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Cristallight Software - Professional Mac Business Cards Maker, QR Code on business card available. The Home Business Card is a mac business card software for creating and printing business cards using various built- in label formats, clipart images, and pre- made business card templates provided. Home Business Card helps you to: create impressive professional mac business cards, making people notice you and your business, create a long lasting image of a business and its representative, create and maintain relationships, personally and professionally, save the time and effort to note down one’s professional contact details, socialize with other people. Image libraries and pre- defined business card templates. Hundreds of templates, horizontal and vertical layouts are supplied with the product. More then a thousand images for backgrounds and clipart in a variety of formats, including JPEG, TIFF, PDF, GIF, etc, are in the product image libraries. Business Card Paper formats There are many varieties of business card paper, if you plan to make perfect business cards, pay attention to choosing business card paper.
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Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis- like disease in C5. BL/6 mice. Abstract. BACKGROUND AND PURPOSECannabis extracts and several cannabinoids have been shown to exert broad anti- inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACHWe used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C5. BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG- immunized animals and on MOG- specific T- cell proliferation. KEY RESULTSTreatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T- cell recruitment in the spinal cord of MOG- injected mice. Moreover, CBD inhibited MOG- induced T- cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors. CONCLUSIONS AND IMPLICATIONSCBD, a non- psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T- cell proliferation by CBD appeared to contribute to these beneficial effects. Keywords: cannabinoids, cannabidiol, microglia, T cells, EAE, MOGIntroduction. The anti- inflammatory properties of cannabinoids, constituents of the Cannabis sativa plant, have been appreciated since ancient times and have been supported experimentally, in models of inflammation in vitro and in vivo (Tanasescu and Constantinescu, 2. Indeed, immune cells express the elements of the cannabinoid system including endocannabinoid ligands, endocannabinoid enzymatic machinery and cannabinoid CB receptors, mostly of the CB2 type and many fewer of the CB1 type (Mackie, 2. Alexander et al. Moreover, expression of CB1 and CB2 receptors and the activity of the endocannabinoid system are regulated in response to inflammation, a finding that further confirms the involvement of the cannabinoid system in immune processes (Croxford and Yamamura, 2. Maresz et al., 2. Pietr et al., 2. 00. The clear and promising therapeutic potential of cannabinoids (Klein and Newton, 2. CB1- mediated, psychotropic effects of many of these materials. A good example is . The preferential CB2 receptor expression on immune cells offers an attractive opportunity to regulate the function of the immune system with CB2 receptor ligands. Studies on CB1/CB2 receptor knock- down mice revealed the existence of other possible, receptor and non- receptor, cannabinoid targets (J. Thus, cannabinoid compounds not acting on CB1 or CB2 receptors seem to offer new tools to manipulate inflammation. Cannabidiol (CBD) is one of these compounds and is the major Cannabis- derived non- CB1/CB2 receptor ligand (Showalter et al., 1. This compound is not only devoid of psychotropic effects but also is able to inhibit many central effects of CB1 receptor ligands, for example, the anxiogenic and psychotogenic activities of THC (Zuardi, 2. Interestingly, despite different pharmacological and behavioural effects, CBD shares with . These effects include suppression of humoral responses, such as release of cytokines, chemokines, growth factors, as well as suppression of immune cell proliferation, activation, maturation, migration and antigen presentation (Mechoulam et al., 2. In an earlier publication, we showed that CBD inhibited production of the cytokine IL- 6 and the chemokine CCL- 2 by activated microglial cells and in parallel activated intracellular anti- inflammatory pathways (Kozela et al., 2. Among the many types of neurodegenerative diseases in which inflammation is involved, multiple sclerosis (MS) is one of those clearly induced and driven by dysfunctional immune system activity. In MS, myelin autoreactive peripheral T cells migrate into the CNS and initiate cytotoxic, degenerative processes that include demyelination, oligodendrocyte cell death and axonal degeneration. These effects lead to neurological deficits such as visual and sensory disturbances, motor weakness, tremor, ataxia and progressive disability as the main clinical symptoms (Compston and Coles, 2. Infiltrating T cells are constantly reactivated within the CNS parenchyma by microglia. Microglial cells via chemokine and cytokine release and constant antigen presentation potentiate T- cell recruitment to the CNS and facilitate their polarization into cytotoxic phenotypes (Th. Th. 17). Moreover, via released chemokines such as CCL- 2, microglial cells recruit other immune cells of myeloid origin, specialized in epitope spreading and phagocytosis of myelin including monocytes, macrophages, B cells and dendritic cells (Jack et al., 2. Koning et al., 2. Depletion of microglia or impairment of their function can attenuate disease progression in experimental animal models of MS supporting their role in initiation and development of this disease (Huitinga et al., 1. Heppner et al., 2. Thus, suppression of microglia will potentially reduce inflammatory lesions and limit demyelination within the CNS. One of the best described and commonly used animal models of MS is experimental autoimmune encephalomyelitis (EAE) which is induced when the animals are immunized with myelin components, for example, myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein (MOG) or by passive transfer of autoreactive myelin specific T cells to produce demyelination and MS- like neurological and clinical signs (Shevach, 1. Using the MOG- induced EAE mouse model, we investigated if systemically given CBD at the time of symptomatic disease onset could affect the progression of the disease. We observed that CBD ameliorated the severity of the EAE in MOG- injected mice. Moreover, CBD attenuated microglia activation in MOG- immunized animals and inhibited MOG- induced proliferation of encephalitogenic T cells. Methods. Animals and experimental design. All animal care and experimental procedures complied with and were approved under the guidance and regulations of the Weizmann Institute of Science and Tel Aviv University. EAE was induced in 3. C5. 7BL/6 mice (Harlan Laboratories, Rehovot, Israel) by two subcutaneous injections of MOG3. MOG) on days 1 and 8, injected in the left and right flanks respectively. Each injection contained 3. Control mice received only CFA without MOG (Ctrl, n = 1. CBD (5 mg. Clinical disease scores were recorded daily until 3. At this time point, spinal cords were collected for further pathological and immunological studies. The signs of EAE were scored as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, partial frontal limb paralysis; 5, total paralysis; 6, death (Lev et al., 2. In parallel, we used control mice which received only CFA (Ctrl) and were also injected with CBD on days 1. Ctrl + CBD group, each group n = 1. The days of EAE onset and respective CBD injections were chosen based on our previous experience with this model (Lev et al., 2. CBD (National Institute on Drug Abuse, Rockville, MD, USA) solution was prepared freshly before each of the treatments. The dose of CBD was chosen based on previous studies with systemic administration of the drug in which CBD at 5 mg. MOG synthesis was carried out by the Weizmann Institute Synthesis Unit, using a solid- phase technique on a peptide synthesizer (Applied Biosystems Inc., Foster City, CA, USA). Histology and immunocytochemistry. Histological analysis of spinal cord sections was used to define severity of inflammation and demyelination and the effect of CBD treatment on these parameters. Spinal cords were dissected 3. The presence of axonal pathology was supported by immunohistochemistry with anti- non- phosphorylated neurofilament H (SMI- 3. Sternberg Antibodies, Emeryville, CA, USA) on 8 . Subsequently, 4 . The assessment of immune cell infiltration was performed using immunocytochemistry on 4 . Thus, in further description of the stainings we refer to microglia/macrophage populations. The Iba- 1 protein is specifically expressed in microglia/macrophages and becomes up- regulated during the activation of these cells. Mac- 2/Galectin- 3 is another microglial/macrophage marker whose expression reflects specifically the activation state of these cells. For example, Mac- 2/Galectin- 3 expression is known to be increased in these cells following phagocytosis of damaged myelin and cell particles (Reichert and Rotshenker, 1. The immunostainings were visualized with respective secondary antibodies conjugated to Cy. Cy. 3 fluorochromes. Spinal cords from control, healthy mice that received 3 daily injections of either CBD or its vehicle were dissected and stained in parallel. Assessments of intensity and differences in immunofluorescence staining for Iba- 1 and Mac- 2/Galectin- 3 were performed using the Image Pro Plus analysis software (Media cybernetics, Bethesda, MD, USA) and collected as arbitrary units representing optical density per area. The statistics was carried out on per cent values calculated from optical density values and is given in the appropriate figure legend. In the case of T cells, CD3+ cells were counted (three areas per mouse, three to four mice per treatment) and the cell numbers were subjected to statistical analysis. Encephalitogenic T- cell line. The MOG3. 5- 5. 5- specific T- cell line was established from lymph node cells of C5. BL/6 female mice that had been primed 1. Activation of caspase- 1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function. Abstract. Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram- positive bacteria is regulated by the NLRP3- inflammasome and caspase- 1. Active caspase- 1 accumulates on phagosomes and acts locally to control the p. H by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3- inflammasome and caspase- 1 in host defense. A particularly important process in host defense is phagocytosis, the internalization of particles into organelles called . Despite this crucial role in immunity, the molecular mechanisms that regulate the functions of phagosomes remain poorly understood. What is known is that phagosomes in macrophages are dynamically remodelled during their . An important aspect of the maturation process is vacuolar acidification, which regulates the activity of the p. H- sensitive enzymes that are delivered from lysosomes and required to digest internalized cargo 5. The timely delivery of microbes into a mature and acidified phagolysosome is essential not only for microbial killing 6, but also to facilitate activation of certain innate immune signalling pathways 7. Moreover, the rate of phagosome acidification also regulates antigen processing and presentation by macrophages and dendritic cells (DCs) 8. Because of these pivotal roles in immunity, understanding the mechanisms that allow the host to remodel phagosomal compartments to optimize their microbicidal and hydrolytic activity is of fundamental importance. Acidification is key to many facets of phagosome function. It is a regulated process that begins almost immediately after the phagocytic cup has closed 9,1. H can drop from 7 to 4 in a matter of minutes. These rapid changes precede the fusion with acidic compartments and instead early acidification requires delivery of the vacuolar- H+- ATPase (V- ATPase)1. This proton transporting holoenzyme is recruited from endosomes and lysosomes, and assembled on the membrane of the nascent vacuole 9,1. However, how the p. H is then regulated remains poorly defined. V- ATPase activity in mammalian macrophages and DCs can be . Additionally, the NADPH oxidase has been suggested to counteract the V- ATPase and neutralize the phagosome p. H in certain cells 1. A number of pathogens have evolved mechanisms to evade these processes, including buffering their local environment in an attempt to maintain a beneficial neutral p. H. The nature of the mature phagosome is therefore determined by the complex interplay between the internalized microbe and the rapid remodeling of the organelle by the host in response to the different cargo. The observation that Toll- like receptors (TLRs) are recruited to some phagosomes 1. TLRs might survey the luminal contents and control vacuole maturation in a cargo- dependent and organelle autonomous manner to mediate these rapid changes. Although attractive as a mechanism for sensing and remodeling the phagosome depending on the type of internalized material, this function of the TLRs remains controversial, especially as the molecular details of how they might regulate this process remains obscure 2. The rapidity of the changes that occur after microbial engulfment argues against the fate of the phagosome being controlled at a transcriptional level. Rather we reasoned that local, post- translational modifications induced by innate immune signals were more likely to underlie the prompt remodeling of the phagosome that occurs after internalization of different microbes. To investigate this possibility, we chose to focus on the inflammasome, as it is an innate immune pathway whose terminal effectors are proteases that can rapidly modify select host components. We found that caspase- 1 is rapidly activated upon phagocytosis of Gram- positive microbes. Moreover, we demonstrate that activation of the NLRP3 inflammasome and its effector, caspase- 1, are instrumental in enabling the microbicidal activity of Staphylococcus aureus- containing phagosomes. Caspase- 1 acts on the NADPH oxidase (NOX2), and by controlling its activity it modifies the p. H of the vacuole. These findings identify an essential role of caspase- 1 in locally coordinating the environment of phagosomes. We propose that this cell autonomous defense function of caspase- 1 acts in concert with its role in pro- inflammatory cytokine release and pyroptosis and thus positions it as a master regulator of innate immunity. Results. Caspase- 1 accumulates on phagosomes containing S. While investigating inflammasome activation during S. Caspase- 1 exists as both an inactive pro- enzyme and, after auto- proteolytic cleavage in an inflammasome platform, an active protease. As these methods could not distinguish pro- caspase- 1 from active caspase- 1 we used the Fluorescent Labelled Inhibitor of Caspases (FLICA) reagent, which forms a covalent bond with the active protease 2. Figure 1c- f). Active caspase- 1 accumulated around bacterial phagosomes (Figure 1c). The FLICA staining required internalization of S. The localization of caspase- 1 after S. The accumulation of active caspase- 1 on phagosomes was cargo- dependent; latex bead phagosomes did not stain with FLICA, despite the presence of total caspase- 1 as detected by antibody staining (Supplementary Fig. Gram- negative bacterium Escherichia coli was not associated with detectable caspase- 1 activation in unprimed cells (Supplementary Figure 1d,e). Together these data indicate that caspase- 1 was activated and then specifically recruited to S. Activation was dependent on the NLRP3 inflammasome, as cells deficient in Ice (caspase- 1), or the inflammasome components Asc and Nlrp. Figure 2a). A similar reduction of FLICA staining was observed when confocal microscopy was used to assess both the total FLICA staining and the amount of phagosome- associated active caspase- 1 (Figure 2b). These data confirmed that the FLICA staining was specific and indicated that the inflammasome was required for the phagosome- associated caspase- 1. Phagocytosis of S. Although caspase- 1 could not be detected in cell supernatants at early time points, we did observe the appearance of caspase- 1 p. Figure 2c). To further define the kinetics, we used flow cytometry to monitor YVAD- FLICA. Phagocytosis of both live and heat- inactivated (HI) S. Early caspase- 1 activation did not require priming initiated by NF- . Similarly, it did not require TLR signaling as it was unimpaired in Myd. Early caspase- 1 activation was also not attenuated by Bafilomycin A, an inhibitor of the V- ATPase, indicating that vacuolar acidification did not play a role at this early point (Figure 2g- h). Instead we observed that phagocytosis of S. However, and consistent with previous reports, caspase- 1 activation was normal in gp. ROS that activates the inflammasome was not the phagocyte oxidase (NOX2) (Supplementary Figure 2a). Notably, addition of DPI, either alone or with Bafilomycin A, did not completely block caspase- 1 activation indicating that there are other additional, but currently unidentified factors, that contribute to activate the inflammasome early during phagocytosis. Further arguing against a role for ROS in early E. Thus phagocytosis triggered ROS and early caspase- 1 activation occurs during the uptake of select microbes. In contrast, late or sustained caspase- 1 activation (> 1 hour after infection) was observed only with live and not heat killed S. Consistent with previous reports, S. The sustained activation of caspase- 1 and IL- 1. Late caspase- 1 activation and IL- 1. Together these data indicate that caspase- 1 activation begins immediately after internalization of S. The p. H of phagosomes containing certain live bacteria including S. Therefore we first measured acidification using HI bacteria which are unable to neutralize the phagosome. For this purpose the rate of acidification was monitored using a highly sensitive and accurate ratiometric assay (Figure 3a,b) in cells pretreated with either a pan- caspase (ZVAD) or caspase- 1 specific inhibitor (YVAD). Both ZVAD and YVAD inhibited acidification of S. Similar results were observed when p. H was measured by quantifying delivery of p. Hrodo. Caspase inhibition reduced phagosome acidification in all macrophages tested including bone marrow- derived macrophages, peritoneal macrophages, and the macrophage cell lines J7. RAW 2. 64. 7 (Figure 3d). Additionally, ZVAD also retarded acidification of phagosomes containing live bacteria (Supplementary Figure 3b). Caspase- 1 inhibition blocks the acidification of S. As an alternative, we explored whether caspase- 1 might directly regulate the accumulation of protons in S. To test this we used a phagosome p. H dissipation assay 2. Supplementary Figure 3e). This assay can estimate the dynamic regulation of p. H by monitoring the acute changes induced after pharmacological manipulation of macrophages containing phagosomes that have already acidified. As an example, and similar to previous reports using other bacteria 9,2. V- ATPase with Bafilomycin A resulted in rapid and total dissipation of the phagosome p. H (Figure 3e), confirming that the p. H of the S. Addition of ZVAD (data not shown) and YVAD (Figure 3f) resulted in partial dissipation of the p. H of phagosomes containing both live and HI S. This is a list of PC games (including Microsoft Windows, Mac OS X, and Linux) that have sold or shipped at least one million copies. Subscription figures for. Now You Can Download PS2 XBox Nintendo PC Games More For Free the number one full PC game downloads site on the internet. Once registered you will gain unlimited game. Kongregate free online game Hack Slash Crawl - Enter the dungeons of Hack Slash Crawl to find your fortune. Be prepared for anything, because. Play Hack Slash Crawl. 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When activated, it will kill all enemies on the map, no matter where they are, as well as disable the enemy's equipment, electronics, and pointstreaks for one minute. Additionally, the team with the person who calls in a M. O. A. B. Create a class with the Hardline perk and use the Specialist Killstreak package. You will get extra points at kills 1, 3, 5, and 7. It will also help you complete the perk's challenges and the Specialist challenges for each perk. You will be completing the perks, and each perk has a Specialist perk challenge to it. For example, if at one kill you unlock Assassin, you will be completing Assassin challenges while also completing the . You will get a lot of XP as you complete these challenges in addition to the 2.
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Hardened Package (title and emblem; reach 5th Prestige)7. Veteran Package (title and emblem; reach 1. Prestige)8. Reset All Stats (reach 1. Prestige)Perks. The following is a list of all perks and their effect: Tier 1. Recon. Recon: Explosive damage shows target on mini- map. Recon Pro: Bullet damage shows target on mini- map. Sleight Of Hand. Sleight Of Hand: Faster reloading. Sleight Of Hand Pro: Switch weapons quicker. Blind Eye. Blind Eye: Undetectable to air support and sentry guns. Blind Eye Pro: Faster lock- on for launchers, and increased bullet damage to air support and sentries. Extreme Conditioning. Extreme Conditioning: Increased sprint time. Extreme Conditioning Pro: Increased climbing speed. Scavenger. Scavenger: Get ammo from dead enemies. Scavenger Pro: Get more ammo from dead enemies. Tier 2. Quickdraw. Quickdraw: Quicker aiming (ADS)Quickdraw Pro: Quicker recovery from using grenade/equipment. Blast Shield. Blast Shield: Increased explosive resistance. Blast Shield Pro: Resistance to flash and concussion grenades. Hardline. Hardline: Killstreaks require 1 fewer kill. Hardline Pro: Two assists count as kill towards killstreaks, and deathstreaks require 1 fewer death. Assassin. Assassin: Undetectable by UAV, portable radar, thermal, and heartbeat sensors. Assassin Pro: Immunity to CUAV and EMP, and no red crosshair or name appears when targeted. Overkill. Overkill: Carry two primary weapons; second primary weapon cannot have attachments. Overkill Pro: Second primary weapon can have two attachments. Tier 3. Marksman. Marksman: Identify enemy targets at long range. Marksman Pro: Hold breath longer while sniping. Stalker. Stalker: Increased movement speed while aiming down the sights (ADS)Stalker Pro: Delay enemy claymore explosions. Sit. Rep. Sit. Rep: Detect enemy explosives and tactical insertions. Sit. Rep Pro: Louder enemy footsteps. Steady Aim. Steady Aim: Increased hip fire accuracy. 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That explodes. DESCRIPTIONSaints Row The Third is a relief, like stretching after being locked in a box for hours. It’s an end- point for the evolution of a series that began life as a straightfaced and somewhat crass GTA clone, but with this iteration finally emerges from that shadow. Saints Row The Third feels fresh, and the reason for that is the value developers Volition place on three little letters: f- u- n. The game doesn’t so much do its own thing as attempt to do everything (except be serious). It’s why the dictionary definition wouldn’t have helped anyway – what Saints Row is all about is unique to videogames. Every single game depends on the player’s agency, but this thing is obsessed: encouraging it, indulging it, rewarding it. Includes downloads, cheats, reviews, and articles.It wants you to do, well – anything. Let’s be clear: this isn’t a revolutionary game, and . But this is a special game. 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