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Innate Inhibition of Adaptive Immunity: Mycobacterium tuberculosis-Induced IL-6 Inhibits Macrophage Responses to IFN-γ
Vijaya Nagabhushanam, Alejandra Solache, Li-Min Ting, Claire J. Escaron, Jennifer Y. Zhang and Joel D. Ernst
J Immunol November 1, 2003, 171 (9) 4750-4757; DOI: https://doi.org/10.4049/jimmunol.171.9.4750
Vijaya Nagabhushanam
Division of Infectious Diseases, University of California, and Loewenstein Laboratory for Tuberculosis Research, San Francisco General Hospital, San Francisco, CA 94110;
Alejandra Solache
Division of Infectious Diseases, University of California, and Loewenstein Laboratory for Tuberculosis Research, San Francisco General Hospital, San Francisco, CA 94110; Trudeau Institute, Saranac Lake, NY 12983;
Li-Min Ting
Department of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461;
Claire J. Escaron
Jennifer Y. Zhang
Department of Dermatology, Stanford University, Stanford, CA 94305; and
Joel D. Ernst
Division of Infectious Diseases, University of California, and Loewenstein Laboratory for Tuberculosis Research, San Francisco General Hospital, San Francisco, CA 94110; Departments of Medicine and Microbiology, New York University School of Medicine, New York, NY 10016
In humans and in mice, control of the intracellular pathogen, Mycobacterium tuberculosis (Mtb), requires IFN-γ. Although the adaptive immune response results in production of substantial amounts of IFN-γ in response to Mtb, the immune response is unable to eradicate the infection in most cases. We have previously reported evidence that Mtb inhibits macrophage responses to IFN-γ, suggesting that this may limit the ability of IFN-γ to stimulate macrophages to kill Mtb. We have also observed that uninfected macrophages, adjacent to infected macrophages in culture, exhibit decreased responses to IFN-γ. Here we report that IL-6 secreted by Mtb-infected macrophages inhibits the responses of uninfected macrophages to IFN-γ. IL-6 selectively inhibits a subset of IFN-γ-responsive genes at the level of transcriptional activation without inhibiting activation or function of STAT1. Inhibition of macrophage responses to IFN-γ by IL-6 requires new protein synthesis, but this effect is not attributable to suppressor of cytokine signaling 1 or 3. These results reveal a novel function for IL-6 and indicate that IL-6 secreted by Mtb-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection.
Infection with Mycobacterium tuberculosis (Mtb)3 induces a cellular immune response including CD4+ and CD8+ T cells that secrete IFN-γ (1, 2). IFN-γ, the predominant activator of microbicidal functions of macrophages (3), is detectable at sites of Mtb infection (4) and is essential for prevention of uncontrolled progression of infection (5, 6). However, despite the development of a cellular immune response and local production of IFN-γ, the immune response is unable to eradicate the infection in humans or mice. Insight into one mechanism by which a cellular immune response might be unable to eradicate Mtb infection was gained by in vitro experiments that demonstrated that IFN-γ, while capable of activating macrophages to kill diverse intracellular pathogens such as Toxoplasma gondii, Leishmania donovanii, and Legionella pneumophila, was unable to activate macrophages to kill virulent strains of Mtb (7, 8, 9, 10). We and others (11, 12, 13, 14) have previously presented evidence that this may be due at least in part to the ability of Mtb to selectively inhibit macrophage responses to IFN-γ.
In addition to a direct effect of Mtb infection of macrophages on responses to IFN-γ, we have also noted an apparent bystander effect in which uninfected macrophages adjacent to Mtb-infected macrophages were also unable to respond normally to IFN-γ. Here we report that Mtb-infected macrophages synthesize and secrete one or more polypeptides that inhibit the responses of uninfected macrophages to IFN-γ. Moreover, we provide evidence that this activity is not attributable to IL-10 or to TGF-β, but rather to IL-6, indicating that the secretion of IL-6 by infected macrophages may contribute to the inability of IFN-γ to eradicate Mtb infection.
C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions.
RAW264.7 cells and L929 cells (both obtained from American Type Culture Collection, Manassas, VA) were maintained in complete DMEM (DMEM supplemented with 10% FCS and 2 mM l-glutamine; Life Technologies, Gaithersburg, MD). L cell-conditioned medium was collected from confluent L929 cultures.
Abs, cytokines, primers, and other reagents
PE-labeled anti-IA/IE (BD PharMingen, San Diego, CA), FITC-labeled anti-FcγRI (Ancell, Bayport, MN), anti-TGF-β (1D11; R&D Systems, Minneapolis, MN), anti-IL-10 (JES5-2A5; BD PharMingen), anti-IL-6 (MP5-20F3; BD PharMingen), anti-STAT1α (Zymed, South San Francisco, CA), and anti-phospho-STAT1 (Tyr701; Zymed) were obtained from the respective manufacturers. IFN-γ was obtained from BD PharMingen, and IL-6 from BioSource (Camarillo, CA). The IL-6 ELISA was purchased from R&D Systems. Cycloheximide was obtained from Sigma-Aldrich (St. Louis, MO). Oligonucleotide primers were purchased from Invitrogen (Carlsbad, CA). The sequences of primers used for real-time RT-PCR analysis are shown in Table I⇓.
Sequences of primers used for quantitative RT-PCR
Isolation and culture of bone marrow-derived macrophages (BMDM)
Mice were euthanized using CO2 narcosis and cervical dislocation, and femurs and tibias were removed aseptically. Bone marrow was flushed from either end with cold, complete DMEM (Life Technologies), and the crude cell suspension was filtered through a cell strainer (Falcon; BD Biosciences, Franklin Lake, NJ) and centrifuged at 500 × g to pellet cells, which were then washed twice in complete DMEM. Cells were plated in 150 × 25-mm bacterial grade petri dishes (Falcon) at 4 × 106/plate, in DMEM supplemented with 20% FCS, 30% L929-cell conditioned medium, 1 mM sodium pyruvate (Life Technologies), and 2 mM l-glutamine (Life Technologies). The cells were incubated at 37°C in 5% CO2 for 4 days, after which nonadherent cells were gently washed away, and the medium was replaced. On day 6 adherent cells were scraped, washed, and replated at 5 × 105/well in 12-well tissue culture plates for further use.
Isolation and culture of human monocyte-derived macrophages
Human monocytes were isolated from buffy coats as previously described (14) with a minor modification: to improve adherence, cells were cultured in RPMI 1640 with 2 mM l-glutamine (Life Technologies) and 1% autologous serum for 1 h after plating. Culture medium and nonadherent cells were removed by aspiration, and monolayers were subsequently incubated with fresh culture medium supplemented with 2.5% autologous serum. Human monocyte-derived macrophages were infected with Toxoplasma gondii (RH strain) as previously described (14).
Infection of macrophages and preparation of conditioned medium
Mtb suspensions were prepared as described previously (14). Cells were incubated with the bacterial suspension, calculated to yield a multiplicity of infection of 10, for 4 h and then were washed to remove uningested bacteria. Alternatively, cells were treated with gamma-irradiated Mtb (500 μg/ml) for 4 h. Cells were then washed three times and cultured in fresh medium for 48 h. Conditioned medium (CM) was collected 48 h following infection, filtered through a 0.22-μm pore size filter (Millipore, Bedford, MA), and stored at −20°C for further analysis.
Assay of conditioned medium
CM was serially diluted and added to macrophages cultured at 5 × 105 in 12-well plates (Corning, Corning, NY). Macrophages were incubated in CM for 24 h and stimulated with IFN-γ (20 ng/ml), and surface expression of MHC class II (mouse macrophages) or FcγRI (CD64; human macrophages) was measured 24 h later by flow cytometry as described below.
Neutralization of cytokines
RAW264.7 cells were plated at a concentration of 5 × 105 in 12-well plates and treated with serial dilutions of CM from either infected or uninfected BMDM. To neutralize cytokines, mAbs to IL-6 (MP5-20F3), IL-10 (JES5-2A5), TGF-β (1D11), or their respective isotype controls were added at 2 μg/ml. Following 24-h incubation, IFN-γ (20 ng/ml) was added, and surface expression of MHC class II was determined by flow cytometry as described below.
Mtb-infected and uninfected macrophages were left untreated or were treated with IFN-γ (20 ng/ml) for 24 h at 37°C. Cell monolayers were washed twice on ice with cold PBS containing 0.5 mM EDTA, scraped from the wells, and transferred to FACS tubes (Falcon). The cells were washed, resuspended in PBS with 2.5% FCS and 0.1% NaN3, and incubated for 45 min on ice with PE-conjugated anti-I-A/I-E (anti-MHC class II; mouse macrophages) or FITC-conjugated anti-FcγRI (human macrophages) using the concentrations recommended by the manufacturer. Cells were then washed with PBS and fixed overnight in 1% paraformaldehyde, if infected, or resuspended in PBS. Ten thousand cells were analyzed for MHC class II or CD64 expression on a FACSort flow cytometer and analyzed using CellQuest software (BD Biosciences, Mountain View, CA). The experimental data were expressed as the mean fluorescence intensity (MFI) of triplicate samples ± SD unless otherwise stated.
Total RNA was extracted using RNeasy (Qiagen, Valencia, CA), treated with DNase (Ambion, Austin, TX) to remove contaminating genomic DNA, and quantitated fluorometrically using the RiboGreen assay (Molecular Probes, Eugene, OR). RNA (1 μg) was reverse transcribed to cDNA under standard protocols using the RT system (Promega, Madison, WI). Standard cDNA, prepared from RNA extracted from RAW264.7 cells following an 8-h stimulation with IFN-γ was used at 50, 10, 2, and 0.4 ng in PCR reactions. For the amplification of individual genes, samples were used at a concentration of 10 ng, with the exception of IFN-γ-inducible class II trans-activator (CIITA; 100 ng) and the housekeeping gene, GAPDH (1 ng). Reactions were set up using SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA), and samples were subjected to thermal cycling on an ABI 7700 thermal cycler (PE Applied Biosystems) under the following conditions: stage 1, 95°C for 10 min; and stage 2, 94°C for 45 s, 60°C for 15 s, and 72°C for 30 s. Stage 2 was repeated for 40 cycles. Controls included a control without template to determine the presence of primer-dimers as well as controls without RT to determine the potential presence of residual genomic DNA. Dissociation curve analysis was performed to evaluate potential contribution to fluorescence from primer dimers or nonspecific amplicons. The data were analyzed with the Sequence Detector version 1.7a software (PE Applied Biosystems). To maintain consistency, the baseline was set automatically by the software, using data collected from cycles 3–15. In some cases it was necessary to over-ride this setting to optimize analysis. The increase in fluorescence intensity of SYBR Green (ΔRn) was plotted against cycle number. The threshold cycle (CT) was calculated by the software as the cycle number at which ΔRn crosses the baseline. For each gene examined, a standard curve was generated by plotting CT values against input cDNA concentration, and samples were quantified by interpolation from the standard curve. Data obtained for individual genes were normalized to the corresponding mean GAPDH levels. Fold increases were calculated taking the untreated sample as the baseline reference.
Production of pGrl-CIITApD1 stable transfectants and assay for luciferase activity
RAW 264.7 cells were stably cotransfected with 1 μg of pGrl-CIITApD1 (15) and 0.1 μg of pcDNA3.1-neo (Invitrogen)/106 cells, using the Trans-IT reagent (Mirus Technologies, Madison, WI) and protocols recommended by the manufacturer. Cells were selected in complete DMEM supplemented with 400 μg/ml G418 (Invitrogen), and clones stably expressing pGrl-CIITApD1 were selected for utilization in additional experiments on the basis of a >5-fold increase in luciferase activity in response to IFN-γ. To determine the effect of IL-6 on CIITA promoter activity, stably transfected RAW264.7 cells were treated with 4 ng/ml IL-6 and stimulated with 20 ng/ml of IFN-γ 24 h later. Cells were lysed 8 h later using passive lysis buffer (Promega), and the lysates were examined for luciferase activity using the luciferase reporter system assay (Promega). Firefly luciferase induction driven by the CIITA promoter was normalized to the input protein levels as determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL).
Immunoblots for STAT1 and phosphorylated-STAT1
For detection of total and phosphorylated STAT1, RAW264.7 cells were pretreated with IL-6 (4 ng/ml) for 24 h and then with 20 ng/ml IFN-γ for various time points as determined in pilot experiments. Total cellular protein lysates were prepared by washing cells with ice-cold PBS and then scraping them in ice-cold RIPA buffer (50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A) supplemented with protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (Calbiochem, San Diego, CA). The lysates were transferred to microfuge tubes, incubated on ice for 20 min, and clarified by centrifugation at 10,000 × g at 4°C for 20 min. Proteins were separated by electrophoresis on a 12% reducing polyacrylamide gel, transferred to nitrocellulose membranes, blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20, and incubated with either anti-STAT1 (Zymed) at a 1/250 dilution or anti-phospho-STAT1 (Tyr701; Zymed) at a 1/1,000 overnight at 4°C. Blots were washed, incubated with the appropriate HRP-labeled secondary Ab, and developed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ).
Statistical analysis was conducted using the software application PRISM (GraphPad, San Diego, CA). The statistical significance of experimental data was determined by two-tailed, unpaired, Student's t test. Differences with p < 0.05 were considered significant.
CM from M. tuberculosis-infected macrophages inhibits human and mouse macrophage responses to IFN-γ
IFN-γ activation of macrophages effectively controls the growth of the intracellular parasite, Toxoplasma gondii. However, when macrophages are infected first with Mtb and subsequently with T. gondii, activation with IFN-γ fails to control the replication of T. gondii (14). This effect of Mtb is not limited to directly infected macrophages. Uninfected macrophages in culture with Mtb-infected macrophages were also unable to control T. gondii replication on activation with IFN-γ (L.-M. Ting and J. D. Ernst, unpublished observations), suggesting that infected macrophages secrete one or more factors that block IFN-γ-induced responses. To further investigate this observation, human macrophages were treated with fresh medium, medium from uninfected macrophages, or medium from macrophages infected with Mtb for 48 h, and then infected with T. gondii. As shown in Fig. 1⇓A, macrophages treated with CM from Mtb-infected macrophages were unable to control replication of T. gondii on activation with IFN-γ, while addition of CM from uninfected macrophages had no such inhibitory effect. The effect of Mtb-CM on IFN-γ-inducible responses was not confined to inhibition of restriction of T. gondii growth. Mtb-CM also inhibited IFN-γ-induced surface expression of FcγRI (CD64) in a dose-dependent manner (Fig. 1⇓B), suggesting a broader effect on IFN-γ-inducible responses. We also found that murine BMDM exposed to live (not shown) or gamma-irradiated (Fig. 2⇓A) Mtb exhibit reduced responses to IFN-γ up-regulation of surface MHC class II, and in addition, CM from Mtb-infected BMDM inhibited IFN-γ-induced MHC class II expression in a dose-dependent manner (Fig. 2⇓B). Inhibition of IFN-γ-induced MHC class II by CM was also observed in the murine macrophage cell line, RAW 264.7. As RAW264.7 cells are easily maintained and manipulated, additional experiments were performed using these cells.
Mtb-CM inhibits IFN-γ-induced responses in human monocyte-derived macrophages. A, Monocyte-derived macrophages were treated with fresh medium, CM from uninfected macrophages, or CM from Mtb-infected macrophages for 48 h, and then infected with T. gondii. One hour after infection, some cells were fixed for time zero enumeration, while others were treated with IFN-γ (100 ng/ml) or medium. After an additional 24 h the remaining cells were fixed with methanol/acetone, and Toxoplasma were enumerated after staining with 4′,6-diamido-2-phenylindole hydrochloride. Values shown are the mean ± SD of the number of T. gondii nuclei per 300 macrophage nuclei, counted in triplicate. The p values shown are the comparison of the number of parasites in untreated vs IFN-γ-treated cells. B, Human monocyte-derived macrophages cultured in 96-well nonadherent plates were treated with CM from Mtb-infected monocyte-derived macrophages for 24 h, then stimulated with 100 ng/ml IFN-γ or left untreated. Surface expression of CD64 was analyzed by flow cytometry. Data shown are representative of three experiments, using monocytes isolated from different donors.
Gamma-irradiated Mtb and Mtb-CM inhibit IFN-γ induction of MHC class II on murine macrophages. BMDM were treated with increasing concentrations of gamma-irradiated Mtb for 48 h (A) or with increasing dilutions of CM for 24 h (B) and then stimulated with IFN-γ (20 ng/ml) or left untreated. Surface expression of MHC class II was measured 24 h later.
IL-6 contributes to inhibition of responses to IFN-γ
Infection of macrophages with Mtb induces the production of multiple cytokines, including IL-10 (16), TGF-β (17), and IL-6 (18). To determine whether any or all of these cytokines were responsible for the inhibition of IFN-γ-induced responses, neutralizing Abs to IL-6, TGF-β and IL-10 or their respective isotype controls were added to CM when used to treat macrophages before stimulation with IFN-γ. Neutralization of IL-6 resulted in a significant recovery of IFN-γ induction of MHC class II expression (Fig. 3⇓A), while neutralization of neither IL-10 nor TGF-β had a measurable effect (Fig. 3⇓, B and C). To determine whether IL-6 alone could antagonize macrophage responses to IFN-γ, RAW264.7 cells were incubated with increasing concentrations of recombinant murine IL-6 for 24 h and then stimulated with IFN-γ. As shown in Fig. 4⇓, IL-6 inhibited the up-regulation of MHC class II by IFN-γ in a dose-dependent manner, with half-maximal inhibition occurring at 2 ng/ml. BMDM showed similar responses, although inhibition of IFN-γ-induced expression of MHC class II occurred at higher concentrations of IL-6, with half-maximal inhibition occurring at 8 ng/ml (data not shown).
Neutralization of IL-6 partially restores IFN-γ induction of MHC class II expression in Mtb-CM-treated macrophages. IFN-γ induction of MHC class II expression was measured in RAW264.7 cells treated with increasing dilutions of CM and a constant concentration (2 μg/ml) of neutralizing Abs to IL-6 (A), IL-10 (B), or TGF-β (C).
Recombinant IL-6 inhibits IFN-γ induction of MHC class II. RAW264.7 cells were treated with increasing concentrations of IL-6 for 24 h, then stimulated with IFN-γ or left unstimulated. Surface expression of MHC class II was assayed by flow cytometry 24 h later.
To determine whether Mtb induces the production of sufficient IL-6 to account for the activity in Mtb-CM, we assayed IL-6 by ELISA. This revealed that 5 × 105 BMDM treated with 500 μg/ml gamma-irradiated Mtb for 24 h produced 12 ± 3 ng/ml of IL-6, a concentration sufficient to account for substantial inhibition of responses to IFN-γ.
IL-6 inhibits induction of selected IFN-γ-responsive genes
Since IL-6 and/or Mtb could inhibit surface expression of MHC class II by disrupting transcription, translation, post-translational modification, or intracellular trafficking, we first investigated whether IL-6 inhibits IFN-γ-induced gene expression. As the expression of MHC class II is under the control of the CIITA (19), we examined the effect of IL-6 on steady state levels of CIITA mRNA in IFN-γ-stimulated RAW264.7 cells. Treatment with IL-6 alone had no effect on CIITA mRNA levels. Pretreatment with IL-6 reduced IFN-γ-induced CIITA mRNA by 50–80% in separate experiments, at peak levels of CIITA expression (8 h; Fig. 5⇓A). The reduction in mRNA levels was not limited to CIITA, but was also observed for the chemokine monokine induced by γ (55–70% reduction at peak expression) and the transcription factors, IFN regulatory factor-1 and IFN consensus sequence binding protein (50% reduction; Fig. 5⇓B). IL-6 did not inhibit induction of all IFN-γ-responsive genes examined; the IFN-γ-inducible GTPases IFN-inducible GTPase and LRG-47, and the chemokine inducing protein-10 were marginally affected or unaffected by pretreatment of macrophages with IL-6 (Fig. 5⇓B), indicating that the inhibitory effects of IL-6 are exerted on a subset of IFN-γ-responsive genes.
Pretreatment with IL-6 inhibits a subset of IFN-γ-responsive genes. RAW264.7 cells, treated with IL-6 or left untreated, were stimulated with IFN-γ. Total RNA was extracted at intervals as indicated, and transcripts of CIITA (A) or other IFN-γ-regulated genes (B) were quantitated by real-time RT-PCR as described in Materials and Methods.
IL-6 inhibits transcription of the class II trans-activator
Although IFN-γ induction of CIITA depends on the induction of IFN regulatory factor-1 as well as on a γ-activated sequence element that binds STAT1 (15, 20), we performed additional studies of the mechanism of IL-6 inhibition by focusing on CIITA, because of its more marked inhibition. As IFN-γ induction of CIITA mRNA in macrophages is predominantly under the control of CIITA promoter IV (21), we examined the effect of IL-6 on RAW264.7 cells stably transfected with a plasmid containing a 560-bp promoter IV fragment driving luciferase. Pretreatment with IL-6 inhibited IFN-γ-induced activity of the CIITA promoter IV (Fig. 6⇓A). Although promoter IV is the predominant IFN-γ-responsive CIITA promoter, CIITA promoters I and III are also IFN-γ-inducible in RAW264.7 cells (21, 22). We also found that CIITA promoters I and III are IFN-γ-responsive in RAW264.7 cells, and that IL-6 decreased IFN-γ-induced CIITA mRNA driven by all three promoters (Fig. 6⇓B).
IL-6 inhibits transcriptional control of CIITA. A, RAW264.7 cells stably transfected with pGrl-CIITApD1 were treated with IL-6 or medium alone. Following stimulation with IFN-γ 24 h later, the cells were lysed at the time points indicated for detection of luciferase activity. Log luminescence units driven by the CIITA promoter for each sample was normalized to the respective protein input. Data are expressed as the fold increase over the untreated sample and represent the mean ± SD of triplicate samples. B, RAW264.7 cells were either treated with IL-6 for 24 h or were left untreated. Total RNA was extracted 0 or 8 h following the addition of IFN-γ, and transcripts of CIITA driven by promoters I, III, and IV were quantitated by real-time RT-PCR.
To determine whether IL-6 also affected the stability of CIITA mRNA, we examined the rate of decay of IFN-γ-induced CIITA mRNA. RAW264.7 cells, treated for 24 h with either IL-6 or control medium, were stimulated with IFN-γ. At the point of maximal expression of CIITA mRNA (8 h), actinomycin D was added to halt further RNA synthesis, and CIITA mRNA transcripts were quantitated by real-time RT-PCR at 2-h intervals over the course of 6 h. The rate of decay of CIITA was the same in IL-6-treated and control RAW264.7 cells (data not shown), indicating that IL-6 does not affect the stability of CIITA transcripts.
IL-6 inhibition of macrophage responses to IFN-γ requires new protein synthesis
To further understand the mechanism of IL-6, we examined the time course of its effect on macrophages. Brief (15–60 min) pretreatment of macrophages with IL-6 had no effect on subsequent IFN-γ induction of MHC class II (Fig. 7⇓A). However, after 24 h, inhibition by IL-6 was apparent (Fig. 7⇓A), which suggests that the effect of IL-6 on responses to IFN-γ requires the synthesis of new protein, rather than inhibition of post-transcriptional modification of constitutive proteins. Indeed, cycloheximide, a reversible inhibitor of protein synthesis, blocked the effect of IL-6 on IFN-γ induction of surface MHC class II (Fig. 7⇓B) and CIITA mRNA (Fig. 7⇓C).
Inhibition of IFN-γ responses by IL-6 requires the synthesis of new protein(s). A, RAW264.7 cells were treated with IL-6 for varying lengths of time as indicated. The cells were either treated with IFN-γ or left untreated, and the surface expression of MHC class II was assayed 24 h later. B, RAW264.7, treated with IL-6 or medium alone were either simultaneously treated with 1 μM cycloheximide or left untreated. Cells were washed 24 h later, allowed to recover for 1 h, and then stimulated with IFN-γ or incubated in medium in the absence of cycloheximide. The surface expression of MHC class II was measured 24 h later. C, Alternatively, total RNA was extracted at 0 and 8 h poststimulation with IFN-γ. CIITA transcripts were quantitated by real-time PCR.
Suppressors of cytokine signaling (SOCS) proteins do not account for the effects of IL-6 in macrophages
SOCS proteins are inducible by IFNs and other cytokines and mediate the inhibition of responses to IFN-γ by blocking phosphorylation of STAT1 (23, 24). Since IL-6 inhibition of macrophage responses to IFN-γ required new protein synthesis, we examined the possibility that SOCS-1 and/or SOCS-3 accounted for the effects of IL-6. As shown in Fig. 8⇓A, LPS induced the expression of SOCS-1 and SOCS-3 mRNA in RAW264.7 macrophages. However, IL-6 did not induce SOCS-1 expression and induced only marginal amounts of SOCS-3 at concentrations that markedly inhibited IFN-γ induction of CIITA and surface MHC class II (Fig. 8⇓A). IL-6 was also unable to induce SOCS-1 or significant SOCS-3 expression in BMDM from C57BL/6 mice (data not shown). As further evidence that the action of IL-6 is not mediated by SOCS proteins, IL-6 did not inhibit tyrosine phosphorylation of STAT1 in response to IFN-γ (Fig. 8⇓B).
IL-6 does not induce SOCS-1 or SOCS-3. A, RAW264.7 cells were treated with IL-6 (4 or 8 ng/ml) or LPS (100 ng/ml) or were left untreated. Total RNA was extracted 5 h later, and SOCS-1 or SOCS-3 mRNA were quantitated by real-time RT-PCR. B, RAW 264.7 cells, either treated with IL-6 or medium alone, were stimulated with IFN-γ, and the cells were lysed at intervals as indicated. The presence of total STAT1 or phosphorylated-STAT1 was detected by Western blotting.
Mtb is a highly successful intracellular pathogen that is able to survive and cause disease despite the induction of innate and adaptive immune responses. While IFN-γ is clearly required for the prevention of rapidly progressive Mtb infection (5, 6), even the development of an immune response with abundant local production of IFN-γ is unable to reliably eradicate Mtb infection. We have previously reported that Mtb is able to block macrophage responses to IFN-γ by inhibiting the transcription of IFN-γ-responsive genes, indicating that this phenomenon may contribute to the inability of an adaptive immune response to provide complete protection against tuberculosis (14). We extend our observations to report here that one mechanism for Mtb to render macrophages refractory to activation by IFN-γ is to stimulate macrophages to secrete an inhibitory factor(s), one of which is IL-6. Furthermore, our observations indicate a novel role for IL-6, in that it inhibits IFN-γ signaling in macrophages.
IL-6 is a pleiotropic cytokine that is produced by a variety of cells, including macrophages (25), T cells (26), endothelial cells (27), and fibroblasts (28), and also exerts its effects on multiple cell types. IL-6, recognized for its role as a proinflammatory cytokine, induces the production of acute phase proteins in response to infection and injury (29) and synergizes with IL-1 in inducing acute phase responses such as pyrexia and leukocytosis (30). In addition, IL-6 is both a B cell growth and differentiation factor (25, 31). In vivo, IL-6 has also been shown to play a role in the initiation of T cell activation (32). More recently, IL-6 induced by microbial components has been observed to support effector T cell proliferation in vivo by suppressing regulatory T cells (33).
In contrast to these observations, IL-6 has also been implicated in inhibiting T cell responses. VanHeyningen and coworkers (34) observed that exposure to macrophages infected with M. bovis BCG or M. avium inhibited Ag-specific proliferation of a CD4+ T cell hybridoma. In addition, they noted that both CM from mycobacterium-infected macrophages and purified IL-6 were capable of inhibiting T cell hybridoma proliferation. As this phenomenon was observed even when APCs were fixed with paraformaldehyde, T cells were probably the predominant target of the action of IL-6 in their studies. Additionally, IL-6 has been been found to block IFN-γ induction of Th1 differentiation in naive CD4+ T cells (35). In these cells IL-6 induced expression of SOCS-1, thereby inhibiting IFN-γ-induced tyrosine phosphorylation of STAT1 and, therefore, IFN-γ-dependent Th1 differentiation. In contrast, our studies of macrophages reveal that IL-6 can also inhibit cellular responses to IFN-γ by a SOCS-1- and SOCS-3-independent mechanism, implying that the effects of IL-6 on IFN-γ responses can be mediated by distinct mechanisms in different cell types.
IL-6 has been previously observed to have an inhibitory effect on macrophage function. It is capable of suppressing the production of LPS-induced TNF-α in human peripheral blood monocytes (36). Additionally, murine peritoneal macrophages pretreated with IL-6 were found to be incapable of restricting Toxoplasma replication in the presence of IFN-γ (37). We observed that IL-6 was induced by infection of macrophages with Mtb and contributed to inhibition of macrophage responses to IFN-γ. Our findings thus concur with these observations and additionally provide insight into the mechanism of action of IL-6 in inhibiting the effects of IFN-γ.
Although infection with Mtb induces IL-6, the role of IL-6 in the immune response to Mtb in vivo is not well understood. Significant quantities of IL-6 are produced in response to Mtb infection; human and murine macrophages secrete IL-6 in response to Mtb in vitro (38, 39), and elevated concentrations of IL-6 are present in plasma of patients with tuberculosis (40). Studies of Mtb infection of IL-6−/− mice suggest that IL-6 may play a role in the early stages of T cell activation (41), in contrast to our results indicating that IL-6 inhibits macrophage activation. We have also observed, however, that Mtb inhibits IFN-γ induction of MHC class II on BMDM from both wild-type and IL-6−/− mice and that CM from M. tuberculosis-infected macrophages from IL-6−/− mice contains some residual inhibitory activity (data not shown), indicating that Mtb inhibits IFN-γ activation of macrophages in both an IL-6-dependent and independent manner.
The induction of IL-6 production is not unique to Mtb. Indeed, stimulation of TLR4 is a predominant mechanism for the induction of IL-6 (42), and numerous bacteria possess components that activate TLR4 signaling. For instance, bacteria such as Escherichia coli (43) and Neisseria species (44) induce the production of significant quantities of IL-6. However, these bacterial species are responsible for acute infections and have not developed mechanisms to survive intracellularly. Consequently, the effects of IL-6 on the immune system do not impact the progress of infection induced by these bacteria in a manner similar to that observed in tuberculosis. Mtb, on the other hand, has developed multiple survival strategies, such as prevention of phagosome maturation (45, 46), prevention of phagosomal acidification (45), and alteration of the capacity of dendritic cells to serve as APCs (46). Our results suggest Mtb has also taken advantage of the induction and activity of IL-6 as another strategy to ensure its survival in the face of an apparently robust cellular immune response. In addition, our observations reveal a novel inhibitory function for IL-6 with respect to IFN-γ signaling, and we suggest that intracellular organisms such as Mtb that lead to chronic infection exploit this inhibitory property of IL-6 to evade eradication by a cellular immune response.
We thank Drs. G. M. O'Keefe and E. N. Benveniste for the pGrl-CIITApD1 plasmid. We also thank Ivan Gomez for his contribution to the work described in this paper.
↵1 This work was supported by National Institutes of Health Grants HL51992 and AI46097.
↵2 Address correspondence and reprint requests to Dr. Joel D. Ernst, Departments of Medicine and Microbiology, New York University School of Medicine, 550 First Avenue, NB16S8, New York, NY 10016. E-mail address: joel.ernst{at}med.nyu.edu
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; BMDM, bone marrow-derived macrophage; CIITA, IFN-γ-inducible class II trans-activator; CM, conditioned medium; CT, threshold cycle; JAK, Janus kinase; MFI, mean fluorescence intensity; RLU, relative light units; SOCS, suppressor of cytokine signaling; TLR, Toll-like receptor.
Received May 19, 2003.
Ladel, C. H., C. Blum, A. Dreher, K. Reifenberg, S. H. Kaufmann. 1995. Protective role of γ/δ T cells and α/βa T cells in tuberculosis. Eur. J. Immunol. 25:2877.
Mogues, T., M. E. Goodrich, L. Ryan, R. LaCourse, R. J. North. 2001. The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J. Exp. Med. 193:271.
Nathan, C. F., T. J. Prendergast, M. E. Wiebe, E. R. Stanley, E. Platzer, H. G. Remold, K. Welte, B. Y. Rubin, H. W. Murray. 1984. Activation of human macrophages: comparison of other cytokines with interferon-γ. J. Exp. Med. 160:600.
Barnes, P. F., S. Lu, J. S. Abrams, E. Wang, M. Yamamura, R. L. Modlin. 1993. Cytokine production at the site of disease in human tuberculosis. Infect. Immun. 61:3482.
Cooper, A. M., D. K. Dalton, T. A. Stewart, J. P. Griffin, D. G. Russell, I. M. Orme. 1993. Disseminated tuberculosis in interferon γ gene-disrupted mice. J. Exp. Med. 178:2243.
Flynn, J. L., J. Chan, K. J. Triebold, D. K. Dalton, T. A. Stewart, B. R. Bloom. 1993. An essential role for interferon γ in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249.
Sibley, L. D., J. L. Krahenbuhl. 1987. Mycobacterium leprae-burdened macrophages are refractory to activation by γ interferon. Infect. Immun. 55:446.
Heath, L., C. Chrisp, G. Huffnagle, M. LeGendre, Y. Osawa, M. Hurley, C. Engleberg, J. Fantone, J. Brieland. 1996. Effector mechanisms responsible for γ interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts. Infect. Immun. 64:5151.
Wang, Z. E., S. L. Reiner, S. Zheng, D. K. Dalton, R. M. Locksley. 1994. CD4+ effector cells default to the Th2 pathway in interferon γ-deficient mice infected with Leishmania major. J. Exp. Med. 179:1367.
Scharton-Kersten, T. M., T. A. Wynn, E. Y. Denkers, S. Bala, E. Grunvald, S. Hieny, R. T. Gazzinelli, A. Sher. 1996. In the absence of endogenous IFN-γ, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.
Sibley, L. D., J. L. Krahenbuhl. 1988. Induction of unresponsiveness to γ interferon in macrophages infected with Mycobacterium leprae. Infect. Immun. 56:1912.
Sibley, L. D., L. B. Adams, J. L. Krahenbuhl. 1990. Inhibition of interferon-γ-mediated activation in mouse macrophages treated with lipoarabinomannan. Clin. Exp. Immunol. 80:141.
Chan, J., X. D. Fan, S. W. Hunter, P. J. Brennan, B. R. Bloom. 1991. Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages. Infect. Immun. 59:1755.
Ting, L. M., A. C. Kim, A. Cattamanchi, J. D. Ernst. 1999. Mycobacterium tuberculosis inhibits IFN-γ transcriptional responses without inhibiting activation of STAT1. J. Immunol. 163:3898.
O'Keefe, G. M., V. T. Nguyen, L. L. Ping Tang, E. N. Benveniste. 2001. IFN-γ regulation of class II transactivator promoter IV in macrophages and microglia: involvement of the suppressors of cytokine signaling-1 protein. J. Immunol. 166:2260.
Hickman, S. P., J. Chan, P. Salgame. 2002. Mycobacterium tuberculosis induces differential cytokine production from dendritic cells and macrophages with divergent effects on naive T cell polarization. J. Immunol. 168:4636.
Hernandez-Pando, R., H. Orozco, K. Arriaga, A. Sampieri, J. Larriva-Sahd, V. Madrid-Marina. 1997. Analysis of the local kinetics and localization of interleukin-1α, tumour necrosis factor-α and transforming growth factor-β, during the course of experimental pulmonary tuberculosis. Immunology 90:607.
Beltan, E., L. Horgen, N. Rastogi. 2000. Secretion of cytokines by human macrophages upon infection by pathogenic and non-pathogenic mycobacteria. Microb. Pathog. 28:313.
Masternak, K., A. Muhlethaler-Mottet, J. Villard, M. Zufferey, V. Steimle, W. Reith. 2000. CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex. Genes Dev. 14:1156.
Hobart, M., V. Ramassar, N. Goes, J. Urmson, P. F. Halloran. 1997. IFN regulatory factor-1 plays a central role in the regulation of the expression of class I and II MHC genes in vivo. J. Immunol. 158:4260.
Pai, R. K., D. Askew, W. H. Boom, C. V. Harding. 2002. Regulation of class II MHC expression in APCs: roles of types I, III, and IV class II transactivator. J. Immunol. 169:1326.
Muhlethaler-Mottet, A., L. A. Otten, V. Steimle, B. Mach. 1997. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16:2851.
Krebs, D. L., D. J. Hilton. 2001. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19:378.
Alexander, W. S., R. Starr, J. E. Fenner, C. L. Scott, E. Handman, N. S. Sprigg, J. E. Corbin, A. L. Cornish, R. Darwiche, C. M. Owczarek, et al 1999. SOCS1 is a critical inhibitor of interferon γ signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98:597.
Van Snick, J., A. Vink, S. Cayphas, C. Uyttenhove. 1987. Interleukin-HP1, a T cell-derived hybridoma growth factor that supports the in vitro growth of murine plasmacytomas. J. Exp. Med. 165:641.
Hirano, T., T. Taga, N. Nakano, K. Yasukawa, S. Kashiwamura, K. Shimizu, K. Nakajima, K. H. Pyun, T. Kishimoto. 1985. Purification to homogeneity and characterization of human B-cell differentiation factor (BCDF or BSFp-2). Proc. Natl. Acad. Sci. USA 82:5490.
Corbel, C., F. Melchers. 1984. The synergism of accessory cells and of soluble α-factors derived from them in the activation of B cells to proliferation. Immunol. Rev. 78:51.
Weissenbach, J., Y. Chernajovsky, M. Zeevi, L. Shulman, H. Soreq, U. Nir, D. Wallach, M. Perricaudet, P. Tiollais, M. Revel. 1980. Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies. Proc. Natl. Acad. Sci. USA 77:7152.
Geiger, T., T. Andus, J. Klapproth, T. Hirano, T. Kishimoto, P. C. Heinrich. 1988. Induction of rat acute-phase proteins by interleukin 6 in vivo. Eur. J. Immunol. 18:717.
Helle, M., J. P. Brakenhoff, E. R. De Groot, L. A. Aarden. 1988. Interleukin 6 is involved in interleukin 1-induced activities. Eur. J. Immunol. 18:957.
Kopf, M., S. Herren, M. V. Wiles, M. B. Pepys, M. H. Kosco-Vilbois. 1998. Interleukin 6 influences germinal center development and antibody production via a contribution of C3 complement component. J. Exp. Med. 188:1895.
Leal, I. S., B. Smedegard, P. Andersen, R. Appelberg. 1999. Interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine. Infect. Immun. 67:5747.
Pasare, C., R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033.
VanHeyningen, T. K., H. L. Collins, D. G. Russell. 1997. IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses. J. Immunol. 158:330.
Diehl, S., J. Anguita, A. Hoffmeyer, T. Zapton, J. N. Ihle, E. Fikrig, M. Rincon. 2000. Inhibition of Th1 differentiation by IL-6 is mediated by SOCS1. Immunity 13:805.
Aderka, D., J. M. Le, J. Vilcek. 1989. IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J. Immunol. 143:3517.
Beaman, M. H., C. A. Hunter, J. S. Remington. 1994. Enhancement of intracellular replication of Toxoplasma gondii by IL-6: interactions with IFN-γ and TNF-α. J. Immunol. 153:4583.
Giacomini, E., E. Iona, L. Ferroni, M. Miettinen, L. Fattorini, G. Orefici, I. Julkunen, E. M. Coccia. 2001. Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response. J. Immunol. 166:7033.
Indrigo, J., R. L. Hunter, Jr, J. K. Actor. 2002. Influence of trehalose 6,6′-dimycolate (TDM) during mycobacterial infection of bone marrow macrophages. Microbiology 148:1991.
el-Ahmady, O., M. Mansour, H. Zoeir, O. Mansour. 1997. Elevated concentrations of interleukins and leukotriene in response to Mycobacterium tuberculosis infection. Ann. Clin. Biochem. 34:160.
Saunders, B. M., A. A. Frank, I. M. Orme, A. M. Cooper. 2000. Interleukin-6 induces early γ interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection. Infect. Immun. 68:3322.
Schilling, D., K. Thomas, K. Nixdorff, S. N. Vogel, M. J. Fenton. 2002. Toll-like receptor 4 and Toll-IL-1 receptor domain-containing adapter protein (TIRAP)/myeloid differentiation protein 88 adapter-like (Mal) contribute to maximal IL-6 expression in macrophages. J. Immunol. 169:5874.
Lee, J. E., J. S. Kim, I. H. Choi, M. Tagawa, T. Kohsaka, D. K. Jin. 2002. Cytokine expression in the renal tubular epithelial cells stimulated by Shiga toxin 2 of Escherichia coli O157:H7. Renal Failure 24:567.
Sprong, T., P. V. Ley, L. Steeghs, W. J. Taw, T. J. Verver-Janssen, M. G. Netea, J. W. Meer, M. V. Deuren. 2002. Neisseria meningitis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4. Eur. Cytokine Network 13:411.
Crowle, A. J., R. Dahl, E. Ross, M. H. May. 1991. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect. Immun. 59:1823.
Mariotti, S., R. Teloni, E. Iona, L. Fattorini, F. Giannoni, G. Romagnoli, G. Orefici, R. Nisini. 2002. Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells. Eur. J. Immunol. 32:3050.
You are going to email the following Innate Inhibition of Adaptive Immunity: Mycobacterium tuberculosis-Induced IL-6 Inhibits Macrophage Responses to IFN-γ
Vijaya Nagabhushanam, Alejandra Solache, Li-Min Ting, Claire J. Escaron, Jennifer Y. Zhang, Joel D. Ernst
The Journal of Immunology November 1, 2003, 171 (9) 4750-4757; DOI: 10.4049/jimmunol.171.9.4750
Early Self-Regulatory Mechanisms Control the Magnitude of CD8+ T Cell Responses Against Liver Stages of Murine Malaria
Sublethal Hyperoxia Impairs Pulmonary Innate Immunity
Dependence of IL-4, IL-13, and Nematode-Induced Alterations in Murine Small Intestinal Smooth Muscle Contractility on Stat6 and Enteric Nerves
Show more Host Defense


text:
The Indian Medical Gazette is an Indian medical journal established in 1866. In its early days, it was closely associated with the Indian Medical Service.

Editors
The following is a partial list of the editors:
 David Boyes Smith (1866)
 John Purefoy Colles (1867)
 Charles K. Francis (1868)
 James Tyrell Carter Ross (1869–70)
 Nottidge Charles MacNamara (1871–73)
 Kenneth McLeod (1871–92)
 John Gay French (1875–76)
 Laurence Austine Waddell (1884–85)
 William John Simpson (1889–97)
 Alexander Crombie (1892–93)

References

External links

https://www.ncbi.nlm.nih.gov/pmc/journals/2470/

Publications established in 1866
1866 establishments in India
Monthly journals
General medical journals
History of medicine in India
19th-century publications

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Why does Google's Keyword Planner updates have SEOs up in arms?
You are reading: Why does Google's Keyword Planner updates have SEOs up in arms?
For time immemorial, SEOs have been using Google's Keyword Planner as their go-to tool to get an idea of traffic volumes for keywords and phrases. If you're not in the know, Keyword Planner is a free tool you can use to search for keywords (e.g. Essex wedding venues, wedding venues in Essex etc.) and ad group ideas relevant to your product or service. Using historical data and traffic forecasts like search volume, you can identify keywords with the most traffic or those with lower volume.
Google's Keyword Planner now combines keywords for search volume, a major change which has made search volume figures somewhat inaccurate and has SEOs up in arms.
Instead of showing individual keyword estimates for a particular keyword or phrase, Google has decided to combine data. The result being the same estimate for similar keywords or phrases. No longer will SEOs be able to choose keywords to use in their campaigns based on individual volume.
The result being that increased figures are an amalgamation and not entirely accurate – thanks Google!
This isn't particularly good news, and in all honesty I can't see what the benefit is to the end user or Google, can you? (comments welcome below).
This feels like another step by Google to make marketers' and website owners' jobs more difficult. Being even more secretive about keyword volumes means that we don't know what variation is most important / most searched upon. It also means that if people want to find out true search volumes for different variations of keywords, then they'll have to run paid ads to do so.
To counter this, we should all be writing content that is optimised for human reading and not for search engines, but keyword research can help with more than just website optimisation.


The X-BOARD - XB 500 is a professional device for winding in/out cable drums including single core lines, cable rings, cable drums, empty conduits, ropes and rope drums ensuring trouble-free and efficient working without the tangles.
The X-BOARD - XB 500 can bear loads of up to 800 kg and is suitable for almost all drum types including damaged cable drums. Twelve rubberised feet ensure a non-slip secure standing position.


text:
Counter-strike:...
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Counter-Strike: Global Offensive review
Counter-Strike: Global Offensive is an extremely popular competitive shooter. You will spend some time to master it and get the skills you need to climb to the top. The game is enjoyable with a lot of variabilities. It has a growing community of players which can play against other players for a price. Anyway, playing will provide a great experience for anyone who prefers competitive gaming.
Storyline 10/10
There are two opposing sides in the game: Terrorist and Counter-Terrorists. It is clear that choosing one of these sides you will get the particular objectives and tools. Terrorists are expected to mine one of the two sites marked on the map. The opponents must prevent this event. If they are late and the bomb has been planted already, they should defuse it. There is a timer showing the time remaining until the bomb explodes. If you want to avoid such a scenario, try to kill all players before they manage to plant a bomb.
There are several modes and the standard one is the Bomb Scenario with most competitive matches. This is a multiplayer. Here you will defend and use a bomb site. There are 15 rounds developed in an elimination style. Each of them will be over when one player wins more rounds.
There is also the mission-based mode, Hostage, where the Terrorists will defend a hostage. The opponents will try to perform the rescue mission – in keeping with the best traditions of the mission-based modes. Also, you can try the Wingman, and Weapons Expert modes added later and introducing twists on the regular gameplay.
Choosing any mode, you will be involved in the active teamwork. You will develop the plans and make decisions together and that will help you to behave effectively and win. Be ready to communicate verbally with other players, discuss the maps you are going to play and the ways to navigate the map efficiently.
There are two ranking systems: Matchmaking Rank and XP Rank. The former is based on your skills. When you advance, you will get better performance and a higher ranking. The latter is based on various things including your success in any of the game's official game modes.
Graphics and Sound 9/10
The graphics are not the best effort here. Still, the improved interface will allow you to feel more convenient and confident while navigating it. Also, you will enjoy the number of cosmetics like weapon skins which can be obtained by playing the game as a reward for your achievements or purchased with money in real life. The price is quite different from a dollar to $3 000. You can refuse these elements because they don't offer any advantage for you to win.
The game can boast of the perfect sound design. The effects are of high quality and you will hear all sounds produced by enemies including their footsteps. The soundtracks can be replaced with the Music Kits which should be paid for.
Counter-Strike: Global Offensive has a lot of maps, modes, and characters with various weapons. It can be regarded as the updated classic CS due to the similar content but there are tons of absolutely free ideas of the gameplay which is still a little bit repetitive. You definitely agree that this free title is one of the best FPS.
Author: Valve
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text:
Alaska orders Boeing jets
— Sep 20th, 1996
SEATTLE (AP) — Alaska Airlines has ordered 12 Boeing 737-400 jetliners and taken options to acquire up to a dozen more, the airline said Thursday.
The firm orders are valued at $540 million, based on the manufacturer's list price.
Delivery of the 12 new 737-400s will be phased in over the next three years, with the first plane scheduled to enter the fleet in June 1997, said John F. Kelly, chairman, president and chief executive officer of Seattle-based Alaska Airlines.
The new planes will replace 12 older McDonnell Douglas MD-80s.
Under provisions of the agreement, Alaska may substitute some or all of its options for the newest derivatives of the Boeing 737 — the 737-600, 700 and 800 aircraft.
The Boeing 737-400 is a mid-range twin-jet, configured in 140 seats and two classes for Alaska.
"This order moves us one step closer toward fleet simplification without relinquishing our flexibility regarding future fleet decisions," Kelly said.
"Moreover, we'll be replacing aircraft on a one-for-one basis, making the youngest fleet of any major carrier in the U.S. that much younger."
Alaska's current fleet averages 7.5 years old.
The airline presently operates 75 airplanes, including 25 Boeing 737-400s, 42 McDonnell Douglas MD-80s and eight Boeing 737-200Cs. The 737-200Cs are specialized passenger/cargo combination airplanes used almost exclusively for service to, from and within the state of Alaska.
Easy Jet may buy 30 Boeing jets
LONDON -EasyJet Airline Co. said it will buy as many as 30 Boeing Co. planes, carrying a list price of $1.2 billion, potentially doubling its fleet as the U.K. no-frills carrier seeks to beat rivals into Europe's low-fare market. The carrier, based at Luton ... [Read More...]
Continental orders 92 jets Continental Airlines' order is good news for Boeing, even though most of it represents earlier purchases that were then canceled. SEATTLE (AP) -- Continental Airlines today gave a boost to Boeing Co., announcing plans to order 92 jetliners valued at $4.5 billion ... [Read More...]
Qantas orders 3 Boeing 747-400s
SEATTLE -- Qantas has ordered three Boeing 747-400s worth approximately $475 million, Boeing Commercial Airplane Group said Thursday. The Australian airline will take delivery of the planes in 1999 and 2000. Qantas said it is buying the planes to expand its fleet, and will use ... [Read More...]
BOEING: American's troubles jeopardize jet orders
Feb 21st, 1997
The airline's dispute with its pilots must be settled before it proceeds with the purchases from Boeing. his is one of those two line feature things that This is one of those two line feature things that I keep forgetting to put into ... [Read More...]
Boeing gets early Christmas gift
SEATTLE -- Taiwan's China Airlines Ltd. has placed the year's largest order for Boeing jumbo jets, agreeing to buy 10 747-400s worth a total of $2 billion at list prices, Boeing Commercial Airplanes confirmed Tuesday. China Airlines also placed orders with Europe's Airbus for 12 ... [Read More...]


text:
Q: SWIG for C++ code to Python , is this the valid way to wrap a std::array the same as a C-style array? SWIG newbie here. Let's say I have some typemaps defined for a C-style array:
%typemap(in) double[ANY] (double temp[$1_dim0]) {
  ...
}

// Convert from C to Python for c-style arrays
%typemap(out) double [ANY] {
    ...
}

If I want these to use this same exact logic for a std::array of doubles instead of a C-style array. However, I am not sure what the correct syntax is for this typemap to apply to an array of arbitrary dimension. Would the following logic be sufficient?
%apply double[ANY] { std::array<double, ANY> };

Thanks!

A: SWIG has support for some C++ templates including std::array, but each instance of a template used in an interface must be declared.  For example:
test.i
%module test

%{
#include <array>
%}

%include <std_array.i>
%template(DoubleArray10) std::array<double, 10>;
%template(DoubleArray5) std::array<double, 5>;

%inline %{
double func(const std::array<double, 10>& arr) {
    double sum = 0.0;
    for(auto n: arr)
        sum += n;
    return sum;
}

std::array<double, 5> outfunc() {
    return {5, 4, 3, 2, 1};
}
%}

Python demo:
>>> import test
>>> test.func([1, 2, 3, 4, 5, 6, 7, 8, 9, 10])
55.0
>>> test.outfunc()
(5.0, 4.0, 3.0, 2.0, 1.0)

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While Petra remains central to Jordan's cultural attractions, the country's leisure industry is now advancing in other areas, such as sports and health and fitness.
The recent naming of Petra as one of the new Seven Wonders of the World is improving international perceptions of Jordan. It has given the country a new focus, detracting from setbacks to tourism and the inextricably linked leisure industry caused by a series of Al Qaeda linked suicide bomb attacks on three hotels in Amman in November 2005 and the conflict in neighbouring Lebanon last year.
Otto Steenback, property manager at the Grand Hyatt Hotel Amman - one of the three hotels bombed in 2005 - estimates that the extra exposure of Petra will lead to a 100% increase in tourism numbers over the next five years.
Jordan Hotel Association president Michel Nazzel agrees, adding that being on the list is a major attraction for Petra and Jordan, with the associated publicity being worth millions of dollars.
"This should make the task of the Jordan Tourism Board (JTB) easier for many years to come," Nazzel adds.
Tohama Nabulsi, communications manager for JTB, says: "In light of Petra's well deserved success by being crowned as one of the New 7 Wonders of the World, the Jordan Tourism Board plans to incorporate a new focus in its marketing plans for next year.
"As new ideas are being explored, innovative marketing methods being studied and tested, and unified campaigns being designed, Petra remains constant in all discussions as one of the main focuses for next year. The main trend we are utilising is promotion of Petra through online activities," she adds.
The predicted influx of tourists to the country means that leisure operators may well need to raise their game. Indeed, the JTB acknowledges several challenges facing the growth of leisure.
Nabulsi says one of the key issues is a lack of trained human resources.
"We try to overcome this by getting staff from outside Jordan and we already have the University of Hospitality from which we can recruit well-trained, experienced graduates," she says.
Other challenges are the very high competition across the region and the geopolitical situation, which Nabulsi says impacts Jordan despite it being a very safe country.
To help overcome these challenges, the government and JTB need to capitalise on the momentum raised by Petra's accolade and use it to focus on the wider leisure industry. So, aside from the cultural and historic attractions that Jordan is famous for and the lure of the Dead Sea (see page 44), what else does the leisure industry in Jordan have to offer?
More than 200 archaeological sites have been discovered in Jordan, and there are believed to be many more. However, even before the Swiss-based New Open World Corporation named Petra as one of the New Seven Wonders list, it was Jordan's most valuable treasure and JTB's greatest tourist attraction. To preserve the UNESCO World Heritage site, Petra Archaeological Museum and Petra Nabataean Museum are located in the town of Wadi Musa, next to the site.
There are many other museums in Jordan and not all of them are linked to the historic sites in Petra, Amman, Wadi Rum and Umm Qays. For example, there is the Alpha Marine Science Station and the Aqaba Birds Observatory in Aqaba and the new National Children's Museum of Jordan in Amman.
The museum aims to create interactive learning experiences for children and families. It comprises a 4400m² interactive garden and a 2600m² internal exhibit area, with the interiors and exhibits created by UK-based Haley Sharpe Design and Beck Interiors.
The attraction features 150 interactive displays based on the themes of Humankind, Technology and the Natural World. Additional facilities include a children's library and IT centre, auditorium, planetarium, activities centre and an outdoor theatre.
The educational resource, which supports the national curriculum for schools, aims to encourage children to explore the arts, sciences, technology and industry in an interactive learning environment.
Jordan is not just a haven for fans of culture. The natural habitat affords a range of opportunities for the adventure lover too.
There are opportunities for horse riding, 4x4 safaris, rock climbing, canoeing and hiking. For example, treks through the Wadi Mujib gorge from the highlands above Mujib to the Dead Sea are organised by The Royal Society for the Conservation of Nature (RSCN). Its most extensive trek, known as the Lost Trail to the Dead Sea, is a full day expedition that the JTB describes as "not for the faint hearted".
In Aqaba, hotels and beachfront operators specialise in scuba diving, water sports, snorkelling, fishing and sailing. Other sports such as horse riding, cycling, golf, basketball, football and running are available at various sports clubs in Amman, which also offers the Waves Waterpark and The Park. This overlooks the city and features an auditorium hosting regular events and festivals, a large sports training facility and a children's playground.
The health and fitness industry, meanwhile, is still developing in terms of size, structure and professionalism. Most gyms and health clubs are privately-owned standalone facilities in Amman.
One of the newest and most impressive of these is Vy, which was launched in January by owner Gravity Athletic Investment Company and fitness manager Chris Farley, who moved to Jordan specifically to manage the club following seven years working in the UK fitness industry.
Vy is a 8000m² wellness centre, which features a wealth of facilities in the Vy Gym including: an indoor 25-metre pool and an aqua fitness pool; 200-metre suspended running track; two squash courts; four fitness studios; a group cycling studio; a Technogym and Nautilus equipped gym; a Kinesis personal training studio; Vy Juice and separate male and female relaxation zones with steam, sauna and spa pool.
Additionally, the club offers a spa called Vy Chi; a gelato ice-cream store called Vy Ice; and the Vy Café restaurant, all of which are open to non-members.
Of other standalone clubs in the country Farley says: "Most have adequate facilities but lack the direction and drive to make an impact on the entire fitness industry here."
Hotel gyms, he continues, are well established within the fitness industry in Jordan, providing members with a more rounded approach to exercise, health and fitness.
Farley adds: "The leading health and fitness operators in the world are just breaking through into Jordan and the rest of the Middle East and I predict that in the next three to six years you will see these major businesses dominating the market throughout the region and taking over from many of the locally-owned clubs."
Challenges with growing the industry, however, stem from two main issues.
"Awareness of health and fitness is throughout Jordan is low, with people still believing that looking good is a priority over feeling good. The process of awareness needs to start from a young age, as educating our current members is a challenge. It's proving successful though, with members finding new targets being achieved through realistic goal setting and education," says Farley.
Even with an increase in awareness, Farley says that only a very small percentage of Jordan's six million population will be able to afford a health club membership.
"Prices of memberships to gyms and health clubs are so high that it will be many years before the majority of the population will allow themselves to have such a luxury of expenditure," he asserts.
Despite this, Farley is positive about the future of the Jordanian fitness and wellness industry, with the owners of Vy potentially looking for a second site.
"The industry needs time to develop; time to set higher standards which will be met by industry professionals who are qualified and ready to take on the huge responsibility of educating customers.
"Many opportunities will arise because of the speed at which the industry is growing," continues Farley. "Positions will appear and salaries will rise; and this will open the door to new club operators who can hopefully see the potential of this market and take it upon themselves to lead the way for the fitness industry in Jordan."


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courtney henninger net worth
Her success in the film industry has also seen her clinch a few roles in television shows such as "Empire." Although her pay is not disclosed, it should be a massive sum from all her roles, helping her reach her net worth of $100 million. By collecting data from the most accurate and reputable resources, we've compiled a collection of the richest celebrities and their net worths. She debuted as the character during the series' pilot episode in 2013.
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Widely known for her role as Claudia on the TV sitcom Mom. She appeared as Susan Beth. Widely known for her role as Claudia on the TV sitcom Mom. In the year 1969, Love parents got separated and ended up in divorce. She gained widespread popularity for her 2008 appearance as Missy Cooper in an episode of The Big Bang Theory. Maybe you know about Courtney Love very well But do you know how old and tall is she, and what is her net worth in 2020? Apart from her acting career, she has also excelled successfully in being a songwriter along with a melodious singing voice, respectively.
– In 2020, Alona Tal's net worth was estimated to be…. https://www.youtube.com/watch?v=zTDbge68pcM, Your email address will not be published. After some time, Love's mother got remarried to Frank Rodriguez. In the year 1997, Love won the 'CFCA Award' for 'The People vs. Larry Flynt' in the category of the most promising actress. Courtney Henggeler Net Worth: US$ 2-2.5 million approx. She took acting lessons in LA. How much money is Courtney Henggeler worth at the age of 39 and what's her real net worth now? Courtney Love is a popular actress who got a notable presence in the field of acting and singing. Courtney Henggeler (born December 11, 1981) is an American actress known for her starring role as Amanda LaRusso in the Netflix comedy-drama web television series Cobra Kai. Courtney is a Capricorn. Her singing and acting attributes were immensely loved by all wholeheartedly.
She credits her childhood best friend Patti for making her audition for a high school production of Carousel when she was in 9th grade. 'Courtney Love' was born (9 July 1964) and brought up in California, the United States, and hence is an American citizen by birth.
The couple together got two daughters and an only son, who soon died of a heart defect. Courtney Henggeler age, Birthday, Height, Net Worth, Family, Salary. Are you Looking for Courtney Henggeler Age, Birthday, Height, Net Worth, Family, Salary, Weight etc..? https://www.wealthypersons.com/courtney-love-net-worth-2020-2021 Thus, she has set a new benchmark for herself with a successful career over the past years. Courtney Henggeler Wiki Biography Michelle Beisner is a feature reporter for ESPN and will be a member of the Sunday NFL Countdown Crew.
Courtney Henggeler is an actress, model and singer. A Troubled Childhood. Bosworth starred in the television series… – In 2020, Kate Bosworth's net worth was estimated to be…, Shailene Diann Woodley is an American actress. Two years later, she made a guest appearance in the medical drama House.
Talking about her age, according to her date of birth she is currently at the age of 32. Courtney Henggeler, Actress: Cobra Kai. Therefore, the total net worth of Courtney Love is about $180 million approximately as of November 2020. Are you Looking for Seth Lee Age, Birthday, Height, Net Worth, Family, Salary, Weight etc..? Capricorn is represented by the sea-goat, a mythological creature with the body of a goat and tail of a fish. While it's relatively simple to predict her income, it's harder to know how much Courtney has spent over the years.
Her debut was as Susan Beth in the film The Bog Creatures (2003). $500 Thousand. Courtney Henninger: Net Worth. Love then went to attend her college at "Nelson College," but she was soon expelled from there. As of 2020, Courtney Henggeler's net worth is, Courtney Henggeler is a Capricorn and was born in The Year of the Rooster. She also had a recurring role as Robin on the TV series Faking It and starred as Amanda LaRusso on the web series Cobra Kai. Find the latest complete information.
Their can-do attitude leads to success in pretty much any industry. Continue to the next page to see Katie Henninger net worth, estimated salary and earnings. Love has also performed varied live shows with a remarkable performance on stage with huge crowded audiences. Courtney Henggeler Net Worth 2020: Wiki Biography, Married, Family, Measurements, Height, Salary, Relationships. https://www.wealthypersons.com/courtney-love-net-worth-2020-2021 She is undoubtedly a versatile lady who is blessed with multiple talents to achieve her dream life of starlight. Edward Norton. Courtney Love, an American established superstar, has become an inspiration for all the women across the world. Her extraordinary talent fetched her multiple awards and recognition with huge net income. She is also known as playing the adult version of Sheldon Cooper's twin sister, Missy, in The Big Bang Theory.
She gained widespread popularity for her 2008 appearance as Missy Cooper in an episode of The Big Bang Theory. In the year 1980, Love first gave a kick-start to her musical career with utmost hope and faith in herself. Find the latest complete information. Continue to the next page to see Courtney Henggeler net worth, estimated salary and earnings. Courtney Henggeler was born in United States on Saturday, January 17, 1981 (Millennials Generation). She pretended to eat an apple while singing about clams for the role.[5]. Meanwhile, the couple had conceived a daughter named Frances Bean Cobain.
While it's impossible to calculate the exact amount Courtney Henggeler is worth, we can use publicly available information such as salary, investments, businesses, endorsements, and other income to estimate a net worth for 2020. Courtney Henggeler net worth and salary: Courtney Henggeler is a TV Actress who has a net worth of $500,000 USD. She appeared as the fictional twin sister of Jim Parsons ' popular character Sheldon Cooper on The Big Bang Theory. Widely known for her role as Claudia on the TV sitcom Mom. Courtney Henggeler's source of wealth comes from being a tv actress.
Undoubtedly, she has excelled well with remarkable performances in various genres. She has an estimated net worth of around 450k USD. She… – In 2020, Mélissa Theuriau's net worth was estimated to be…, Ariana Grande-Butera, known professionally as Ariana Grande, is an American singer and actress. Courtney Henggeler was born in in January 17, 1981. She studied Journalism and became a newscaster on TV. She began with the "Pagan Babies," which was her first musical project. As of 2020, Courtney Henggeler's net worth is $100,000 - $1M. However, there are several factors that affect a celebrity's net worth, such as taxes, management fees, investment gains or losses, marriage, divorce, etc. Her height is 1.78 m tall, and her weight is 55 kg. The fame and name have given much popularity to her at the right time of her career with a number of fan followings. Her first roles included Amy Juergens in The Secret Life of the American… – In 2020, Shailene Woodley's net worth was estimated to be…, Mélissa Theuriau is a French journalist and news anchor for M6. – In 2020, Clayton Moore's net worth was estimated to be…, Alona Tal is an Israeli-American singer and actress. As a child she was definitely theatrical. Lori Loughlin Starts Two-Month Imprisonment for College Admission Scam, Blake Shelton and Gwen Stefani Gets Engaged After Five Years of Dating, Hollywood stars are in love with leggings, Arnold Schwarzenegger Heart Operations: Says He is Feeling 'Fantastic', Instagram Tips for Growing Your YouTube Channel, House [2005, as Server]The Bog Creatures [2003, as Susan Beth], Family Practice [2011 TV film, as Chelsea], Friends with Benefits [2011, as Flight Attendant]. She began her career in the… – In 2020, Ariana Grande's net worth was estimated to be…, Victoria Dawn Justice is an American actress and singer-songwriter. However, there are several factors that affect a celebrity's net worth, such as taxes, management fees, investment gains or losses, marriage, divorce, etc.
Courtney Henggeler net worth and salary: Courtney Henggeler is a TV Actress who has a net worth of $500,000 USD. She currently resides in United States. She earned her on-screen debut in the 2003 film The Bog Creatures. =), Copyright © 2020 CelebrityNetWorth123.com | Contact Us | Privacy Policy. Courtney Henggeler (born January 17, 1981) is famous for being tv actress. [6] After playing various guest roles in several movies and TV shows over the years and a recurring role on the CBS sitcom Mom, Henggeler finally made her breakthrough in 2018 when she landed a starring role on Cobra Kai as Amanda LaRusso, the wife of the original Karate Kid Daniel LaRusso. DETAILS BELOW. Courtney Henggeler also got huge fame with her starring role of Hillary in the 2018 comedy and drama film "The List". Love has got full support from her mother to live her dream life extravagantly. Inheritance Money Online estimates of Katie Henninger's net worth vary. Baby, Husband, Married life. Katie Henninger's net worth. The initial stage of Courtney Love has been very struggling with her single mother's only support. They are currently expecting their second child together. Love has also featured in a movie named 'Straight to Hell.'. Save my name, email, and website in this browser for the next time I comment.
While it's relatively simple to predict her income, it's harder to know how much Katie has spent over the years. She is an American famed star who won critical acclaim for her major role as Missy Cooper in the CBS network's television sitcom "The Big Bang Theory". Well, if you're ready, let's start. Courtney Henggeler is a member of Richest Celebrities and TV Actresss. In the year 1997, Love also won the 'Golden Satellite Award' in the category of best actress in the supporting role.
Know about Bill Kristol Age, Bio, Parents, Net Worth, Wife, Children, Twitter (Opens in a new browser tab) Is Courtney Henninger married? Widely known for her role as Claudia on the TV sitcom Mom. [8], Learn how and when to remove this template message, "INTERVIEW: COBRA KAI'S COURTNEY HENGGELER", "Cobra Kai: First trailer for Karate Kid sequel is here", "Courtney Henggeler – (Friends With Benefits – 2011)", "Baby Registry of Courtney Henggeler and Ross Kohn", "Courtney Henggeler Is Pregnant! If you feel differently, please submit DMCA request for immediate review and amicable resolution. In 2014, she appeared as the recurring character Robin on the TV series Faking It. Advertisement Fees: Unavailable . 10 Facts on Courtney Khandabi: Courtney Khandabi is famous American on-air host currently associated with QVC. Tia Dashon Mowry-Hardrict, known by her maiden name Tia Mowry, is a German-born American actress and model. After some time, her mother and her step-father also ended up in divorce. If you do not know, We have prepared this article about details of Courtney Love's short biography-wiki, career, professional life, personal life, today's net worth, age, height, weight, and more facts.
https://networthpost.org/net-worth/jennifer-courtney-net-worth In the year 2001, Love won the Grand Jury Award. Fans, please take into consideration that we didn't break into Courtney Henggeler's bank accounts.
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courtney henninger net worth 2020


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Q: inverse laplace using partial fractions and completing square what is the inverse Laplace transform of this equation $$\frac{1}{(s+1)(s^2+s+1)}$$
I know that completing the square for the quadratic term is required to avoid complex roots and then I need to use partial fractions. After completing the square the denominator becomes:
(s+1)((s+0.5)^2+0.75)
how shall I proceed from here on?
many thanks

A: Try this way:
$$\frac{1}{(s+1)(s^2+s+1)} = \frac{A}{s+1}+\frac{Bs+C}{s^2+s+1}$$
Notice that the second term has complex solutions, so it's a good idea if you try to transform it in something like $\frac{s+a}{(s+a)^2+b^2}$ and $\frac{b}{(s+a)^2+b^2}$
The solution is:
$$e^{-t}-\frac{e^{-t/2} (\sqrt3 \cos(\frac{\sqrt3 t}{2})-\sin(\frac{\sqrt3 t}{2}))}{\sqrt3}$$

$1)\frac{s+a}{(s+a)^2+b^2} \to e^{-a}\cos(bt)$
$2)\frac{b}{(s+a)^2+b^2} \to e^{-a}\sin(bt)$
Example:
If $B=1$ and $C=1$
$$\frac{s+1}{s^2+s+1} = \frac{s+1}{(s+\frac12)^2+(1-\frac14)} = $$
$$\frac{s}{(s+\frac12)^2+(\frac34)} + \frac{\sqrt{\frac34}}{(s+\frac12)^2+(\frac34)}*(\sqrt{\frac43})    $$
In this case you have to add $\sqrt{\frac43}$, so the numerator can be equal to $1$, but you need $\sqrt{\frac34}$ to use the  second transform (obviously you will add $\sqrt{\frac43}$ to the result of this transformation).
...
In your case is $A=1, B=-1, C=0$, so:
$$\frac{1}{s+1}-\frac{s}{(s+\frac12)^2+\frac34}$$
Now, to use the transformation $1)$ you need something to add to the fraction like that:
$$\frac{{\frac12}}{(s+\frac12)^2+\frac34}$$
So:
$$\frac{1}{s+1}-(\frac{s}{(s+\frac12)^2+\frac34} + \frac{{\frac12}}{(s+\frac12)^2+\frac34} - \frac{{\frac12}}{(s+\frac12)^2+\frac34}) $$
So:
$$\frac{1}{s+1}-(\frac{s+{\frac12} }{(s+\frac12)^2+\frac34}  - \frac{{\frac12}}{(s+\frac12)^2+\frac34}) $$
That becomes:
$$\frac{1}{s+1}-(\frac{s+{\frac12} }{(s+\frac{1}{2})^2+\frac34}  - \frac{{\frac{\sqrt{3}}{2}}}{(s+\frac12)^2+\frac34}*\frac{2}{\sqrt{3}}*\frac12 )$$


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Camden Reeves's three motets Spells, Remedies and Potions inspired by the Pendle Witch Trials of 1612 will be premiered at the Pendle Heritage Centre on Saturday 13 October 2018. The motets are for 13 sopranos, symbolizing a kind of inverted and demonic Last Supper. The words are taken from the Malleus Maleficarum – the 16th century witch hunter's manual, outlining methods for the detection, trial, torture and execution of witches. Boiling bodies to make soup, copulating with Satan, twisting limbs and cracking bone, drinking from skin, eating babies and more.
We look forward to publishing Spells, Remedies and Potions soon.


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John Parris, Parris Cues
Forest Hill Society support, promote and encourage local businesses. In each newsletter we plan to interview successful businesses in the community who are helping to make Forest Hill a vibrant and successful town centre.
Helen Wicks from the Forest Hill Society recently met local businessman John Parris, owner of Parris Cues who manufacture snooker cues in Church Vale Forest Hill.
Q. How did you get into the snooker cue trade?
It started as a hobby, I played snooker and started experimenting with repairs/ fiddling with snooker cues. I was asked by others to repair their cues and it progressed from there. It's quite a niche market and there are not a lot of competitors just small 'one man bands'. Obviously larger shops sell mass produced cues much more cheaply, but like any other sport, if you are serious and/or talented, bespoke equipment is recommended.
Q. Why Forest Hill?
I started in Bromley about 30 years ago. We outgrew our premises in Bromley and were looking for a larger site, and then found this building in Church Vale SE23 which was bigger with good transport links by road and rail into central London. Our premises in Forest Hill are very modest and many of our distinguished customers expect to see a grand showroom are surprised to find the business tucked away in the back streets of Forest Hill in an unglamorous shop. We are established here now, we like it – we have no plans to move.
Q. How many people do you employ?
It's a family business run by myself and my wife and my son looks after the IT side of things. We employ 8 people including an apprentice and I will soon be looking for a second apprentice to work on a new leather snooker case line that I am introducing. I am fortunate to have a skilled and loyal workforce with very little staff turnover producing high quality cues.
Q. Who are your customers?
At the beginning we were basically providing cues to the UK market. During the 1980's snooker was very popular in England…..thanks to Steve Davis and Jimmy White.
We are now a global business and 60% of our business is export. Our biggest customers are from China particularly Hong Kong, and also Brazil, Canada and Australia. The Crown Prince of Brunei has bought cues and flew me over to deliver them. He has even visited the showroom arriving in 3 limos …blocking Church Vale!
The internet has facilitated this success. We have invested a lot in our website and this has been an important way to reach the world market.
The fortunes of Parris Cues prospered during the 1980's snooker boom and were further lifted by the Sporting Events Promoter Barry Hearn, who as chairman of the World Professional Billiards and Snooker Association and the subsequent controlling interest in the commercial arm World Snooker Limited, revitalized the game from 6 tournaments a year to tournaments somewhere in the world every week.
Q. Any well known customers?
Yes, snooker players; Ronnie O'Sullivan, Jimmy White, Steve Davis, Stephen Hendry, Stephen Maguire, John Higgins, and Neil Robertson. Other celebrities who have bought cues include: Ronnie Wood, Damien Hirst and Ian Wright.
Have a look at our photos on the website!
www.parriscues.com/gallery
Q. So how did you manage to get such big names on your books?
It all started when I cornered Steve Davis at a tournament… I offered to produce a replica of a cue that he damaged in a tournament…and he liked it! From this stroke of good luck, I built up my reputation on recommendations and to add to this Steve has become a good friend.
Q. How many snooker cues do you make on average every year?
We manufacture about 1500 cues a year.
Q. What materials do you use?
I source wood for the cues from all over the world, exotic wood mainly, ash and maple from North America in addition to British plum and pear tree wood.
Q. Snooker was very popular - Why do you think are there no clubs in Forest Hill?
There were three clubs within walking distance of Forest Hill station, two of which were in Perry Vale behind the station and one at Brockley Rise... a real shame they closed…I believe this was because of high rents, combined with drop in popularity, possibly also because smoking ban and gaming regulations took their toll on profits. However most pubs are not big enough for snooker tables but do have pool tables – incidentally, we also make cues for pool and American Pool!
Q. Can you give any advice to a budding entrepreneur wanting to start up a business in Forest Hill?
Yes, find a niche market product that you are passionate about and spend as much as you can on a website to promote it!
at Tuesday, May 06, 2014
Labels: newsletter0514, shops
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From Web Analytics Land: Why do we need to care about this? It lies in an inherent deficiency of Internet Explorer. IE can not accept a URL that is longer than 2083 characters. If your implementation is pretty complex and you are passing in a lot of variables, your image request can easily approach if not exceed that amount. Since Microsoft still commands the lion's share of the browser marketplace, this is something we need to keep an eye on. If this was a problem with Opera or another third or forth tier browser then we may not worry about it. But this is not the case with Internet Explorer.
I have made no effort to conceal my hatred of IE 6.
My plan was to using ColdFusion, my preferred development language, take the nasty url and convert it into a Structure and then build an XML document to post to Omniture.
First I needed to just copy the url string into a ColdFusion variable to begin the process.
What a mess! Next I needed a Struct to be able to reference later that held the accepted XML tags for the Data Insertion API and the codes from the url.
NOTE: This is not meant to be a complete list of all options!
I have not done any testing to see what impact this process would have on site performance or how it performs against the normal JS beacon. But if you have a situation where the amount of data that had to be passed to Omniture exceed the magic 2083 characters, this is an approach you could take.
Thanks to Web Analytics Land for his original post, and thanks to Craig for bouncing some ideas back and forth on this.
Yeah, more than likely you need to update the year on a plugin that you are using like Time parting.
The code above was pulled from another blog post. So I do not know if time parting was used at all.
1[...] Build Custom XML for Data Insertion [...] - Rudi Shumpert : Code By Numbers » Omniture & ColdFusion: Why you should care.
Next post What are you willing to pay for free analytics?


Situated in close vicinity to Tour Montparnasse, the Vandamme mixed-use block, designed in the early 1970s by the French architect Pierre Dufau, was as one of the largest urban projects implemented in Paris at the time. As a design driven by the ideal of the automobile, it appeared as a triangular urban island surrounded by the traffic loaded Rue Mouchotte, Avenue du Maine and the rail tracks to Gare Montparnasse opposite the site. Dufau's design is characterized by a clearly defined horizontal plinth, interrupted only by the verticality of the slender, 30-storey tower of the Hotel Pullman. Once a landmark of the era, over time the complex has failed to adapt to the changing needs of an urban society, resulting in an introverted and self-contained block which lacks urban connectivity, discourages pedestrian activity and neglects any sense of identity.
MVRDV's proposal aims to reintroduce the lost human scale and bring back a sense of place within the Montparnasse district: breaking the solid, horizontal volume up into fragments and making the mixed-use program inside the plinth more extroverted allows each part of the program to distinguish itself through a unique identity. Each façade is opened up to light and access as much as possible, and replaced by a collection of 'boxes' inserted into the existing structural frame, which differ in size, program, activity, colour and materials. Each box accommodates a different part of the program while being flexible with regards to future demands. Based on the existing structural grid, the majority of boxes are suspended from the façade revealing the wide range of functions such as bars, restaurants, shops, a library, exterior gardens, living and working spaces. Density is increased carefully while respecting the architectural language of the original design and refreshing the run down details and organisation of the block.
In order to improve accessibility and increase permeability at ground level, additional entrances are created on Avenue du Maine and Rue Mouchotte. The shopping centre is extended; the offices, which are currently stretched over the entire width of the plinth are replaced by a six -storey office block which includes accessible roof terraces creating an 'address' of sorts on Avenue du Maine. The public library Bibliothèque Vandamme is moved from its current underground location to the top of the plinth for improved daylight conditions and direct access to and from the station Gare Montparnasse. A community of 62 social housing units, and a kindergarden of 500 m², are added onto the plinth between the hotel tower and the office block Le Héron, while maintaining the required distance from its neighbours for light and views. Rue Vercingétorix, the southern plot boundary, will be upgraded by opening up the current back face of the building. In order to relieve on-street parking spaces, 150 extra spaces for scooters are created within the six-storey underground car park.
The project was commissioned by Unibail-Rodamco and has been conceived by MVRDV in collaboration with local architect SRA, engineers SCYNA4, installation consultant LAFI + INEX, cost consultant VANGUARD, façade engineers RFR, acoustic consultant LASA and BATISS for fire safety.