Biol Chem 1989; 264: 12025–8.
Hwang I, Shen X, Sprent J. Direct stimulation of naive T cells by membrane vesicles from antigen-presenting cells: Distinct roles for CD54 and b7 molecules. Proc Natl Acad Sci U S A 2003; 100: 6670–5.
Andreola G, Rivoltini L, Castelli C, et al. Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles. J Exp Med 2002; 195: 1303–16.
Abusamra A, Zhong Z, Zheng X, et al. Tumor exosomes expressing Fas ligand mediate CD8(+) T-cell apoptosis. Blood Cells Mol Dis 2005; 35: 169–73.
Taylor D, Gerçel-Taylor C. Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects. Br J Cancer 2005; 92: 305–11.
Altieri S, Khan A, Tomasi T. Exosomes from plasmacytoma cells as a tumor vaccine. J Immunother 2004; 27: 282–8.
Dai S, Wan T, Wang B, et al. More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells. Clin Cancer Res 2005; 11: 7554–63.
You are going to email the following Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2
Cancer Res August 1 2007 (67) (15) 7458-7466; DOI: 10.1158/0008-5472.CAN-06-3456
Abstract SY30-02: NK cells remember: Engineering NK cell memory-like responses for cancer immunotherapy
Abstract PL01-04: Epigenetic mechanisms of malignant clonal dominance and immune evasion
Abstract SY26-01: Targeting natural ligands for TCR engineering
Show more Immunology


text:
Q: Symfony 3, include a css library with some dependencies From a personal symfony 3 project, I want use the font-awesome library. In the config.yml file I added an asset like that :
    awesomefont_css:
        inputs:
            - '%kernel.root_dir%/../web/library/font-awesome/css/font-awesome.css'

I call the asset from my index.php page :
{% stylesheets output="stylesheet.css"
'@bootstrap_css'
'@awesomefont_css'
"@style_css"
%}
<link rel="stylesheet" href="{{ asset_url }}"/>
{% endstylesheets %}

I cleared caches (via php bin/console cache:clear) but when I load my php page, I get the errors :

I understood the library font-awesome need use some another files. My question is : how can/must I add properly theses anothers files in a symfony 3 ?
Thanks !

A: I add samples symbolic links from mywebsite/web/library/font-awesome/fonts/* to mywebsite/web/fonts . I don't know if there is a better solution but I don't find another and that work

A: I included fortawesome/font-awesome via composer.
Then, you have to add additional entries to your config.yml:
        fontawesome_css:
            inputs:
                - '%kernel.root_dir%/../vendor/fortawesome/font-awesome/css/font-awesome.css'
            filters: [cssrewrite]
        font-awesome-otf:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/FontAwesome.otf'
            output: 'fonts/FontAwesome.otf'
        font-awesome-eot:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/fontawesome-webfont.eot'
            output: 'fonts/fontawesome-webfont.eot'
        font-awesome-svg:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/fontawesome-webfont.svg'
            output: 'fonts/fontawesome-webfont.svg'
        font-awesome-ttf:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/fontawesome-webfont.ttf'
            output: 'fonts/fontawesome-webfont.ttf'
        font-awesome-woff:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/fontawesome-webfont.woff'
            output: 'fonts/fontawesome-webfont.woff'
        font-awesome-woff2:
            inputs: '%kernel.root_dir%/../vendor/fortawesome/font-awesome/fonts/fontawesome-webfont.woff2'
            output: 'fonts/fontawesome-webfont.woff2'

then only include the fontawesome_css (as you did already):
    {% block stylesheets %}
        {% stylesheets '@bootstrap_css' '@fontawesome_css' '@custom_css' %}
            <link rel="stylesheet" type="text/css" media="screen" href="{{ asset_url }}"/>
        {% endstylesheets %}
    {% endblock %}

Finally, run
bin/console assetic:dump

and you're done! It is not necessary to mention the font files in your template, they are copied automatically.

A: I should add that the webpack/encore package is new and much better in my opinion! It replaces assetic.
You can also easily customize your bootstrap theme on the fly with this tool.

redpajama_set_name: RedPajamaStackExchange


text:
Posts Tagged 'Terrorism'
Terrorists: Publish Their Photos or Not?
29July London
A terrorist slits the throat of a French priest. Newspapers and broadcasters throughout the world publish and show high definition photographs of the murderer and his partner. We know what they look like, their names, ages, something of their history and a nice quote from the knifeman's mother who says that at heart he was a good boy.
The leading French daily Le Monde has announced (after the Nice terrorism) that it will not give terrorists publicity and so will not publish their photographs. The murder of Father Jaques in Normandy confirms that decision according to Le Monde.
The argument at Le Monde is that by publishing photographs the paper is in some way glorifying the terrorism. The terrorist becomes a celebrity.
By taking such a decision Le Monde's editors have stepped aside from the code of so-called journalistic impartiality. Most societies regard the hounding, capture and even the destruction of terrorism as a role for the military, the intelligence agencies, the policy and the elected politicians.
By banning pictures – in theory starving terrorism of the oxygen of publicity – Le Monde has joined the fight against terrorism or at the very least changed editorial policy in the hope of helping to capture those who killed Father Jaques.
Le Monde's editor Jerome Fenoglio says "We have to do this for all victims of the criminal organisation known as the 'Islamic State'."
The decision of an admired newspaper to publish or not sets it aside from other papers in democratic society. The Turkish government has this past week ordered the shut down three news agencies, 16 television channels, 23 radio stations, 45 daily newspapers, 15 magazines and 29 publishing houses. It is not uncommon for a government to restrict the media in a crisis. It is very uncommon for a newspaper to impose self-censorship. Moreover, the Le Monde decision is not a one-off. There could be more to come because the editorial board recognise IS and other groups are not passing ideologies.
As Fenoglio puts it, "After the Nice Attack, we are publishing no more images of terrorists, perpetrator of killings and massacres, to avoid potential posthumous glorification. Other debates about our practices [as a newspaper] are ongoing."
What about other news outlets? Le Monde has not been followed. Le Figaro says it shall wait and see. British papers will publish everything it can other than the act of killing. Equally, the head of the Quilliam Foundation (a think tank of radicalisation analysts) says it has been a great decision.It reduces the propaganda value of the terrorist.
So what are the practical ambitions and consequences?
It is a purpose of IS to gain publicity from an act of violence. Publicity tells a global audience that IS can do something and is willing to do something and that no one is safe from such acts. This induces a varying stage of terror – a large part of any terrorist ambition.
Secondly, by running pictures, names and backgrounds of the perpetrators the news outlet may spread among readers further disgust about what had happened but identity of the terrorist suggests a cause rather than an anonymous event of violence.
Withdrawing names etc reduces any possibility of a neutral public understanding of why as opposed to what has happened. In a bizarre sense, no picture no name removes an imagined chance of hero worship.
Yet all this is an argument of times past.
Le Monde's decision is taken in an internet age where all is revealed and where there are few rules of what is right and wrong to publish. The importance of the decision of Fenoglio and his editorial board is that a great newspaper is attempting to take part in what is a state venture – the prevention and eventually the destruction of terrorism. It is not enough to rant in a newspaper editorial. Le Monde in its honourable way is being counted. Very few in these times will have the moral debate with themselves.
Tags:Daesh, Father Jaques, Figaro, IS, Le Monde, Terror, Terrorism
Terror is Just a Train Ride Away
Ever since the attacks in Paris last year, most cities within a train ride have been anticipating another atrocity.
The Belgians caught Salah Abdeslam last Friday. He was part of the Paris attacks. But the second man they wanted, everyone wanted, Najim Laachraoui, the bomb maker slipped through the net. So did the getaway driver in Paris, Mohamed Abrini. The two on the run meant others were free covering them. That meant another attack was "on the clock" – likely.
The security alert in London and other cities is Severe – the counter-terrorism agencies have covered all known bets. But the Belgian authorities have warned at the highest levels that something was about to happen.
This morning it was Zaventem airport. An hour later Maelbeek metro station was hit. Brussels Midi train station was closed. Eurostar from London was terminating at Lille in Northern France. What is special about Lille? Modern terror is just a train ride away.
Sickenly, terror means more than corpses and wounded.
Terrorism also means the bringing to a standstill or diversion of systems we take for granted. So this morning (22 March) flights were cancelled or diverted. Railroads halted. People did not get where they expected to be.
But for most who were not in the airport and not on the metro there are two concerns: am I all right and when can I get to wherever I was heading?
Therefore the greater unlikelihood is the relationship between the Western urbanite and the poor wretches trapped in the origins of terrorism, Iraq, Syria and wherever. A Syrian child observes then plays in the rubble of that morning and a late for conference all expenses paid executive checks the board for the next flight to wherever she or he was supposed to be that afternoon.
We have now a world between the longitudes of the Tigris and Euphrates and the Iberian Peninsular that is fighting for its identity. That is what terrorism is.
Every police and security agency this day in Western Europe (hardly ever Eastern Europe) is running double checks on the terror state of its capital. Terrorists like capitals. They are less obvious and the target is more terror-efficient. Anyone, especially the nervous who avoids the carriage with the guy with a Muslim look and a backpack, who rides any subway knows this.
So if we know all this and more, what now? Now is the test of a counter-terror adage. When a terrorist team strikes they are for 48 hours on the run. When they run they are, if the intelligence report system is in place, vulnerable to capture. Najim Laachraoui and Mohamed Abrini prove some escape the net, especially the Belgian net. The Belgians are particularly bad at counter-terrorism. But the rule is reasonable sound. The terrorist team is vulnerable. But if one goes down another will form. This morning's terror attack was successful.
Many dead. Many wounded. Many scattered. The best we can think is that more attacks have been prevented than have got through. The worst we can think is that Najim Laachraoui and Mohamed Abrini are still out there. The network that supports them is still out there. For all our habit of "carrying on" we should remind ourselves that the next act of terrorism is rarely more than a train ride away.
Tags:Brussels, Terror, Terrorism
Terrorism & War – Looking Good says Colonel Steve
27 February 2016. London.
The colonel from the Pentagon looked the part. Cleaner cut than a saint, with more medal ribbons than a pantomime hero. He landed in the UK this past week with a word perfect mission.
He had come to to every newspaper and broadcaster that the war on IS (he uses Daesh) is right on schedule and IS/Daesh is taking a beating. He says the US-led coalition is killing terrorists leaders most days. Their bases are taking big hits, the armoured vehicles are been wasted.
Colonel Steve Warren was here to tell us that Daesh was beginning to lose. "We see them in a defensive crouch" he told us. No journalist mentioned that a panther springs from a crouch.
The fact that IS is on a killing high hit and having nauseating fun using beheaded cadavers as road blocks is a side issue for the colonel with a message. He says we've got it wrong.
Who sent the colonel? Answer: the people who believe that too many media reports from Syria, Iraq, Turkey and Libya are telling a different story. They are undermining the reports, especially those relayed in Washington, that it is not long now before IS collapses.
About five or six months ago the same US sources were briefing British and French TV and newspaper hacks that ISIS was a second rate bunch of no-hopers that given political guts could be wiped out inside the Christian winter holiday. The message is dulled. Hence the colonel's visit. A regional scan shows why. From the Bosphorus to Tripoli via the Gulf there is a tragedy that just a few years back would not have got off the staring block.
The Turks are hitting the Kurds, the Kurds are hitting the Turks. Sunni and Shia states and peoples are at war face to face or by proxy. The coalition is hymning the Syria ceasefire but the two main teams – IS and Bashar al-Assad are not signatories. IS does not care about a ceasefire. Assad is bombing rebel backyards and is ready to go the extra 100 meters with I Told You So banners when the ceasefire crumbles. The Russians are happy to help them out.
Across the almost non-existent border in Iraq on Friday about 20 people were killed in a bombing of a Shia mosque (the government is Shia seeking revenge). Who did it? Surely not IS who is on the run according to the colonel. Yup. The very same IS. Ask the mourners at the Rasul al-Zam mosque.
In Lebanon, Hezbollah are training Houthi rebels to fight in Yemen and the Saudi are looking at target planning into Hezbollah. Imagine the death toll. Imagine the knock-on. Meanwhile the Saudi bombing in Yemen appears as casual as cruelty can be in warfare. Hitting civilian targets is good terrorist bombing. It causes confusion and terror.
Before we leave that bit of the dusty world, two pipe bombs were found in Jerusalem at Herod's Gate. Lots of shooting. Lots of dying. Also Friday, a prediction that Gaza will be a death bed scene by 2020. Next door in Egypt, President Sisi is telling his people, do not listen to others. Just do as I say.
And in Libya, there is a meeting Monday to see if Libya may be split into three. The West recognises one group, detests and will fight the other while everyone will be hit by the third. It is a simple example of bloodbath created when the British, the French and the colonel's employers go into something blazing saddles without guaranteeing the result they claim to be promising. Other examples? Viet Nam, Iraq and Afghanistan with the dankness of Syrian dead joining the tragic tapestry of failure.
So best the colonel goes home. Best someone who writes the propaganda tours gets to understand that what we call the Middle East is seeking a new identity that is different from the one the West created for it in the 1920s. IS emerged because the Western-led coalition got it wrong.
And, let us hope and those who do these things, let them pray that the crouching IS not about to leap with another Paris. When a well turned out colonel tells them they are losing, they may just have to prove they are not in the crudest manner.
Tags:Daesh, Egypt, IS, ISRAEL, Libya, Syria, Terrorism, Turkey, Yemen
New Terrorism Threat to UK? Beware Snake-oil politicians
EU Vote & Terrorism: Beware Cameron & Co selling snake-oil politics
The theatre of David Cameron's assault on the EU is done. A UK referendum will be held on 23 June when the British will vote to stay in the EU or leave.
Mr Cameron's people are looking for ways of guaranteeing a Yes Vote. So watch for a change of the electoral roll to include 16+ year-olds. Teenage voters will vote to stay in Europe.
If the government does not get its way then another referendum will be held to check that the people knew what they were saying. Corrupt? It is the way of these things.
Meanwhile the texture of the debate is immediately felt. Cameron is saying that anyone who votes No will be jeopardising national security. Terrorists will be aiming a strike on the UK after 23 June if we all vote to quit the EU. The Prime Minister is suggesting that by staying in the EU we are very much part of the Intelligence information supply line that helps UK counter terrorism. Downing Street says that pulling out of the EU will mean that many of the European agencies would not include the UK in that ring.
According to Cameron logic present non-EU states do not get security information and warnings. Rubbish. As for EU protection, ask the French.
Iain Duncan-Smith MP (who wants to quit) says that if we stay terrorists will be aiming a Paris-type strike on the UK after 23 June largely because it would be easier to get into the country. The fact that most radical action is home grown or home based bins that argument. Paris would have happened whatever French membership status of EU.
We imagine Mohammad al-Baghdadi sitting in Daesh council reviewing attack projections based on a referendum result in a country that has trumpeted a major airstrike operation on Daesh but has yet to have had much more than a car-load's success in spite of using missiles that cost £300,000 a time. It is a bigger story than that and it defies scare tactics from two leading and supposedly trusted members of government.
Both the Cameron and Duncan-Smith camps are blatantly playing on fears. Worse, it takes only a couple of seconds to work out that they are unconvincing. Therefore both Cameron and Duncan-Smith are insulting the intelligence of the electorate. The terrorist threat is real enough. The agencies in most countries are aware of information exchange. The UK in or out of the EU is the second strongest member of NATO and would continue to be even outside the EU and therefore wired into every country with an A1 Intelligence Analysis System.
The vote on 23 June is important enough and the counter-terrorist debate sensitive to the extreme without the people trusted as leaders copying the Trump line in political rhetoric.
Ironically, if there is a Cameron plan to pack the voting by including 16+ year-old teenagers then he should be reminded by his carpet bagging aides that those same young people are not tired out voters. They have sharp brains. They can smell political snake-oil salesman a mile off.
Tags:Cameron, EU & British, Terrorism
Terrorists Don't Sign Peace Agreements
There are two things we have to understand about fighting terrorism:
A terrorist does not sign peace agreements
An insurgent wins if he does not lose
Take those two points on board then you can put the grand alliance announced in Riyadh this week in some perspective.
You will also get an idea why American led anti-IS policies have the finger prints of a whole lot of pretty dumb Washington analysts written all over them.
The announcement in Riyadh says this: 34 mainly Muslim nations have agreed to join a Middle East, Asian and African axis to fight terrorism.
The group is largely Sunni. The three missing states from this agreement -Iran, Syria and Iraq – are largely Shia.
The only significance of the announcement is that Gulf States and others others accept that they must do what they should have done in October 2014 – take it upon themselves to destroy the threats from ISIS instead of waiting for the Americans to do it for them.
The military tone poem coming out of all this is that the Saudis understand what ISIS terrorism is all about but cannot do too much about it and the US-led lot do not understand ISIS and they too cannot do much about it.
Talking to American policy wonks it is sadly clear they do not understand that rebellion is a greater power than ever because the means of making rebellion attractive and successful has never been greater.
Until autumn 2014 and with ISIS advancements the average Arab never believed it was possible to change society into something different. ISIS said we can and we have.
Moreover most at Riyadh understands what America and her allies like the UK do not understand: an insurgency wins if it does not lose. Unlike state to state warfare, a rebellion can slink away and therefore cannot be beaten. It can be contained as post-World War II America attempted to contain emerging USSR. But it need never be beaten as long as the will remains.
The further reason for doubting coalition understanding of what they are into is their continuing belief in signing agreements. That is a very western way of doing things. Making the other side sign means you have won. Terrorism has no political signature that obliges those coming later to observe such a signature.
Western nations sign as states. Terrorists have no such structure nor authority.
All this says that whatever the plans of Riyadh, of Washington, of Moscow or of any other state, terrorism cannot be beaten because a signature is not a symbol of having lost.
It is a hard thought to follow, but it is the only one to be understood if ISIS is to be contained, which it will have to be because it has nothing to lose and therefore will for ever win.
Tags:ISIS, Riyadh, Terrorism
ISIS Can Be Beaten – But Not This Week
ISIS is beatable. It is not a mystical zealotry wielding an Islamist excalibur. It is not invincible.
ISIS is not a supremely organised and state supported military organisation
ISAS is in military terms terrorist organisation that could not withstand a 21st century centrally command-led onslaught from, say, the United States and the Global Coalition.
ISAS has very little natural support in the Middle East. Most governments and people of all Islamic persuasion want ISIS put down.
That is the message that the G20 leaders meeting in Turkey should be giving out and then backing that up with all their military and most importantly political resources. They have to agree what they are going after, when they are doing that and those without major military roles should support diplomatically, financially and territorially those that have.
They should be making it clear that today starts new planning to re-establish a joint service military command to destroy ISIS in Iraq and Syria. There will be subordinate commands to do the same in the ISIS diaspora in Afghanistan, Gaza, (more powerful by the day), Libya and sub-Saharan Africa.
This is not let's-get-'em redneck reactionary rhetoric.
Compared with the political and economic authority of the major nations against ISIS and most certainly the military assets of those same powers (ISIS does not have, an air force, a drone capability, satellite intelligence, elint etc) ISIS's only advantage is that coalition powers are relying mostly on air attacks and Kurdish ground forces. Even now an apparent success against ISIS in reality means fast withdrawal having raped and wasted the town they then leave behind the booby traps and images of misery because the locals assume they will return. This means that something has to come out of Turkey that will lead not only to chasing ISIS out of town but then going in hot pursuit.
But that does not make ISIS unbeatable.
The International Syria Support Group who will be in Turkey insist that ISIS will be turned back and then talks on both sides to bring peace to Syria can proceed. Maybe. Maybe not.
The anti-Assad rebels will not agree to an election and anyway you cannot have an election in rubbled homeland. All the time there is that condition then ISIS will be there until the high table in Turkey or wherever next they meet take a decision to go beyond a handful of nations with bombings and drone attacks.
A single command system with a four star general and five star resources must be established. It should have regional and UN plus domestic legal authority to throw electronic, satellite, human and special forces intelligence systems into locating and then applying the military hardware in unrelenting bitterness and purpose against ISIS. It must too have a follow-on plan what to do in regions where ISIS is defeated and the resentment of its followers festers.
None of this is a job for the week, next week or long weeks after that. Wars do not work that way. But the near truth is that with all the advantages of scattered and sleeping anonymity ISIS has not been so successful.
If the suicide bombers had got into the Stade de France on Friday, then the dead list would be even more gruesome and the mood and shock nationally depressing. But the bombers did not get in. There too is the fact that although 132 deaths occurred on Friday, that is not a big number.
That number is killed on a daily basis in the Middle East. Now we may appreciate that – for the moment.
So put in perspective the Global Coalition with hugely superior intelligence gathering and firepower can defeat ISIS. Whether or not it has the political will (the Paris atrocity will quickly lose its effect) is another matter.
Tags:ISIS, Terrorism

redpajama_set_name: RedPajamaCommonCrawl


text:
Biologically Sensitive Area
Fig. 2. The Biologically Sensitive Area in the Celtic Sea including the Irish Exclusive Economic Zone and the continental slope. (From EC Request - Review of the Biologically Sensitive Area/Irish Box)
The continental shelf off the southwest coast of Ireland is an important spawning area for many commercially important species especially mackerel, horse mackerel, blue whiting, hake, megrim herring, haddock and cod. In 2003 a biologically sensitive area was established off the southwest coast of Ireland with the boundaries being decided upon mainly in a political context whilst considering areas with high concentrations of juvenile hake.
While this area is deemed especially important for hake nursery it does not correspond exactly to nursery grounds for other fish as their grounds are found further to the west along the continental slope. Nevertheless it is still an important nursery area for anglerfish, mackerel and horse mackerel, a spawning and nursery area for herring, whiting, and haddock, a spawning area for megrim and cod and includes a large area of nephrops ground. The area also has important stocks of scallops, brown crabs and spider crabs. The development of thermal fronts and phytoplankton blooms in the spring time are important for the growth and development of larvae species in the area.
Ireland is the most significant fisher in the area followed by France and Spain. The French and Spanish fleets have been found to focus their fishing efforts along the continental slope to the west and around the Goban Spur. Management measures in the BSA focus on reducing discards, protecting spawning areas, increasing the use of environmentally friendly fishing gear and establishing protected areas. Limits on the demersal fishing effort and an increase in the mesh size for towed and fixed gear are some measures that have been introduced. The implementation of the management measures has not been entirely successful due to poor communication of changes in regulation and the necessary recording of effort needed to determine the regulations was poor due to low compliance. The biomass of hake, megrim and anglerfish has increased in recent years although it is unclear how much the management measures have contributed to this as the boundaries of the BSA do not always match the spawning and nursery grounds of the stocks.
The Beaufort Research team is using the Biologically Sensitive Area as a test-case to look at the success of using ecologically meaningful boundaries in management. The boundaries of the BSA are not prohibitively strict as they need to consider the co-existence of key-species, bathymetric and other environmental variables which define a 'fuzzy' box with a useful degree of biological homogeneity. The behaviour of marine mammals is also an important focus in the area.
EC request - Review of the Biologically Sensitive Area/Irish Box
Marine Institute - Biologically Sensitive Area
Proposal from KFO to manage crab effort in the Biologically Sensitive Area
Biological Region
Inshore Fishery
Local Inshore
Hosted by Queen's University School of Biological Sciences


During my private tours in Florence, I often see bored tourists with whispers at their neck following a tour guide and absorbing the information that the Florence's guide decides to offer them. I do sometimes group tours in Florence but I have to be honest, I prefer to work as a private tour guide, as for me, a tour of Florence dedicated entirely to a couple, a family or a group of friends is way more enthusiastic and enjoyable for the tour guide but first of all for the travelers.
To really comprehend the spirit of Florence in all its various aspects and above all, to get to know the things you're most interested in, you need a private tour guide who can unveil the secrets of this amazing city and make you want to come back and see more.
Florence is like Pandora's box, but instead of containing all the evils in the world, it contains all the treasures a traveler might dream of: magnificent Renaissance buildings, famous paintings and statues, breathtaking views, unforgettable food and wine experiences, hidden artistic gems and fascinating cobble stone streets where the Medici, Michelangelo, Leonardo da Vinci, Galileo Galilei and other glorious personalities walked on. We can follow their steps and discover the best that Florence has to offer during a pleasant stroll throughout the city, enjoying it in slow motion and learning about its great past and present.
Almost every day I wonder on Florence's streets and I realize how lucky I am to work here. That's why I do my best to offer to all of my guests a unique experience that will make them decide to come back for another Florence tour with me, not only as their tour guide, but as a new friend in Florence.


BEIJING. December 4. KAZINFORM. /Ruslan Suleimenov/ Kazakhstan attaches big significance to the supplies of its grain to China, joint construction of a near-border grain terminal. First Vice Prime Minister of Kazakhstan Umirzak Shukeyev said it during the V sitting of Kazakh-Chinese Cooperation Committee in Beijing on Friday. As Kazakh Minister of Agriculture A. Kurishbayev commented upon the situation, during the sitting the sides discussed the issues of Kazakh grain transit in the volume of 3 million tons via the territory of China as well as the supplies of Kazakhstan's grain to China. "South Korea, Japan and other countries are interested in Kazakhstan's grain. Our grain in the ports of the East Cost is competitive both on price and quality. We can supply it to these countries provided that China allows transit", the Minister said. He noted that the date of supplies would be known after the talks of the correspondent departments. Besides, the sides agreed on promotion of trade in livestock products between Kazakhstan and China as well construction of Kazakh-Chinese agricultural technologies exhibition center.


text:
Cheung Kong Graduate School of Business (Cheung Kong GSB), (chiń.长江商学院, pinyin: Chángjiāng Shāng Xuéyuàn) – wyższa szkoła biznesu w Chinach, finansowana przez Fundację Li Ka Shing. Główna siedziba mieści się w Pekinie; oddziały w Szanghaju i Kantonie. Szkoła ma obecnie około 400 studentów EMBA i 60 MBA rocznie.

Linki zewnętrzne
 Cheung Kong Graduate School of Business

Uczelnie w Chinach

redpajama_set_name: RedPajamaWikipedia


The Neuromorphic Computing Platform allows neuroscientists and engineers to perform experiments with configurable neuromorphic computing systems. The platform provides two complementary, large-scale neuromorphic systems built in custom hardware at locations in Heidelberg, Germany (the "BrainScaleS" system, also known as the "physical model" or PM system) and Manchester, United Kingdom (the "SpiNNaker" system, also known as the "many core" or MC system). Both systems enable energy-efficient, large-scale neuronal network simulations with simplified spiking neuron models. The BrainScaleS system is based on physical (analogue) emulations of neuron models and offers highly accelerated operation (104 x real time). The SpiNNaker system is based on a digital many-core architecture and provides real-time operation.
These large-scale neuromorphic systems are currently in their commissioning phase, which is accompanied by short technology and software switchover times. In order to ensure that obtained results are up to date and valid, we kindly request all users to review all material intended for dissemination (websites, papers etc.) with the involved groups.


This is a sponsored post by General Mills. All opinions are my own.
I may not be the best cook in the world but I can rock the baking and the no-bake recipes like no other! The holidays are my favorite time of the year and it excites me to think about all the cookie swaps I'm a part of. My family often eats everything before I have the chance to do a cookie exchange so making these tasty Pillsbury™ Peanutty Pie Crust Clusters gave me a sigh of relief! This easy, 'barely bake' recipe rocked my house this weekend.
This time around we picked out our fun pieces for the holiday goodie swaps and went to town on the Pillsbury™ creations. He was fab at heating up the white chocolate chips, peanut butter, butter and dropping in all the pieces to the pie crust clusters pie! I let my nine-year-old do the cookie drops on the waxed paper and my four-year-old was the official taste-tester.
Cut them into cubes with a pizza cutter.
Toss the cubes in the oven to lightly brown.
Heat the peanut butter, white chocolate, and butter.
Drop in peanuts & tiny baked pie crust pieces.
Easy right? What are some of YOUR favorite holiday treats??
For additional recipes and inspiration visit Sam's Club!
This post presented by General Mills. All thoughts & opinions are our own.
Winter Warm-Up: Creamy Sausage Tortellini Soup!!


Andrew Weaver is BC politics' kingmaker, with a government secretariat dedicated to ensuring "good faith and no surprises" between his Green Party caucus and the minority NDP government.
And yet, he found out the details of the speculation tax in the newspaper – some of which he had just assured his constituents wouldn't happen.
"We were told by the finance minister and Premier that it wouldn't affect British Columbians. [Then] I was sent a Vaughn Palmer article, and read that I was sending out incorrect information," said Weaver.
Despite holding what amounts to a veto – assuming the opposition BC Liberals also vote against the government – Weaver's Greens have swallowed a steady stream of compromises, including Site C, LNG, and the speculation tax.
Weaver says those days are over.
"Times have changed. That wouldn't happen now," said Weaver.
"I like John [Horgan]. I think it was some people who were advising him that he probably shouldn't be listening to as much," said Weaver.
Weaver points to a number of items he got the NDP to alter, including moving up GHG emissions target legislation to the spring session, and softening the speculation tax to focus solely on urban areas.
"It was bad public policy. We able to get it to a position to make three further amendments. [Finance Minister] Carole [James] didn't want to do them; it was tough but very positive," adds Weaver.
That said, Weaver allows that Woodfibre has already gone through the environmental assessment process – and "I'm not so worried – it's tiny." Since then, the assessment process has become more rigorous.
Weaver vigorously disputes that First Nations and Indigenous support for either or both projects should make any difference for public – or crucially, NDP government – support.
"If you're creating emissions, it's affecting my ability to do business, because now I gotta reduce more emissions," adds Weaver.
Another potential storm cloud ahead – an issue where Weaver has publicly disagreed with stated NDP policy – is in labour relations.
Premier John Horgan and Labour Minister Harry Bains have both been clear: they want to do away with secret ballots for union certification, and move to a card check system, where everyone knows whether and how everyone else has voted.
Weaver also pointed to the hybrid model in Ontario, where building trades have a form of card check.
In other words, his commitment to a secret ballot is at least somewhat negotiable.
We closed our interview with two issues the Greens and NDP have been in lockstep: the Speaker's Office scandal, and Proportional Representation.
On the still-unfolding events in the legislature and Speaker's Office, Weaver pleads for patience.
"I have a lot of questions that I'm going to be asking, but I don't think now's the time to be asking," says Weaver.
"It is what it is. Love for it to be done before Christmas," says Weaver, admitting he has no idea if that's reasonable.
On PR, Weaver predicts a Yes win (naturally), on a 52-48 margin – and says B.C. had a good public debate, despite some hyperbole on both sides – which may have kept turnout down.


Every year, around this time, I look longingly at all the beautiful Advent Calendars in the shops. I know my daughter would ADORE one but I really don't like the idea of giving her chocolate every day (I am trying to ensure she doesn't end up a chocoholic, like me!).
Well, this year, my dilemma is over. I just came across a fantastic idea over on Read Aloud's Facebook page. They suggest wrapping 25 children's books and placing them under the Christmas tree, along with a special blanket. Every night, before bed, the children select one book to unwrap and then read together, snuggled up under the special blanket.
I will definitely be adopting this suggestion in lieu of an Advent calendar, with an extra special one for Christmas morning. I suspect it will become an annual tradition. I think my kids will love it even more than chocolate – especially seeing as the baby can't eat yet. Best of all, it gives me a great excuse to indulge my book-buying habit, guilt-free!
One of the mums over on the brillkids.com forum recommended that instead of buying new books, you could use 25 Christmas-themed books which could then be recycled each year #recessionbustingmoms. And, here's a link to another mum's idea of wrapping up 25 winter-themed books http://dancingwithdinos.blogspot.co.uk/2011/12/advent-calendar.html.


classified staff member, and four parent or community members. Each representative is elected from their constituency for a term of two years. The group represents the parents, community and staff members, soliciting input, suggestions, and feedback from all three.
Some of the issues addressed by the council may include budgetary concerns, dress code, fundraisers, field trips and comments. The council also reviews literature and data, makes recommendations for school improvement and provides local leadership and representation in the school decision-making structure. In essence, the council has the "ear" of the school administration and as a result of that, can voice the concerns, suggestions, and input provided by parents, staff, and community members. However, it must be emphasized that voices of all three groups cannot be heard if they elect not to provide council members with their views, concerns and suggestions. Parents, community members, and staff are invited to attend council meetings as their dates and locations will be announced in this website.


text:
The Only Way is Essex: Glass House Retreat
Baglioni Hotel London: La Dolce Vita
Through the Emerald of the Equator: Cruising Indonesia
Burns Night, Two Ways
Getting Reflective in Gstaad
Home from Home: Deal
White Gold: Truffle Hunting in Alba
Le Corsaire with English National Ballet
Spirited Away on the Isle of Jersey
Spa of the Month: QMS at The Lowry
UK Restaurants
Tipples
The Culturist
The Stylish
The Tailor
The Socialite
The Idler
In Other Matters
The Arb Club
Arb TV
You are at:Home»The Culturist»Theatre»The Girl on the Train
By Rebecca Lipkin on	 April 10, 2019 Theatre
The Anthony Banks stage production of The Girl on the Train, based on the hit 2015 psychological thriller novel by Paula Hawkins, 'arrived' at Theatre Royal Bath on Monday for a whistle stop week-long run before continuing an extensive UK tour. Made into a Hollywood film in 2016 starring Emily Blunt as the complex heroine Rachel Watson, whilst the film transferred the drama to suburban New York, the stage version follows the original British setting, complete with references to Waitrose and Bargain Booze. You get the sense that the audience consists of punters who aren't regular theatre-goers, proving that the broad appeal of adaptations such as this are vital for the future of British theatre.
Samantha Womack leads the cast as the dishevelled, greasy-haired Rachel, a woman whose alcohol dependence – as illustrated by the countless empty bottles of wine and spirits lying around in the opening scene at her grotty apartment – is causing her to have regular memory black-outs. Her life has taken an increasing nose dive since failed IVF prompted the break-up with her husband Tom (Adam Jackson-Smith) who is now married to Anna (Lowenna Melrose), with whom he committed adultery. Tom still drops in on Rachel to check she's alright, further adding to her humiliation and low self-esteem. James Cotterill's costume and revolving set design is crammed with detail that draws us in, while Womack delivers a desperately convincing portrait of a woman who has lost everything. Or almost.
Tom and Anna not only live at the same house Rachel once inhabited, but have a new baby, something the childless, isolated Rachel naturally finds difficult to comprehend. It seems everyone has the perfect life apart from her, something ever more relevant in this deeply unhealthy alcohol-fuelled age of voyeuristic and aspirational social media scrolling. A couple of doors down from her old house, which she passes on the commuter train to work every day, Rachel begins observing and obsessing over another young couple – making up names for them and fantasising about their lives. She sees the blonde Meghan (Kirsty Oswald), whom she has called 'Jess', on the terrace with her husband and then, on another occasion, with a mysterious Asian man who kisses her just before she goes missing.
Was Meghan cheating on her husband? And who was that man? All that Rachel has observed might unravel the mystery of her disappearance. Or does Rachel herself have something to do with it? She can't remember her precise movements on the night of Meghan going missing, nor how she got a cut on her head and blood on her hands, matters of concern to D.I. Gaskill (John Dougall) who is investigating the case. Avoiding alcohol in the hope of recalling vital information, Rachel goes about trying to piece everything together for herself, claiming to Meghan's husband Scott (Oliver Farnworth) that she was a friend of his wife. Revealing that she saw Meghan from the train window embracing another man, Scott works out that this was his wife's therapist Kamal Abdic (Naeem Hayat), prompting Rachel to book herself a 'session' in order to glean more. Womack and Hayat's meeting is conducted facing the audience, with chairs at either side of the stage, a clever device which allows us to study their body language in compelling detail – Rachel, all the while, clutching a water bottle containing neat vodka.
Integral to this slick Simon Friend, Amblin Partners and Josh Andrews production is Andrzej Goulding's ingenious projection design, allowing for gripping, fast-paced modern storytelling and the audience to experience the confusion of Rachel's flashbacks and the dizzying landscape speeding past the train window. Turns out Rachel lost her job six months ago but was still taking the same morning train, transfixed by Meghan's life in order to escape her own. We're all in suspense when Gaskill announces that Meghan has been found dead at the close of the first act, although this play might make you think twice about ordering a large vodka in the interval. The twists and turns continue, keeping us on the edge of our seat throughout. Without giving you spoilers, The Girl on the Train arrives at a climatic destination with electrifying energy and an audience that are absorbed right up until the murderer is revealed. Don't 'delay' in seeing it when it calls at a theatre near you, for sheer entertainment it's just the 'ticket'.
The Girl on the Train at Theatre Royal Bath until 13 April 2019. Running time approximately 2 hours 10 minutes including an interval. Production images by Manuel Harlan. For more information and tickets please visit the website.
Lungs at The Old Vic


A very few achievements are account-only.
Our intent is that any rewards granted from achievements (pets, mounts, titles, and tabards) will be shared at the account level.
I'm not going to promise this yet, because a lot of magic has to happen for that to work, but it's our intent. We have discussed having a character level requirement for some rewards, so that your level-2 gnome couldn't walk around with the "Defender of a Shattered World" title just because your level-90 shaman earned it. If the gnome made it to level 80, though, you could proudly show off your title. You also won't be able to use a faction-specific pet, mount, or title on the wrong faction. The achievement wouldn't go away -- you just wouldn't be able to show it off on that character. There are probably additional exceptions and details we'll find as we dig deeper into the system.


Greetings, and thanks for coming by.
We can create beautiful 3D Rendering Designs for any Style or Size of Kids Bedroom.
I'll be pleased to help you 3D'ing any space/furniture idea or project you want. I've worked before with kitchens, bathrooms, closets, music and recording studios, ventilation systems, remodeling and more!
Apart from design and build, we also do collaborations with companies, it shows what you have in some of our designs. If you want to collaborate with us or you have any project, we are happy to work with you.
We can create beautiful 3D Rendering Designs for any Style of Kitchen.
Apart from design and build, we also do collaborations with companies, it shows what you have in some of our designs. If you want to collaborate with us or you have any project, we are happy to work with you. from here I send a greeting.


Spirals of vibrations are constantly whirring through our souls, our cells, and our cell phones. Changing times induce time releasing change, and we have all noticed, I'm sure, that time is moving differently than before – traveling much much faster. So much is happening in every moment and again, change is everywhere. Our brains, genetically wired to be controlled and afraid, are now releasing old programs opening our inner selves to the new possibilities. The possibilities of understanding one another, of attuning to folks and life all over the world, of being able to extricate ourselves from the training that dominates our systems of politics, religion, the economic grid, gmo'd reality, and the media constructed ideas on our appearance, safety, and so on.
Cells and change – I often chuckle at the synchronicities of all the cell/DNA research and all the inherent health possibilities. As the proliferation and research of cell phones and fuel cells, and even groups like Al Quaida community cells race through our consciousness, we literally become connected to the awakening within the collective.
Apparently, and I didn't know this until I met Dr. Julia Assante author of The Last Frontier: Exploring the Afterlife and Transforming Our Fear of Death, the same communication foundations that apply to the telephone and Edison, and the magically infinite properties of free energy from Tesla can also be used in connecting folks with what is in store for us on the other side of life's ultimate barrier.
So today I am musing again about the internet, cell phones, the mysteries of instantaneous connection round the world, and how all these things can combine to be the training wheels to Oneness. 7 billion sparks of one higher mind? We could truly shift to being able to know love and empower ourselves on a mass level. And does that shift include many "on the other side" with the masses of folks currently living in this mundane reality known as the third dimension?
Many of us experience tremendous connections with people who have died. It has made a huge difference in my family and the families of my clients when our connectins to the after life unlock old lineage cellular/DNA patterns through explanation, apologies, and messages of love and knowing.


Home > News > News > Boogie Wonder Band Concert Cancelled!
Boogie Wonder Band Concert Cancelled!
We are sorry to announce that due to a scheduling conflict, the Boogie Wonder Band concert scheduled for Tuesday, October 5, has been cancelled.
Ticket holders may choose between switching their tickets to a performance of The AMT Band Plays ABBEY ROAD with no additional costs, or transferring the money to a customer credit or gift card for a future purchase at AMT. Switching tickets to The AMT Band Plays ABBEY ROAD would be a savings of $6 per ticket off the regular price.


Where Noted, Cyclists Use of Full Lane Now Legal in MD | Citizens Planning and Housing Association, Inc.
Drive safely! Allow bikes full lane where posted! On certain roads and in certain circumstances, cyclists are now permitted to use the full lane of roads in Maryland. The Maryland Department of Transportation (MDOT) has begun to install special signs, called R4-11 signs, around the state, which read: "NOTICE: BICYCLES MAY USE FULL LANE." Better yet, there are already twenty such signs installed in Baltimore City.
Keep an eye out for these new signs, and drive safely! For more cycling news and resources, along with opportunities to help improve cycling in Maryland, go to Bike Maryland's page. For information about all current cycling laws, visit MDOT's Biking and Walking page.


My daughter is self-employed with a Bachelor's degree currently living in Hosur. We come from a middle class, nuclear family background with orthodox values.
My sister has completed her Bachelor's degree. She is currently not working and she lives in Hosur. We come from a middle class, joint family background with traditional values.
Looking good.very talented, knowledgeable, calm, respective, friendly,adjustable in all situations,highly tackling capacity.
My daughter is a Software Professional with a Bachelor's degree currently working in Private sector in Bangalore. We come from an Upper middle class, Nuclear family background with Moderate values residing in hosur.
I have made this profile on behalf of my daughter. She is currently working as an officer in private sector, after having completed her bachelor's degree. We come from a middle class, nuclear family with traditional values presently living in Bangalore.
My friend has a Bachelor's degree and she is employed in Private Sector as a Sales Professional currently based in Hosur. She belongs to a Middle class, Joint family with Orthodox values.
I am creating this profile on behalf of my sister. She is currently working in the private sector as an executive, after having completed her bachelor's degree and she is pursing CMA(cost accountant). We come from a middle class family with moderate family values. We belong to nuclear family currently living in hosur.