text:
The induction of ectopic lymph node structures (ELNs) holds great promise to augment immunotherapy against multiple cancers including metastatic melanoma, in which ELN formation has been associated with a unique immune-related gene expression signature composed of distinct chemokines. To investigate the therapeutic potential of ELNs induction, preclinical models of ELNs are needed for interrogation of these chemokines. Computational models provide a non-invasive, cost-effective method to investigate leukocyte trafficking in the tumor microenvironment, but parameterizing such models is difficult due to differing assay conditions and contexts among the literature. To better achieve this, we systematically performed microchemotaxis assays on purified immune subsets including human pan-T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells, with 49 recombinant chemokines using a singular technique, and standardized conditions resulting in a dataset representing 238 assays. We then outline a groundwork computational model that can simulate cellular migration in the tumor microenvironment in response to a chemoattractant gradient created from stromal, lymphoid, or antigen presenting cell interactions. The resulting model can then be parameterized with standardized data, such as the dataset presented here, and demonstrates how a computational approach can help elucidate developing ELNs and their impact on tumor progression.
Despite advances in immunotherapy and other treatment options, melanoma remains an increasing concern for caregivers, with over 60,000 new diagnoses of invasive melanoma per year in the United States1, and over 112,000 cases projected per year by 20302. If detected early, surgical resection offers the best outcome and can often be curative. However, once the disease becomes metastatic, the prognostic outlook is bleak with only 16% of patients surviving 5 years3. For metastatic disease, immunotherapy can offer a handful of attractive options, which display potent but often incomplete clinical responses4,5,6. The use of cytokines and the more recent implementation of antibodies against immune checkpoint receptors CTLA-4 or PD-1 all display dramatic and durable clinical responses in a minority of patients7. Juxtaposed to this group of biologics is the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL) expanded ex vivo from patient tumors. TIL therapy was first pioneered at the NCI Surgery Branch8,9, and is now available at several institutions in the U.S. and abroad10,11,12,13. When combined with lympho-depleting, non-myeloablative chemotherapy prior to adoptive transfer, TIL therapy can display clinical response rates approaching 50%14,15.
The initial presence of lymphocytes in the tumor microenvironment is presumptive to the success of any immunotherapy. The prognostic association of immune infiltrate in metastatic melanoma was at first contested, with some reports that TIL presence serves as an independent prognostic indicator16,17,18, and others reporting no association with clinical outcome or lacking independence as a prognostic factor19,20,21. More detailed investigation suggests that taking into account the activation state or proliferation rate of TIL can better indicate positive prognosis22. Of importance, recent observations suggest that the presence of tumor-localized, ectopic lymph node structures (ELNs) is associated with better prognosis across a broad spectrum of tumor types including metastatic melanoma23, breast cancer24, colorectal carcinoma25, and non-small cell lung cancer26,27. ELNs are highly organized aggregates of leukocytes, often displaying distinct T cell and B cell zones, as well as, in some cases, clearly defined marginal zones with activated antigen presenting cells28. Structural features of ELNs, such as the de novo generation of lymphatic vessels, can greatly enhance the infiltration of TIL deeper into the tumor parenchyma29. Such dissemination away from the vasculature is highly associated with better clinical outcome30. While ELNs may be beneficial for the majority of cancer types, this is not universally true. The presence of ELNs serves as a negative prognostic indicator for a few cancer types such as hepatocellular carcinoma, and was associated with polarized immune cell subsets or suppressed immune response31 demonstrating a clear dichotomy based on different microenvironments. Taken together, the organization, activation state, and polarization of the microenvironment appear just as important as the number of TIL.
Looking forward, the ability to induce or construct ELNs with anti-tumor activity holds great promise to help recruit TIL to the tumor microenvironment and enhance their anti-tumor activity, particularly in solid tumors devoid of these structures. To help develop such a strategy requires the creation of sound preclinical models in which to study ELNs formation. The localization of lymphocytes is largely governed by networks of chemokines, which guide their trafficking to different parts of the body at different stages of development, maturation, and activation. In similar fashion to the trafficking in a conventional peripheral lymph node, lymphocyte involvement in ELNs likely depends on a network of chemokines produced by resident stroma or resident leukocytes such as dendritic cells. Indeed, our previous work identified a tumor gene expression signature associated with the presence of ELNs in certain human solid tumors that encodes for 12 distinct chemokines23,24,25,32.
We are interested in employing these chemokines as leads to construct or induce ELNs in the solid tumor microenvironment with the intent to potentially enhance immunotherapies, particularly in those devoid of such structures. To achieve this goal will first require a series of fundamental biologic studies and modeling. It is known that chemokines and their cognate receptors have been extensively studied, but differing methodologies and sources of responding cells have made comparing chemokine/chemokine receptor axes across publications difficult to interpret. A dataset reporting chemoattractive potential using a single methodology as well as a similar cell isolation technique, migration time, and chemokine concentration range would greatly enhance the parameterization of in silico ELNs models, and inform preclinical in vivo murine models investigating ELN formation and function. Here, we have used a conventional transwell migration assay to first catalogue the chemotactic index (CI) of 48 recombinant murine chemokines on resting pan T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells immunomagnetically isolated from normal C57BL/6 spleen, including the 12 chemokines associated with ELNs formation in humans. We also catalogue the CI for select chemokines for activated B cells and T cells. Using this database, the CI of different chemokines can be directly compared, and may serve as a valuable tool for the parameterization of preclinical animal models. In addition, we introduce a groundwork mathematical model able to make use of standardized chemoattraction data that can serve as a basic infrastructure for more complex models in the future. The model makes use of reticular fibroblast cells (RFC) as a stromal source of chemokine production28, antigen presenting cells as an activating source for RFC and chemokine production, and generalized T cell and B cell populations responding to chemokine gradients32,33.
Materials and Methods
Female C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN). Mice were bred and maintained at the Animal Maintenance Facility at the H. Lee Moffitt Cancer Center and Research Institute (Tampa, FL) for at least 1 week prior to use, and were age-matched at 8 weeks or older before their usage in experiments. All mice were handled and treated in accordance with the institutional guidelines established by the animal review board for animal care at the H. Lee Moffitt Cancer Center and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of South Florida.
Culture Medium
Complete medium (CM) consisted of RPMI 1640 medium with 10% heat-inactivated fetal bovine serum, 1 μM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM fresh L-glutamine, 100 μg/mL streptomycin, 100 U/mL penicillin, 50 μg/mL gentamicin, 0.5 µg/mL fungizone (all from Life Technologies, Rockville, MD), and 0.05 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO).
Recombinant murine CCL2, 3, 4, 5, 6, 7, 8, 9/10, 11, 12, 19, 20, 21, 22, 24, 27, 28, CXCL1, 2, 4, 5, 9, 10, 11, 12 SDF1β, 13, 15, and 16 were purchased from PeproTech (Rocky Hill, NJ), and CCL17, 25, CXCL3, 17, CX3CL1, XCL1 and Chemerin were obtained from R&D Systems (Minneapolis, MN). In some cases, murine chemokines were not available, and human chemokines were used due to their known cross-species activity. Human CCL16, 18, CXCL6 and 8 were purchased from PeproTech, XCL2 was from Life Technologies (Carlsbad, CA) and CCL1, 13, 14, 15, 23, 26, CXCL7 and 14 were provided by ChemoCentryx (Mountain View, CA).
Isolation of Lymphocyte Populations
Spleens and the superficial inguinal, axillary, lateral axillary, mesenteric and cervical lymph nodes of mice were removed under sterile conditions and mechanically dissociated to prepare single-cell suspension with a 100-µm nylon mesh. Erythrocytes were lysed with RBC lysing buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA in sterile water). After incubation for 1 minute, cells were washed in 1x PBS and used for further experiments.
Pan T, CD4+ T, CD8+ T, B and NK cells were then isolated from splenocyte and lymph node cell suspensions using negative immunomagnetic isolation kits as per the manufacturer's instructions: Pan T Cell isolation kit II, CD4+ T Cell isolation kit II, CD8α+ T Cell isolation kit II, B Cell isolation kit and NK isolation kit for mouse (Miltenyi Biotech, Auburn, CA). Isolations were performed using a magnetic cell sorter (autoMACS) (Miltenyi Biotech, Auburn, CA). Pan T and B cell isolation enrichments were greater than 95%, CD4+ T and CD8+ T cells were greater than 90% and NK cells were greater than 80% as determined by flow cytometric analysis. See supplemental Fig. 1.
Flow Cytometry
Cells were washed with flow buffer (0.01% NaN3, 2% FBS in PBS) and Fcγ III/II receptor blocking was performed for B and NK cells by purified anti-mouse CD16/32 Fc blocking antibody (BD Biosciences, San Diego, CA). After incubation at 4 °C for 5 minutes, appropriate antibodies (1 µg/1 × 106 cells) for each cell marker were added to each sample and placed at 4 °C for 30 minutes. After washing with flow buffer, cells were fixed with 1% paraformaldehyde. Data acquisition was performed on FACSCalibur (Becton Dickinson, Mountain View, CA) and data analysis was done using FlowJo (Treestar, Ashland, OR). CD3+ CD19−, CD3+ CD4+, CD3+ CD8+, CD19+ CD3− and NK1.1+ CD3− cells were measured for Pan T, CD4+ T, CD8+ T, B and NK cell, respectively (all antibodies and isotype controls from BD Biosciences). For staining chemokine receptors, cells were incubated with anti-CCR7, -CXCR4 (BD Biosciences) or -CXCR5 antibodies (eBiosciences, San Diego, CA) for 40 minutes at room temperature.
Microchemotaxis Assay
Each chemokine was added to the lower chamber of a 24-well transwell (Costar, Cambridge, MA) at indicated concentrations in 600 μL of CM, and incubated at 37 °C in a humidified incubator with 5% CO2 for 30 minutes. Respective lymphocyte populations were resuspended at 1 × 107 cells/mL in CM, and 100 μL (1.0 × 106 cells) were seeded into 6.5-mm 24-well transwell inserts (Costar, Cambridge, MA), and allowed to incubate at 37 °C for 10 minutes. After pre-incubations, the upper chambers were moved into lower wells to start assay. To determine the actual input amount, 100 μL of cell suspension was added to 500 μL of CM and put into the lower wells directly and incubated without upper chambers. After 3 hour incubation at 37 °C, the upper chambers were removed and the migrating cells were retrieved. Cells were stained with the appropriate antibodies and added 2 × 105 polystyrene beads (Bangs Laboratories, Fishers, IN). Twenty thousand beads were then counted flow cytometrically. Baseline chemokinesis was determined by the fraction of cells moving into the lower chamber in control wells containing no chemotactic agent, and is expressed as a percent of seeded cells. The chemotactic index (CI) is calculated as the fraction of cells migrating in response to the chemotactic agent normalized to the chemokinesis background such that CI = (fraction of cells migrating to condition)/(fraction of cells migrating to control media). Transwell migration through 3 μm, 5 μm, and 8 μm pore sizes was compared (Supplemental Fig. 2), and the 5 μm pore size was selected for use. Data reported are representative of at least two experiments.
Activation Assay
Prior to CD4+ and CD8+ T cell activation, the culture plate was coated with 5 μg/mL of anti-CD3 and 1 μg/mL of anti-CD28 antibodies (BD Biosciences). T cells were resuspended to 1 × 106 cells/mL in CM and cultured at 37 °C for 5 days in the presence of 60 IU/mL of IL-2 (Prometheus, San Diego, CA). For B cell activation, B cells were resuspended in RPMI 1640/1% FBS containing 10 μg/mL LPS (Sigma-Aldrich) and cultured for 3 days. Analysis was performed on CD4+CD25+, CD8+CD25+ and CD19+CD69+ cells for activated CD4+ T, CD8+ T and B cells, respectively (Supplemental Fig. 3A–C).
Enzyme-linked Immunosorbent Assay (ELISA)
ELISA was carried out to evaluate T and B cell activation (Supplemental Fig. 3D). After supernatants were harvested at day 5 in the T cell activation assay or at day 3 in the B cell activation assay, IFNγ and IgM were measured, respectively. Mouse IFNγ ELISA Kit II (BD Biosciences) and Mouse IgG ELISA Quantitation Set (Bethyl laboratories, Montgomery, TX) were used in this experiment according to the manufacturer's instructions.
Statistical Analysis
Unpaired student t-test was performed for each chemokine concentration versus control wells containing no chemokine using GraphPad Prism 5.04 software (GraphPad Software, La Jolla, CA). Microchemotaxis assays were performed on individually isolated/activated wells for each cell population (n = 4).
Mathematical Model
A cellular automaton model was developed to investigate the effect of ELNs on the organization and effectiveness of the immune response to a nearby tumor. The mathematical model is simulated in two dimensions and contains a tumor, a patch of fixed RFC, and four types of motile cells that move in a continuous domain (i.e., off-lattice): inactive T cells, activated T cells, resting antigen presenting cells (APCOFF), and activated marginal zone antigen presenting cells (APCM). The tumor is represented by a circle of radius R, where we assume that the radius grows linearly in time in the absence of any immune response, and that the T-cell response causes a reduction in growth rate proportional to the number of tumor-infiltrating lymphocytes (L). The equation for tumor growth per time step of the simulation is given by:
where g is the innate tumor growth rate, k is the killing rate of TILs, and Δt is the time step used in the simulation. Assuming a disease state in which anti-tumor cytotoxicity can occur, increasing numbers of TILs would slow the growth and potentially cause the tumor to regress if kL > g.
APCOFF are introduced into the tumor area at a constant rate. The new cells are randomly positioned in an annulus centered around the tumor, with inner radius of (R − 500 µm) and outer radius of (R + 1500 µm). Once in the environment, they move with a combination of an unbiased random walk and a directed walk towards the tumor center, following tumor-produced inflammatory chemokine gradients. When an APCOFF is inside the tumor, the antigen collection process is simulated by converting the cell to an APCM, which can present antigen to the T cells. These APCM are also motile, using an unbiased random walk in combination with a directed walk away from the tumor center, to represent the seeking of T cells and/or vasculature for subsequent antigen presentation.
Inactive T cells are added to the simulation with a constant rate, using the same annulus as for APCOFF cells. These cells have an unbiased random walk. When an inactive T cell encounters an APCM (based on the two cells being in proximity to each other at a given time step), the APCM activates the T cell. The APCM can activate a number of T cells before it is removed from the simulation. Active T cells follow a biased random walk in the direction of the tumor. When they cross the tumor boundary as determined by R, they become TIL, which affects the tumor killing rate as shown in Eq. 1. They remain TIL for a set period of time, after which they are removed from the simulation, as expired T cells have no further effect on the results.
To model the effect of ELNs, we randomly place a fixed number of RFC in a circular patch situated away from the tumor. Each RFC begins in the "off state" with no secretion of chemokines, and therefore no influence on the movement of any of the APC or T cells. However, when an APCM migrates and comes into "contact" with an RFC (based on the two cells' proximity to each other), it will activate the RFC, which will in turn start to produce a chemokine gradient. For simplicity here, we model the chemokine gradient produced by an activated RFC as a two-dimensional Gaussian function centered at the cell. The total chemokine gradient for multiple activated RFCs is the sum of these Gaussians. As more RFC are activated, the chemokine signal becomes stronger. This gradient affects two of the motile cells, APCM and inactive T cells. For both of these cell types, when they detect the presence of chemokine gradient produced by RFC, movement is biased toward the ELNs patch. The bias scales with the strength of the gradient. We vary the number of RFC in different simulations to investigate this effect.
The random walks in the model were implemented as follows: the motile cells in the simulation are off-lattice and therefore can take any value for their position. The maximum travel distance for a cell in a time step is its kinetic speed multiplied by the time step. The algorithm selects a random distance between 0 and this maximal distance, and moves the cell in the direction of a randomly chosen angle. When there is an additional chemotactic term, a second random distance is chosen based on the maximum tactic speed, but the angle is fixed, directed towards the chemokine source (ascending the gradient). In this model, we do not consider spatial occupancy, so cells can get arbitrarily close to each other.
To summarize the model, there are two chemokine gradients, one from the tumor and one from the RFCM. APCOFF and active T cells move towards the tumor, and APCM and inactive T cells move away from the tumor. When there are activated RFCM, APCM and inactive T cells are both drawn to the ELNs. The simulation is initialized with a small tumor, a patch where RFCOFF cells are placed, and 30 APCOFF and inactive T cells. Parameters used in the model are shown in Table 1, with those labeled as 'model specific' refer to parameters that are highly variable in practice (e.g., tumor size, ELN-tumor distance, influx rate of immune cells); values were chosen that were in line with biologically reasonable ranges.
Resting pan T cells, B cells, CD4+ T cells, CD8+ T cells, or NK cells were immunomagnetically isolated and chemotaxis assays were performed for the 48 chemokines listed in Table 2 at three different concentrations: 10 ng/mL, 100 ng/mL, and 1,000 ng/mL. Non-directional random motility, or chemokinesis, was assessed for each cell population using control wells containing no chemokine gradient. Resting pan T cells, CD4+ T cells, CD8+ T cells, and B cells all displayed relativity low levels of chemokinesis while NK cells displayed significantly higher non-directional movement (Fig. 1). Directional movement in response to a chemokine concentration gradient was normalized to baseline chemokinesis for each respective cell type, and is reported as a chemotactic index (CI) for each condition. CI was calculated as the fraction of cells that migrated into the lower chamber from the total number of cells seeded in the top chamber for each condition, and then divided by the fraction of cells that migrated into the lower chamber randomly in control wells (i.e. by chemokinesis).
Resting pan T cells displayed significant and concentration dependent chemoattraction toward CCL19 and CCL21, and to a lesser extent CXCL10 and CXCL12. Statistically significant CI was also observed toward CCL16, CXCL9, CXCL11, CXCL13, and CXCL16 at some concentrations, but was of low magnitude or was not concentration-dependent. Resting CD4+ and CD8+ T cells displayed similar concentration-dependent chemoattraction toward CCL19, CCL21, CXCL10, and CXCL12 as pan T cells, and displayed variable low magnitude CI toward CCL13, CCL16, CXCL9, CXCL11, CXCL13, and/or CXCL16 (Fig. 2 and Table 2). Significant and concentration-dependent chemoattraction of resting B cells was restricted to CXCL12 and CXCL13, with low magnitude statistically significant CI observed toward some concentrations of CCL9, CCL11, CCL12, CCL19, CCL20, CCL21, CCL27, CXCL5, and XCL2 (Fig. 3 and Table 2). Resting NK cells displayed statistically significant and concentration-dependent chemoattraction toward a broad range of chemokines including CCL2, CCL3, CCL4, CCL5, CCL7, CCL12, CCL13, CXCL9, CXCL10, CXCL11, and CXCL12 (Fig. 4 and Table 2). However, NK cells did not display CI values greater than 4 toward any chemokine at any concentration, perhaps reflecting the high baseline chemokinesis displayed by resting NK cells (Fig. 1).
When activated, lymphocytes become more motile and often down-regulate expression of chemokine receptors recognizing secondary lymphoid tissue-homing chemokines such as CCR7. This aids in emigration from the follicle and secondary lymphoid tissues34. Here, activation of pan T cells, enriched CD4+ T cells, and enriched CD8+ T cells with plate-bound anti-CD3 resulted in significantly increased chemokinesis. Similarly, but to a lesser extent, activation of B cells with LPS significantly increased chemokinesis (Fig. 5A). Despite higher baseline motility, activated T cells loose chemoattraction to lymphoid-homing chemokines as demonstrated by reduced CI toward CCL19 and CCL21 for pan T cell populations (Fig. 5B,C), and reduced chemoattraction of enriched CD4+ and CD8+ T cells toward CCL21 (Fig. 5D,E). In contrast, B cells displayed significantly increased chemoattraction toward CCL21 and CXCL13 upon activation with LPS (Fig. 5F,G). This likely reflects the divergent roles of B cells and T cells following activation in secondary lymphoid tissues with T cells often emigrating T-cell zones and recirculating into the periphery and with B cells clustering in follicles.
The standardized set of chemotaxis assays presented here demonstrate that chemokines differentially affect random and directed motion of lymphocytes. To investigate the implications of this regarding immune response and subsequent tumor growth depending on the presence of the ELNs we developed a phenomenological mathematical model of the tumor and surrounding environment that is able to make use of chemoattraction data an simulate different scenarios.
Figure 6 shows snapshots of the model at 14 days of simulation, under three different conditions for the RFC: (a) no RFC, (b) 5 RFC, and (c) 30 RFC. Time course plots for these and several other simulations with different RFC counts are shown in Fig. 7, in the left panel. The results suggest that the presence of the RFC has an impact on the immune response to the tumor, both in terms of T-cell activation and tumor reduction. In all cases, the tumor grows for an initial period since the immune system starts in an inactive state. Eventually, APCs present tumor antigens to inactive T cells, causing activation, migration, and accumulation of TILs inside the tumor and eventual tumor regression. The presence of higher numbers of RFC causes a faster immune response to occur: without an ELNs, the peak tumor size occurs at day 9; while for simulations with 100 RFC the peak tumor size is around 4 days, suggesting that the migratory organization of lymphocytes provided by the ELNs significantly accelerates the anti-tumor immune response. In the case with 100 RFC, the tumor also reaches a maximum size that is about 25% smaller than the simulation with no RFC. This advantage persists as tumor growth continues to decline. The right panel of Fig. 7 compares tumor sizes after 30 days of simulation. Of interest, having a small number of RFC cells (5–10) does not produce an immune response as effective as the case with no RFC or a high number of RFC. This nonlinear result is because the weak chemokine signal of the ELNs draws some nearby cells away from the tumor, but a lack of immune cell density limits the effectiveness of rapid activation. In essence, there is a need for critical mass in the ELNs to achieve efficient cell attraction and activation. This can be seen in Fig. 6B, where the dendritic cells are accumulated in the ELNs area, but inactive T cells remain predominantly in the tumor microenvironment due to the weak signaling from the ELNs patch.
We had earlier identified a unique tumor-derived, 12-chemokine gene expression signature that could accurately predict the degree and type of lymphoid infiltrate, organized remarkably as ELNs that comprise - by immunohistochemistry staining - prominent B cell follicles, T cell marginal zones comprised of both CD4+ and CD8+ cell subsets, and associated follicular dendritic cells23. Of importance, there was a highly significant and consistent association between a marked increase in overall patient survival, the value of the mean score of this gene expression signature, and the presence of ELNs in stage IV (non-locoregional) melanoma, colorectal cancer, and stage IV bladder cancer. However, we found that the majority of human solid tumors lacked the presence of these particular ELNs, and patients harboring these 'poorly-immunogenic' tumors have had uniformly poor prognosis (i.e. reduced overall survival). Thus, there is a clear unmet medical need to 'reverse' this tumor microenvironment (that lacks these particular ELNs) by manufacturing de novo 'designer' ELNs.
Lymphocyte recruitment to the tumor microenvironment represents an attractive target to enhance anti-tumor immunity. As we continue to study lymphocyte trafficking, it is increasingly necessary to view chemokine-mediated trafficking as networks of chemokines and chemokine receptors working in concert as opposed to individual chemoattractive axes. Understanding how chemokine receptor expression and lymphocyte responsiveness changes during the course of maturation, activation, and effector response will greatly inform the development of preclinical animal and mathematical models. The chemokine database described herein may serve as a normalized resource to parameterize such models.
Given a direct link between gene expression of certain chemokines within human solid tumor microenvironments, the presence of tumor-localized ELNs, and addressing a clinical unmet need, we embarked on developing a standardized database of chemoattractive potentials of immune cell subsets for a broad range of chemokines to identify candidates for future engineering of ELNs. To approach this goal, we developed a strategy that combined the use of defined, recombinant chemokines, highly enriched resting and activated immune cell subsets, with standardized microchemotaxis assays to provide potential leads to further test in mathematical models. In this regard, we are developing both mathematical and pre-clinical animal models of ELNs formation in which multiple elements are being interrogated. The inclusion of lymph node-derived primary cellular components, which normally provide chemotactic and homeostatic queues in conventional lymph nodes, are being genetically modified to express selected chemotactic (e.g., see Table 3) and lymphoid neogenesis-related genes to enhance ELN formation. These modified cell lines are being combined with tumor antigen-pulsed dendritic cells and then incorporated in biocompatible scaffold materials and administered to tumor-bearing mice as injectable or implantable matrices35. These matrices may provide two potential benefits when delivered to tumor-bearing hosts. First, they may serve as model systems to better understand the factors governing the formation and/or maintenance of ELNs with anti-tumor reactivity. Second, these matrix-based systems may function as a therapeutic platform by delivering, stimulating and expanding transplanted lymphocytes and/or dendritic cells. In addition, the inert nature of bio-scaffolds also allows for the implementation of microparticle or nanoparticle constructs for controlled release of soluble factors. Such measures can provide sustained environmental queues to augment antigen-presenting cell or lymphocyte longevity, maturation, and activation. The initial mathematical modeling presented herein will be further developed to include these parameters.
In regard to translating in vitro migration assays to in silico models, care must be taken to differentiate between non-directional movement known as chemokinesis and directional chemotaxis toward a chemokine gradient. CI, as it is calculated here, normalizes transwell migration relative to baseline chemokinesis. This denominator is greatly increased after lymphocyte activation. Without normalizing to baseline movement, the percentage of cells migrating may appear very high when the majority of migration can be accounted for by random motility. Because this database was constructed using a single methodology, time point, and concentration range, CI can be compared across chemokines. For example, CCL19 and CCL21 are both well studied T cell chemoattractants; however, here CCL21 appears to be a 50% stronger chemoattractant compared to an equimolar amount of CCL19. In addition, this database serves to point out suboptimal chemoattractants whose combined cumulative effects may play an important role for in silico models such as the broad responsiveness observed for NK cells.
As a first step toward integrating this type of chemotactic data into an in silico model of ELNs formation, we developed a simple phenomenological mathematical model that predicts the chemokine gradient created as a result of lymphocyte, APC, and stromal interactions in the tumor microenvironment. Here, we consider secretion of a general chemoattractant for responding lymphocytes, and can serve as the basic groundwork for simulating multiple chemokine/chemoattraction axes involving more detailed cellular phenotypes. Further investigation of relevant chemokine relations between APC, RFC, and T cells would lead to a more mechanistic implementation of the model, which could then inform the design of future in vivo studies. The generalized gradient presented here can be replaced by specific chemokines observed in the microenvironment of tumor samples, or with chemokines that are known to be secreted by activated DC and/or stroma. In this way, multiple gradients can be modeled in concert. In addition, each chemokine gradient can be further developed beyond a two-dimensional Gaussian distribution taking into account extracellular matrix components that can bind to and slow the degradation of chemokines, better representing lymphocyte conduit systems observed in follicular structures in vivo 28. So too can the responsiveness of activated cell populations be adjusted based on lymphocyte activation, and on negative feedback mediated by shingosine-1-phosphate receptor (S1P1) activity36,37,38.
Importantly, this model outlines the framework to simulate chemotactic movement of infiltrating populations in response to chemokine gradients. This alone inadequately represents the physiologic tumor microenvironment, and in particular does not address immune cell polarity or immunosuppression. In addition to representing more defined subpopulations of immune cells, other soluble factors governing tumor-induced immune suppression will likely need to be added to fully capture the dynamic interplay of pro and anti-tumor elements. In particular, TFG-β, IL-10, and regulatory T cell populations are prevalent components of the tumor microenvironment known to suppress anti-tumor immunity39. Concurrently, the production of type I-polarizing factors such as IFN-γ and IL-12 can also be added as a counterbalance to these type II-polarizing factors40. Furthermore, it is now appreciated that the tumor as a system is heterogeneous, composed of numerous dominant and subdominant clones with related but different genetic lesions and mutational loads46. This diversity results in compartmentalized spatial fields across the tumor bead, each with its own variation of environmental queues, grow rates, and immune involvement47. It is reasonable to expect that variations in the type and amount of soluble factors released can also vary across the tumor bed in accordance with clonal diversity. Such variations would directly impact the localization and function of infiltrating immune cells, and may be required to fully model the complex tumor physiology.
The addition of these factors in future models would result in a system whose outcome is not guaranteed, but would rather depend on the balance of pro and anti-tumor forces recruited or induced in the tumor microenvironment. The complexity such models will increase exponentially with the addition of individual chemokine gradients, further subdivision of inflammatory or suppressive cell types, the addition of polarizing soluble factors, and representation of clonal heterogeneity, necessitating the use of standardized datasets such as is presented here. The model introduced here, though simplified, clearly demonstrates the potential benefit of ELNs and suggests that stromal/APC interaction is paramount for effector lymphocyte organization and response. Continued development of integrated ELNs formation models will greatly inform potential points of therapeutic intervention, and generate novel hypotheses regarding anti-tumor immunity.
- 1.
US Cancer Statistics Working Group. United States Cancer Statistics: 1999–2013 Incidence and Mortality. Web-based report. Atlanta, GA: Centers for Disease Control and Prevention; 2017 nccd.cdc.gov/uscs. Accessed February 24, 2017.
- 2.
Guy, G. P. Jr et al. Vital signs: melanoma incidence and mortality trends and projections - United States, 1982–2030. MMWR Morb Mortal Wkly Rep 64, 591–596 (2015).
- 3.
Freeman-Keller, M. & Weber, J. S. Anti-programmed death receptor 1 immunotherapy in melanoma: rationale, evidence and clinical potential. Ther Adv Med Oncol 7, 12–21 (2015).
- 4.
Griffith, K. D. et al. In vivo distribution of adoptively transferred indium-111-labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma. J Natl Cancer Inst 81, 1709–1717 (1989).
- 5.
Rosenberg, S. A. et al. Gene transfer into humans–immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med 323, 570–578 (1990).
- 6.
Wolchok, J. D. et al. Four-year survival rates for patients with metastatic melanoma who received ipilimumab in phase II clinical trials. Ann Oncol 24, 2174–2180 (2013).
- 7.
Balar, A. V. & Weber, J. S. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother, (2017).
- 8.
Dudley, M. E. et al. A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma. J Immunother 25, 243–251 (2002).
- 9.
Rosenberg, S. A. et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J Natl Cancer Inst 86, 1159–1166 (1994).
- 10.
Besser, M. J. et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res 16, 2646–2655 (2010).
- 11.
Ellebaek, E. et al. Adoptive cell therapy with autologous tumor infiltrating lymphocytes and low-dose Interleukin-2 in metastatic melanoma patients. J Transl Med 10, 169 (2012).
- 12.
Pilon-Thomas, S. et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J Immunother 35, 615–620 (2012).
- 13.
Radvanyi, L. G. et al. Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients. Clin Cancer Res 18, 6758–6770 (2012).
- 14.
Rosenberg, S. A. et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 319, 1676–1680 (1988).
- 15.
Rosenberg, S. A., Spiess, P. & Lafreniere, R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318–1321 (1986).
- 16.
Clark, W. H. Jr et al. Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer Inst 81, 1893–1904 (1989).
- 17.
Clemente, C. G. et al. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 77, 1303–1310 (1996).
- 18.
Sondergaard, K. & Schou, G. Survival with primary cutaneous malignant melanoma, evaluated from 2012 cases. A multivariate regression analysis. Virchows Arch A Pathol Anat Histopathol 406, 179–195 (1985).
- 19.
Barnhill, R. L., Fine, J. A., Roush, G. C. & Berwick, M. Predicting five-year outcome for patients with cutaneous melanoma in a population-based study. Cancer 78, 427–432 (1996).
- 20.
Larsen, T. E. & Grude, T. H. A retrospective histological study of 669 cases of primary cutaneous malignant melanoma in clinical stage I. 3. The relation between the tumour-associated lymphocyte infiltration and age and sex, tumour cell type, pigmentation, cellular atypia, mitotic count, depth of invasion, ulceration, tumour type and prognosis. Acta Pathol Microbiol Scand A 86A, 523–530 (1978).
- 21.
Thorn, M., Ponten, F., Bergstrom, R., Sparen, P. & Adami, H. O. Clinical and histopathologic predictors of survival in patients with malignant melanoma: a population-based study in Sweden. J Natl Cancer Inst 86, 761–769 (1994).
- 22.
Palmer, R. H. et al. Effects of Maternal Smoking during Pregnancy on Offspring Externalizing Problems: Contextual Effects in a Sample of Female Twins. Behav Genet 46, 403–415 (2016).
- 23.
Messina, J. L. et al. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy? Sci Rep 2, 765 (2012).
- 24.
Prabhakaran, S. et al. Evaluation of invasive breast cancer samples using a 12-chemokine gene expression score: correlation with clinical outcomes. Breast Cancer Res 19, 71 (2017).
- 25.
Coppola, D. et al. Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol. 170, 37–45 (2011).
- 26.
Goc, J. et al. Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells. Cancefr Res. 74, 705–15 (2014).
- 27.
Dieu-Nosjean, M. C. et al. Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures. J Clin Oncol. 26, 4410–7 (2008).
- 28.
Pitzalis, C., Jones, G. W., Bombardieri, M. & Jones, S. A. Ectopic lymphoid-like structures in infection, cancer and autoimmunity. Nat Rev Immunol 14, 447–462 (2014).
- 29.
Shin, M. S., Ho, K. J., Kirklin, J. W. & Sears, N. J. Metastatic mediastinal neoplasm masquerading as aortic dissection: a skip sign on computed tomography for their distinction. J Comput Tomogr 9, 299–303 (1985).
- 30.
Erdag, G. et al. Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma. Cancer Res 72, 1070–1080 (2012).
- 31.
Finkin, S. et al. Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nat Immunol. 12, 1235–44 (2015).
- 32.
Coppola, D. et al. Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol 179, 37–45 (2011).
- 33.
Katakai, T., Hara, T., Sugai, M., Gonda, H. & Shimizu, A. Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes. J Exp Med 200, 783–795 (2004).
- 34.
Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999).
- 35.
Weber, J. S. & Mule, J. J. Cancer immunotherapy meets biomaterials. Nat Biotechnol 33, 44–45 (2015).
- 36.
Mandala, S. et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346–349 (2002).
- 37.
Matloubian, M. et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427, 355–360 (2004).
- 38.
Schwab, S. R. et al. Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309, 1735–1739 (2005).
- 39.
Mellman, I., Coukos, G. & Dranoff, G. Cancer immunotherapy comes of age. Nature. 7378, 480–9 (2011).
- 40.
Zhu, J., Yamane, H. & Paul, W. E. Differentiation of Effector CD4 T Cell Populations. Annu Rev Immunol. 28, 445–89 (2010).
- 41.
Friberg, S. & Mattson, S. On the growth rates of human malignant tumors: implications for medical decision making. J Surg Oncol 65, 284–297 (1997).
- 42.
Dudley, M. E. et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298, 850–854 (2002).
- 43.
Riol-Blanco, L. et al. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immunol 174, 4070–4080 (2005).
- 44.
Miller, M. J., Wei, S. H., Cahalan, M. D. & Parker, I. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc Natl Acad Sci USA 100, 2604–2609 (2003).
- 45.
Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).
- 46.
Wang, E. et al. Cancer systems biology in the genome sequencing era: Part 1, dissecting and modeling of tumor clones and their networks. Semin Cancer Biol 4, 279–85 (2013).
- 47.
Wang, E. et al. Cancer systems biology in the genome sequencing era: part 2, evolutionary dynamics of tumor clonal networks and drug resistance. Semin Cancer Biol 4, 2869–92 (2013).
This work was funded by NCI-NIH (1R01 CA148995, 1R01 CA184845, U01 CA151924, P30 CA076292, U54 CA193489, P50 CA168536), Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; we thank the staff of the Moffitt Cancer Center Flow Cytometry Core.
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Yagawa, Y., Robertson-Tessi, M., Zhou, S.L. et al. Systematic Screening of Chemokines to Identify Candidates to Model and Create Ectopic Lymph Node Structures for Cancer Immunotherapy. Sci Rep 7, 15996 (2017). https://doi.org/10.1038/s41598-017-15924-2
Further reading
Biological mechanisms of ectopic lymphoid structure formation and their pathophysiological significance
International Reviews of Immunology (2020)
High School Internship Program in Integrated Mathematical Oncology (HIP IMO): Five-Year Experience at Moffitt Cancer Center
Bulletin of Mathematical Biology (2020)
Treatment with an immature dendritic cell-targeting vaccine supplemented with IFN-α and an inhibitor of DNA methylation markedly enhances survival in a murine melanoma model
Cancer Immunology, Immunotherapy (2020)
Cell and tissue engineering in lymph nodes for cancer immunotherapy
Advanced Drug Delivery Reviews (2020)
Illuminating the Numbers: Integrating Mathematical Models to Optimize Photomedicine Dosimetry and Combination Therapies
Frontiers in Physics (2019)
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


text:
Nissan Versa Vehicles with Defective Side Curtain Air Bags Recalled
In the event of a crash, the side curtain air bags May not properly deploy
Nissan North America is recalling certain model year 2017 Versa vehicles that were manufactured between August 1, 2016 and August 16, 2016.
A seam in the fabric section of the side curtain air bags may tear during side curtain air bag deployment, potentially affecting the performance of the air bag.
As such, these vehicles fail to comply with the requirements of Federal Motor Vehicle Safety Standard (FMVSS) number 226, "Ejection Mitigation", and number 214, "Side Impact Protection."
In the event of a crash necessitating side curtain air bag deployment, if the side curtain air bags do not deploy as intended, there would be an increased risk of injury to vehicle occupants.
Approximately 1,750 Nissan Versa vehicles are affected by this recall.
Nissan will notify owners of the recalled vehicles, and dealers will replace the left and right side curtain air bags, free of charge. Nissan has not yet provided an owner notification schedule for this recall.
For further information, contact Nissan customer service at (800) 647-7261.

subset: commoncrawl


text:
Viewing an unwieldy topic through a narrow prism almost always yields greater insights than a sprawling overview, and prisms don't get much narrower than the slant of Eddy Moretti and Suroosh Alvi's arresting doc: the shifting fates and fortunes of Iraq's only heavy-metal band, Acrassicauda, in the wake of the 2003 invasion. The four bandmates shrug off the novelty of Muslim youths moshing to Metallica covers: "We are living in a heavy-metal world," one explains, as bombs rock the Casbah like pyrotechnics at an Iron Maiden show. It only gets heavier, as civil war forces half the band into Syria and a blast obliterates their practice space. Bassist Firas al Lateef and lead singer Faisal Talal, best friends who live 15 minutes apart in Baghdad, go six months without seeing each other because the streets are so deadly. Inspired by a Vice profile, the HD-shot doc covers a lot of ground despite (or perhaps because of) its limited focus. Along with grim data about the "brain drain" flight of Iraq's most educated citizens (and the continuing hemorrhage of refugees into neighboring countries), it offers a gritty travelogue of bullet-riddled Baghdad, which the jittery hipster filmmakers prowl with moonlighting bodyguards whose paranoia surpasses even their own. In the process, the movie reclaims metal's appeal to the powerless as well as its threat—when you can get shot for wearing a Slipknot T-shirt (talk about "Death, be not proud") or speaking the English you learned off Master of Puppets, raising those devil horns isn't an empty act of aggression. Given the courageously downbeat closing note, here's hoping a follow-up catches Acrassicauda rockin' the free world.
This article from the Village Voice Archive was posted on May 20, 2008


text:
Mawarra Street, Surfers Paradise
Ranked 4th best street in Surfers Paradise
Great for
- No ratings yet
Not great for
- No ratings yet
Who lives here?
- No ratings yet
Got a burning question? Why not ask the locals! Simply ask your question below
Mawarra Street
"Chevy Island"
Mawarra St is on the northern side of Chevron Island close to Cronin Island. Many shops, bars, restaurants and more only a short walk to thomas Drive
Mawarra Street
Mawarra Street
"soooooooo close to the water!"
Not officially a suburb of the Gold coast, however part of Surfers Paradise, pain to get over the bridge sometimes however the island has a mix of older homes, newer homes and amazing riverfront properties. Nice areas nearby and on Cronin Island which are set aside for parks/landscaping.


text:
Camellia oil, also known as tea seed oil – but definitely not to be confused with tea tree oil, which is inedible – is commonly used in some Asian dishes. Unlike olive, sesame and peanut oils, camellia oil is known for its light flavor that doesn't interfere with the flavors of the other ingredients. This makes it a good option for cooking simple dishes in which you want to spotlight delicate flavors and fresh ingredients.
Camellia oil lets the flavors of other ingredients shine through in stir-fries, and its lightness means that other fried items maintain their own flavors and seasonings become bolder. Drizzle or brush it on meats to keep moisture in for baking, or spread a light coating on your pans for its nonstick properties without adding unwanted flavors. It works particularly well for cooking fresh vegetables to let the full flavor come through without an oily taste, which might even make your pickiest eaters happy. It also has a higher smoke point than many other common cooking oils, making it suitable for high heat and extended cook times without concern of burning the oil and imparting rancid flavors into your food.
You can substitute camellia oil for any other cooking oil in the exact amount. If you are substituting camellia oil for butter, however, cut back the amount by about one-fifth. Substituting oil for butter or other solid fats can also alter a recipe's texture, so you may need to experiment a bit with cutting back on other liquids if using camellia oil in baked items like breads, cakes or cookies. They'll still taste just as good, but you may hear a few complaints from the kids if their favorite snacks don't look or feel the same as they used to.
There are plenty of oils to choose from, each with different properties, and the selection can be confusing. If you want the lightness and mildness of camellia oil but can't find it in stores, grape seed oil has similar properties. If you're looking for an oil with a stronger flavor, try olive, peanut or sesame oil, or add a little extra seasoning to the camellia oil, such as lemon juice, vanilla or garlic, to make up for its milder flavor.
A Good Source of Good Fat
Camellia oil, like other cooking oils, is 100 percent fat. This means it packs a high caloric punch – 9 calories per gram – so is best used minimally. However, camellia and other nontropical plant-based cooking oils are made up of heart-healthy monounsaturated and polyunsaturated fats, as opposed to less preferable saturated fat and the truly-bad-for-you trans fat. So, camellia oil is a perfectly sensible part of a healthy diet.
- WSU Clark Extension: Garden Mastery Tips, Camellias
- "The Joy of Cooking"; I. Rombauer and M. Rombauer Becker; 1992
- American Heart Association: Dietary Fats


text:
Confidence Self Hypnosis MP3 (Download/CD)
Stephen Armstrong DHP; Faith Waude DHP Acc. Hyp.
- Length: 31:05
- Download: 30MB
Improve your confidence with this hypnosis MP3.*
What kind of things spring to mind when you think of Confidence? A loud, overwhelming person to whom all the attention goes to when they enter a room? Or someone who is able to speak quietly and steadily, but not in a shy way? Our conceptions of confidence vary, and if our impression of a stereotypical confident person is of the overwhelming example above, perhaps it's because we'd like a little of their self-belief to rub off on us.
Confidence is a great asset to possess: it can help us to further our career, by making people believe in what we have to say. It helps us when we're in the spotlight, such as giving a speech at work or in college. Most importantly, it helps us inside, alleviating the worry we have of our own self-image.
Boost your confidence and self-image with this Confidence Hypnosis MP3
Hypnotherapy is a proven tool used daily around the world to increase confidence and help people to convey their messages confidently and effectively in everyday life, and to socialize with ease. Without hypnosis, as we all know, the first steps towards self-confidence are the hardest. Using Hypnotic World's confidence MP3, you'll be able to increase your self-confidence and belief.
Download MP3
Get this hypnosis download and start benefiting today for just $12.95
New York, NY
16th Aug, 2018
"Thanks for an excellent session!"*
I'm very happy with this recording. The induction is unique and very effective. After less than a week using the MP3, I already feel significant shifts occurring. Thanks for an excellent session!
Sawtell, New South Wales, Australia
12th May, 2016
"I really enjoy this one, it does take me quite deep. I use it a lot. When I first started listening I felt I couldn't find a great memory, the more I use it I have found the more great memories come. I use this hypnosis for all occasions it is a great "pick me up!""*
10th Jul, 2014
"I felt a difference after one session so looking forward to great results after further listening!"*
12th Oct, 2013
"I really needed this and I am glad I got it. "*
(Name Supplied)
23rd Sep, 2009
" Great Mp3 I listen and now I am more postive in my own self. "*
11th Sep, 2009
"Very good product, very satisfied"*
Very relaxing MP3 which is very different than other confidence downloads
How do I listen to this MP3?
Once you have ordered an MP3, you will receive an email with a link where you can download the hypnosis recording as an MP3 file and listen to it whenever you wish. You can also 'burn' the MP3 onto a CD for your personal use - this will enable you to listen to the hypnosis MP3 at bed time, for example.
How long does the MP3 take to download?
This depends on the speed of your connection. For broadband users, it should take less than ten minutes to download the hypnosis MP3, while retaining the high quality of the recordings.
What software do I need to listen to the MP3?
Virtually all modern computers are capable of playing MP3s. Software such as Windows Media Player, iTunes, QuickTime or Real Player will enable you to listen to hypnosis MP3s with ease.
For more questions and answers, read the complete FAQ here
Hypnotic World's MP3 offerings are based on the highly popular scripts library that we have been building for many years and perfecting through experience and qualified expertise. The MP3s you download can be listened to from your computer, transferred to an MP3 player or even burned onto a CD for your listening, and you can use the MP3s as many times as you like as you see beneficial.
The Voices behind the Hypnosis
Founder of Hypnotic World, Faith has been practising hypnotherapy for over ten years in Yorkshire, England, and has treated many hundreds of clients for a vast range of addictions, phobias and other issues. Read more
Stephen records many of the hypnosis MP3s on Hypnotic World. In his own words
* Results may vary from person to person. For peace of mind, Hypnotic World offers a 100% money-back guarantee on self-hypnosis MP3s. If you are not entirely satisfied with this MP3, simply contact us for a full refund.


text:
AWS Security Blog
Tag: Amazon DynamoDB
The AWS Shared Responsibility Model and GDPR
The EU's General Data Protection Regulation (GDPR) describes data processor and data controller roles, and some customers and AWS Partner Network (APN) partners are asking how this affects the long-established AWS Shared Responsibility Model. I wanted to take some time to help folks understand shared responsibilities for us and for our customers in context of […]Read More
How to Protect the Integrity of Your Encrypted Data by Using AWS Key Management Service and EncryptionContext
One of the most important and critical concepts in AWS Key Management Service (KMS) for advanced and secure data usage is EncryptionContext. Using EncryptionContext properly can help significantly improve the security of your applications. In this blog post, I will show the importance of EncryptionContext and will provide a simple example showing how you can […]Read More


text:
By Yohannes Gebresellasie (Ph.d)
The issue, expression and state of democracy is relative. Winston Churchill, the late former British prime Minister from (1940-1945) was famous for his inspiring speech regarding democracy. On 11th November 1947 his remark regarding democracy goes like this "Many forms of government have been tried and will be tried in this world of sin and woe.
No one pretends that democracy is perfect or all worst. If I have to sum up the immediate future of democratic politics in a single word, I should say "insurance". That is the future insurance against dangers from scarcely less grave and much more near and constant which threatens us here at home in our own inland".
Different governments exercise acceptable democratic state and thus, they are considered as civilized democratic republic. That means there is no such perfect democracy anywhere. On the other hand however, this does not mean that governments and alike do not have to strive to achieve what is considered basic, fundamental, acceptable, reliable and that encompasses and fulfills standard and civilized democratic norms and citizenship rights to their people as much as possible. Churchill interpreted the idea of democracy giving reference to the common person that casts his/her vote during the election day having no pressure from anybody what so ever as follows: "At the bottom of all the tribute paid to democracy is the little man walking into the little booth with a little pencil, making a little cross on a little bit of paper – no amount of rhetoric in voluminous discussion can possibly diminish the overwhelming importance of that point.
How is that word of democracy to be interpreted? Churchill's idea was that the plain, humble common man, just the ordinary man who keeps a wife and family who goes off to fight for his country when it is in trouble, goes to the poll at the appropriate time and puts his cross on the ballot paper showing the candidate he wishes to be elected to parliament that he is the foundation of democracy. And it is also essential to this foundation that this man or woman should do this without fear and without any form of intimidation or victimization. He marks his ballot paper in strict secrecy and then elects representatives and together decides what government or even in times of stress, what form of government they wish to have in their country. According to Churchill, if that is democracy, he salutes it, responses it and works for it.
Since the downfall of the former dictatorial military regime and despite internal as well as external provocations and conspiracies, Ethiopia continues to maintain agreeable and relative peace, stability and embark on civilized norms and values of democratization, good governance and sustainable development. Although those norms and values can take long time to be fully implemented, from the very outset, the Ethiopian government formulated a constitution that guarantees equality for all nations, nationalities and peoples of the nation.
The constitution guarantees freedom of speech, expression in all aspects, ways and means. There are different independent newspapers with variety of views within the country expressing different social, economic and political views and addressing different issues. Also, there are different political parties operating with different political agendas within the parliament of Ethiopia. Generally, the Ethiopian people have by and large been enjoying relative peace, stability and the fruits of democratic values and norms since this current government came to power over twenty-five years ago. It is to be recalled that these fundamental rights of citizens were absent during the former regimes.
Recent election where over millions of Ethiopians cast their votes to the parties they wished is but one demonstration where the Ethiopian people exercised their democratic rights and responsibilities. During the last elections, many international observers including the renowned Carter Center and over thirty-five thousand local observers declared that the elections were free, fair and democratic.
Democratic nations follow rather strictly, only democratic and legal ways and means to resolve problems that may arise during social or political disagreements and this was the trend that should be followed by some opposition party members to resolve problem arising from the recent unfavorable demonstrations and disturbances in some areas of our nation. However, some extremist individuals and groups located in foreign countries are using tax-payers money to destroy every peaceful democratic of the Ethiopian people thus far gained by choosing violent ways and means instead of peaceful dialogue for achieving their objective i.e. to grasp power by every means including through violence and anarchy.
Further, some extremist leadership have made their number one priority to try to convince the international community that there is no democracy in Ethiopia or that the government is reversing the democratic process in Ethiopia and therefore the international community should penalize the country by denying economic assistance etc.
All these allegations by some internal and external extremist leadership against the government of Ethiopia is simply nonsense. It is non-sense because democracy is a matter of survival and not a matter of choice to the Ethiopian people.
Read More News Here Source link


text:
Get Out Of My Way
Rock N Roll Woman
A good glam record recordered in 1978, they just missed the Glam boat with this song , if it had been released in 1974-75 it would have been a massive hit.
Billy Franks and Lee Herons from Scruff went onto become The Legendary Luton Kippers, then the Faith Brothers (who released two albums and six singles with Siren/Virgin records in the mid-80s). These days Billy Franks a solo artist and has released 6 solo albums. For more information go to: http://www.billyfranks.com
You must be logged in to post a comment.


text:
Keep in Touch
- Announcements Appointments Bishop Cathedral Chaplaincies Children's Ministry Children's Work Christmas Churches in Cork Church in Society Church Music Church of Ireland Church Services Clergy Community Involvement Contemporary Issues Cork Diocese Ecumenism Education Five Marks of Mission Fund-Raising Lay Ministry Parish News People from Cork People from the Diocese Schools in the Diocese Special Events Voluntary Work Youth Work
Category Archives: Summer
Busy Summer Months in Cork, Cloyne and Ross ~ a Season in Photos
In Ireland's most southerly Church of Ireland Diocese, Cork, Cloyne and Ross, the summer holiday months of June, July and August, are one of the busiest times of the year. 'Summer only' churches in holiday villages open up. Tourists drop … Continue reading
Posted in Bishop, Cathedral, Cathedral Choir, CDYC, Charities in the Diocese, Charity Work, Children's Ministry, Children's Work, Concert, Diocesan Registrar, Diocesan Synod, Diocese, Drama, East Cork, Ecumenism, Education, Environment, Environmental Issues, Festivals, Five Marks of Mission, Fund-Raising, Holiday Club, Lord Mayor of Cork, Organist, Parish News, People from Cork, People from the Diocese, Summer, Youth Work
Holiday Club in Blarney Church of Ireland Parish
Another parish in the Diocese which hosted a holiday club during this past summer was the Church of the Resurrection, Blarney in the Parish of Carrigrohane Union. The local secondary school – Scoil Mhuire Gan Smál – was the venue … Continue reading
Posted in Parish News, Summer, Voluntary Work, Youth Work
Summer SING! at St Fin Barre's Cathedral, Cork
Summer SING! is a week-long children's singing festival which encourages children to experience singing in Cork City. Some 200 children and 100 professional and volunteer staff, aged from 3 to 14 years, gather together and they sing in venues across … Continue reading
Children's Holiday Club in Carrigaline and Templebreedy Parishes, Cork
A joint holiday club was organised by the Carrigaline Union and Templebreedy Group of parishes in the Diocese of Cork once again this year. This year it was called: 'Champions!'. 50 children and 20 teen leaders (along with the adult … Continue reading
Posted in Children's Ministry, Diocese, Five Marks of Mission, Parish News, Summer, Youth Work


text: Dr. Hicks is a licensed clinical psychologist, who received her doctorate in clinical psychology from The Chicago School of Professional Psychology. She works with children, adolescents, and adults with a wide array of presenting issues. She has extensive training in working with individuals who have been exposed to traumatic experiences, those with co-occurring substance use issues, as well as those struggling with emotional regulation. Dr. Hicks works actively to achieve understanding of each person's unique circumstances, all while ensuring one's individual values and beliefs are respected within the therapeutic relationship. She believes the ultimate goal of therapy is to empower individuals to confront problematic circumstances in their lives, and to work collaboratively toward furthering health and wellness. Dr. Hicks is an active proponent of multicultural counseling and theory, and works dynamically to understand how individuals make meaning of the world within their various cultural contexts. Dr. Hicks is also a self-proclaimed "Geek Therapist," and incorporates elements of popular culture into treatment, when beneficial.


text:
As a fitness trainer, people are always asking me for the magical ingredient that will melt off fat. While I continue to explain that no 1 magic bullet exists, there are a number of nutrients that will promote faster fat loss.
Vitamin D
The University of Minnesota did a study in 2008 where Dr. Shalamar Sibley observed 38 obese people on an diet that was 750 calories less than their estimated daily need for 11-weeks. Participants with higher levels of vitamin D at the onset of the study lost significantly more weight – 70% more!
Based on the results of this study, it can be concluded that eating vitamin D-rich foods can help the body stay out of a fat-storage state and in a fat-burning state. Simply put, high vitamin D levels can boost weight loss by 70 percent! Over the last 40 years, there are many studies on vitamin D. It's clear that vitamin D is an important and effective nutrient. However, it's not the only vitamin on this fat-melting team. Boost your metabolism and shed body fat by adding vitamin D and these other fat burning vitamins to your diet.
Melts fat by: Calcium is a mineral that combines with vitamin D to help rid you of fat. It is stored in fat cells, and researchers believe that the more calcium a fat cell has, the more fat that cell will release to be burned. In addition, calcium helps weight loss by sticking to fat in your GI tract and preventing some of it from getting absorbed into your bloodstream.
Melts fat by: Protein is the most complete types of food for you body. Regular doses of it can help to keep body composition, the amount of fat relative to muscle, in proportion. Along with calcium and vitamin D, protein helps you to preserve muscle mass as you lose weight. Researchers have found that women who consumed protein twice daily lost almost 4 percent more weight than women who consumed less of it. Also, the women with more protein in their diet got stronger.
Omega-3 Fatty Acids
Melts fat by: Omega-3's enable weight loss by turning on trigger fat-burning enzymes in cells. A definite benefit of this nutrient is it reduces emotional eating by soothing mood. And omega-3's are said to improve leptin signaling in the brain; which causes the brain to turn up fat burning and turn down appetite. It's hard to consume vitamin D without consuming omega-3 fatty acids. Salmon (also high in vitamin D) is one of the richest sources of omega-3's. Nuts and seeds contain a type of fat that can transform into omega-3's after ingestion.
Monounsaturated Fatty Acids (MUFAs)
Melts fat by: MUFA's are a type of fat found in olive oil, nuts, seeds, avocados, peanut butter, and chocolate. It have just one chemical bond, hence the word "mono." In one study, 26 men and women found that a diet that included 20 percent of its calories from MUFA's improved calorie burning by 0.1 percent and fat burning by 0.04 percent after 6 months. That may not seem like a lot of fat and calorie burning, but over time it is. Other research shows that MUFA's targets belly fat. Foods that are high in MUFA's include especially peanuts, tree nuts, and olive oil; and each has been shown to keep blood sugar steady and reduce appetite as well.
Conjugated Linoleic Acid (CLA)
Melts fat by: CLA's are powerful fat burners that are found in dairy products. CLA's are created when bacteria ferments the food in the first part of the stomach of cows, sheep, and other ruminant animals. Through this process, the CLA's makes its way into the meat and milk of these animal; and then to our plates.
When we consume these foods, the CLA helps blood glucose enter body cells. CLA can be burned for energy, not stored as fat and helps to promote fat burning, especially in muscles, where most of our calorie burning takes place.
The skinny on the fat burning nutrients
Now before you start buying supplements with all of these fat melters and waiting for the fat to magically melt away, remember: vitamin D and other fat melters facilitate weight loss, but they are not magic. Taking a bunch of supplements might net you some benefits, but to experience significant weight loss, combine whole foods full of these fat melting nutrients with portion control and regular exercise.


text:
Two fascinating tales of terror. One: the Academy Award short list for feature-length documentaries. Two: an account of a few ... um ... alleged irregularities at the Queens International Film Festival. (Keep in mind, anyone can allege anything. I'm linking to the story as it has been published online -- but you'll definitely want to check out the facts for yourself.)
Ah, what a tangled web we weave, when we try not to deceive. Two somewhat shocking stories....
Oscar Short List of Documentaries Draws Controversy
Pressed for details, Mr. Toback said only that he had experienced something connected with the selections process, "which I put fully in the category of extortion that I did not go along with." Mr. Toback added that he was "furious" at himself for "having chosen to be passive and quiet in the face of that extortion."
Film fest head a fraud, many say
A New York filmmaker who served on the advisory board of the first QIFF and has worked with Castaldo on and off for the past several years, said he was conned out of $20,000 this summer. The filmmaker, who wished to remain anonymous as he is negotiating a big movie deal, said Castaldo offered to distribute a film he and his partner had made earlier this year. According to their agreement, the filmmaker allegedly paid Castaldo $20,000, with the understanding that she would take the film to Cannes. "She never went to Cannes," he said. "It was one total ripoff. ... Everything was phony." Castaldo allegedly tried to convince him that she had been to Cannes, showing him a website with video footage of her there. The filmmaker said he soon discovered that the footage was doctored. The website offers to Photoshop anyone into Cannes, he said.


text:
Galen Carrington is a 27 year old Indiana resident who has classic MSUD. He underwent liver transplantation 5 years ago at Children's Hospital UPMC by Dr. Kyle Soltys as a treatment option for MSUD. Since surgery, Galen's health has been excellent! He graduated from Wilmington College with a Bachelor's degree in Psychology and is currently employed by the Carl Linder YMCA and Cincinnati Recreation Commission as a swim instructor and lifeguard. He has enjoyed swimming in 2 consecutive Transplant Games of America which were held in Houston and Cleveland-he brought home a total of 8 Gold Medals in all his events which included butterfly, breaststroke and freestyle!
This summer, Galen had the privilege of representing
the USA at the World Transplant Games in Malaga,
Spain! With over 30 countries in attendance and a
strong competitor facing Great Britain, he managed
to bring home 1 Gold and 4 Silver Medals! What's
more important than the medals is the global support
for the success of transplantations and the need to
increase awareness of organ donations.
Galen emphasizes how important it is to be a part of
the MSUD Family Support Group as well as the Donate
Life campaign in order to successfully manage his
disorder and belong to a community of others who
can relate to his condition. Galen continues to strive
in his swimming career in honor of his donor family.
Please look for him to represent Indiana in the next
Transplant Games to be held in Salt Lake City, Utah
August 2018!
Galen Carrington Classic MSUD age 27
- Details
- Written by Jody Carrington, Mom


text:
Long information
What is it and how is it used?
Prepandrix is a vaccine for use in adults from 18 years old. It is intended to be given before or during the next influenza (flu) pandemic to prevent flu caused by the H5N1 type of the virus. Pandemic flu is a type of influenza that occurs at intervals that vary from less than 10 years to many decades. It spreads rapidly around the world. The symptoms of pandemic flu are similar to those of ordinary flu but are usually more severe.
When a person is given the vaccine, the immune system (the body?s natural defence system) will produce its own protection (antibodies) against the disease. None of the ingredients in the vaccine can cause flu.
As with all vaccines, Prepandrix may not fully protect all persons who are vaccinated.
|Table of Contents
|What do you have to consider before using it?
|How is it used?
|What are possible side effects?
|How should it be stored?
|Further information
What do you have to consider before using it?
Prepandrix should not be given:
- if you have previously had a sudden life-threatening allergic reaction to any ingredient of Prepandrix (these are listed at the end of this leaflet) or to any of the substances that may be present in trace amounts as follows: egg and chicken protein, ovalbumin, formaldehyde, gentamicin sulphate (antibiotic) or sodium deoxycholate. Signs of an allergic reaction may include itchy skin rash, shortness of breath and swelling of the face or tongue.
- if you have a severe infection with a high temperature (over 38°C). If this applies to you then your vaccination will be postponed until you are feeling better. A minor infection such as a cold should not be a problem, but your doctor will advise whether you can still be vaccinated with Prepandrix.
Do not have Prepandrix if any of the above apply to you. If you are not sure, talk to your doctor or pharmacist before having this vaccine.
Take special care with Prepandrix:
- if you have had any allergic reaction other than a sudden life threatening allergic reaction to any ingredient contained in the vaccine, to thiomersal, to egg and chicken protein, ovalbumin formaldehyde, gentamicin sulphate (antibiotic) or to sodium deoxycholate. (see section 6. Further information).
- if you have problems with your immune system, since your response to the vaccine may then be poor.
- if you are having a blood test to look for evidence of infection with certain viruses. In the first few weeks after vaccination with Prepandrix the results of these tests may not be correct. Tell the doctor requesting these tests that you have recently received Prepandrix.
Using other medicines or vaccines
Please tell your doctor if you are taking or have recently taken any other medicines, including medicines obtained without a prescription or have recently received any other vaccine.
There are no data on the use of Prepandrix given at the same time as other vaccines. Therefore, Prepandrix is not intended to be given at the same time as other vaccines. However, if this cannot be avoided, the other vaccine will be injected into the other arm. Any side effects that occur may be more severe.
If you take any medicines that reduce immunity to infections or have any other type of treatment (such as radiotherapy) that affects the immune system, Prepandrix can still be given but your response to the vaccine may be poor.
Pregnancy and breast-feeding
There is no information on the use of Prepandrix in pregnant or breast-feeding women. Your doctor needs to assess the benefits and potential risks of giving you the vaccine if you are pregnant or breast-feeding. Please tell your doctor if you are/may be pregnant or intend to become pregnant, or if you are breast-feeding and follow his advice.
Driving and using machines
Some effects mentioned under section 4. ?Possible side effects? may affect the ability to drive or use machines.
Important information about some of the ingredients of Prepandrix
Thiomersal (preservative) is present in this product, and it is possible that you may experience an allergic reaction.
This medicinal product contains less than 1 mmol sodium (23 mg) and less than 1 mmol of potassium (39 mg) per dose, i.e. essentially sodium- and potassium-free.
How is it used?
You will receive two doses of Prepandrix. The second dose should be given after an interval of at least three weeks.
If you are older than 80 years of age, you may receive two double injections of Prepandrix. The first two injections should be given at the elected date and the two other injections should preferably be given 3 weeks after.
The doctor or nurse will give Prepandrix as an injection into your upper arm muscle. The vaccine should never be given into a vein or into the skin. The double injections will be given in opposite arms.
If you have any further questions on the use of this product, ask your doctor or nurse.
What are possible side effects?
Like all medicines, Prepandrix can cause side effects, although not everybody gets them.
The side effects listed below have occurred in the days or weeks after vaccination with vaccines given routinely every year to prevent flu. These side effects may occur with Prepandrix.
Very rare (these may occur with up to 1 in 10,000 doses of the vaccine):
- Temporary inflammation of the brain and nerves causing pain, weakness and paralysis that may spread across the body.
- Narrowing or blockage of blood vessels with kidney problems
Rare (these may occur with up to 1 in 1,000 doses of the vaccine):
- Allergic reactions leading to a dangerous decrease of blood pressure, which, if untreated, may lead to collapse, coma and death
- Fits
- Severe stabbing or throbbing pain along one or more nerves
- Low blood platelet count which can result in bleeding or bruising
Uncommon (these may occur with up to 1 in 100 doses of the vaccine):
- Generalised skin reactions including urticaria (hives)
If any of these side effects occur, please tell your doctor or pharmacist immediately.
The side effects listed below have occurred with Prepandrix in clinical studies:
Very common (these may occur with more than 1 in 10 doses of the vaccine):
- Tiredness
- Headache
- Pain, redness, swelling or a hard lump at the injection site
- Fever
- Aching muscles, joint pain
Common (these may occur with up to 1 in 10 doses of the vaccine):
- Warmth, itching or bruising at the injection site
- Increased sweating, shivering, flu-like symptoms
- Swollen glands in the neck, armpit or groin
Uncommon (these may occur with up to 1 in 100 doses of the vaccine):
- Tingling or numbness of the hands or feet
- Dizziness
- Sleepiness
- Sleeplessness
- Diarrhoea, vomiting, stomach pain, feeling sick
- Itching, rash
- Generally feeling unwell
These reactions usually disappear within 1-2 days without treatment.
If any of the side effects gets serious, or if you notice any side effects not listed in this leaflet, please tell your doctor or pharmacist.
How should it be stored?
Keep out of the reach and sight of children.
Before the vaccine is mixed:
Do not use the suspension and the emulsion after the expiry date which is stated on the carton. The expiry date refers to the last day of that month.
Store in a refrigerator (2°C - 8°C).
Store in the original package in order to protect from light.
Do not freeze.
After the vaccine is mixed:
After mixing, use the vaccine within 24 hours and do not store above 25°C.
Medicines should not be disposed of via wastewater or household waste. Ask your pharmacist how to dispose of medicines no longer required. These measures will help to protect the environment.
Further information
What Prepandrix contains
- Active substance: After mixing, one dose (0.5 ml) contains 3.75 micrograms of haemagglutinin from the following influenza virus strain:
A/Indonesia/05/2005 (H5N1) like strain used (PR8-IBCDC-RG2)
- Adjuvant: The emulsion vial contains an ?adjuvant? (AS03). This compound contains squalene (10.69 milligrams), DL-?-tocopherol (11.86 milligrams) and polysorbate 80 (4.86 milligrams). Adjuvants are used to improve the body?s response to the vaccine.
- Other ingredients: The other ingredients are: polysorbate 80, octoxynol 10, thiomersal, sodium chloride (NaCl), disodium hydrogen phosphate (Na 2HPO 4), potassium dihydrogen phosphate(KH 2PO 4), potassium chloride (KCl), magnesium chloride (MgCl 2), water for injections
What Prepandrix looks like and contents of the pack
Suspension and emulsion for emulsion for injection.
One pack of Prepandrix consists of:
- one pack containing 50 vials of 2.5 ml suspension (active substance) for 10 doses
- two packs containing 25 vials of 2.5 ml emulsion (adjuvant) for 10 doses
The suspension is a colourless light opalescent liquid.
The emulsion is a whitish homogeneous liquid.
Prior to administration, the two components should be mixed. The mixed vaccine is a whitish emulsion.
Marketing Authorisation Holder and Manufacturer
GlaxoSmithKline Biologicals s.a.
Rue de l?Institut 89
B-1330 Rixensart
For any information about this medicine, please contact the local representative of the Marketing Authorisation Holder:
BelgiëBelgiqueBelgien GlaxoSmithKline s.a.n.v. TélTel 32 2 656 21 11 LuxembourgLuxemburg GlaxoSmithKline s.a.n.v. TélTel 32 2 656 21 11
. 359 2 953 10 34 Magyarország GlaxoSmithKline Kft. Tel. 36-1-2255300
Malta GlaxoSmithKline Malta Tel 356 21 238131 eská republika GlaxoSmithKline s.r.o. Tel 420 2 22 00 11 11 gsk.czmailgsk.com
Danmark GlaxoSmithKline Pharma AS Tlf 45 36 35 91 00 dk-infogsk.com Nederland GlaxoSmithKline BV Tel 31 030 69 38 100 nlinfogsk.com
Deutschland GlaxoSmithKline GmbH Co. KG Tel 49 089 360448701 produkt.infogsk.com Norge GlaxoSmithKline AS Tlf 47 22 70 20 00 firmapostgsk.no
Eesti GlaxoSmithKline Eesti OÜ Tel 372 667 6900 estoniagsk.com Österreich GlaxoSmithKline Pharma GmbH. Tel 43 1 970 75-0 at.infogsk.com
GlaxoSmithKline A.E.B.E T 30 210 68 82 100 Polska GSK Commercial Sp. z o.o. Tel. 48 22 576 9000
España GlaxoSmithKline, S.A. Tel 34 902 202 700 es-cigsk.com Portugal GlaxoSmithKline, Produtos Farmacêuticos, Lda. Tel 351 21 412 95 00 FI.PTgsk.com
România GlaxoSmithKline GSK SRL Tel 40 021 3028 208 France Laboratoire GlaxoSmithKline Tél 33 0 1 39 17 84 44 diamgsk.com
Ireland GlaxoSmithKline Ireland Ltd Tel 353 01 4955000 Slovenija GlaxoSmithKline d.o.o. Tel 386 0 1 280 25 00 medical.x.sigsk.com
Ísland GlaxoSmithKline ehf. Sími 354-530 3700 Slovenská republika GlaxoSmithKline Slovakia s.r.o. Tel 421 02 48 26 11 11
Italia GlaxoSmithKline S.p.A. Tel 39 04 59 21 81 11 SuomiFinland GlaxoSmithKline Oy PuhTel 358 10 30 30 30 Finland.tuoteinfogsk.com
GlaxoSmithKline Cyprus Ltd 357 22 39 70 00 Sverige GlaxoSmithKline AB Tel 46 08 638 93 00 info.produktgsk.com
Latvija GlaxoSmithKline Latvia SIA Tel 371 67312687 lv-epastsgsk.com United Kingdom GlaxoSmithKline UK Tel 44 0808 100 9997 customercontactukgsk.com
GlaxoSmithKline Lietuva UAB
Tel. +370 5 264 90 00
This leaflet was last approved in {MM/YYYY}.
The following information is intended for medical or healthcare professionals only:
Prepandrix consists of two containers:
Vial A: multidose vial containing the antigen (suspension),
Vial B: multidose vial containing the adjuvant (emulsion).
Prior to administration, the two components should be mixed.
Instructions for mixing and administration of the vaccine:
1. Before mixing the two components, the emulsion and suspension should be allowed to reach room temperature, shaken and inspected visually for any foreign particulate matter and/or abnormal physical appearance. In the event of either being observed, discard the vaccine.
2. The vaccine is mixed by withdrawing the contents of the vial containing the emulsion (Vial B) by means of a syringe and by adding it to the vial containing the suspension (Vial A).
3. After the addition of the emulsion to the suspension, the mixture should be well shaken. The mixed vaccine is a whitish emulsion. In the event of other variation being observed, discard the vaccine.
4. The volume of Prepandrix (5 ml) after mixing corresponds to 10 doses of vaccine.
5. The vial should be shaken prior to each administration.
6. Each vaccine dose of 0.5 ml is withdrawn into a syringe for injection.
7. The needle used for withdrawal must be replaced by a needle suitable for intramuscular injection. Any unused product or waste material should be disposed of in accordance with local requirements.


text:
Sargassum can be seen surrounding the coast of Guadeloupe.
Click on the box in the lower-right corner to view this image at its full 10 m resolution directly in your browser.
This Copernicus Sentinel-2 image, which was acquired on 6 July 2019, has been processed in a way that highlights vegetation in red tones. The pink shades in the ocean show floating Sargassum, while the dark shades are most likely underwater Sargassum. The blue indicates shallow waters.
Read more: Washed up: Sargassum blankets beaches


text:
Dump it in the army's lap!
This piece has been inspired by a picture in a reputed English Daily's front page recently, showing three soldiers in combat gear, SMGs in hand, standing at a gate of the Lahore's Qaddafi Stadium to provide it the necessary security for PSL's cricket match. Two things came to my ...Read More
Horse Trading: Past, Present and Future
If media reports are to be believed, the recently concluded Senate elections seem to have demolished all previous records of vote buying and selling by unimaginable margins. Many candidates contesting for elections came from the exclusive billionaires' club. So-called secular and progressive ...Read More


text:
Copy URL
All entered transactions can be found here. Search criteria include the date, Accounts(Left/Right) and Item(Parenthesis) by default and can be extended to Note and Amount by clicking 'Advanced search.' Transactions can be edited or deleted in the same way as on the Insert page.
Copy URL
Search CriteriaFor other criteria, the result will be the same as the search keyword. However, for Items, the search result may vary depending on the parentheses and the use of asterisk(*).
Copy URL
Searching ItemsBy default, Items with the 'exactly' same name are searched when a keyword is entered, but using * can expand the search range.
- All entries involving the Item John : 'John'
- All entries involving Items of which their names start with John : 'John*'
- All entries involving Items of which their names end with John : '*John'
- All entries involving Items of which their names include John : '*John*'
Copy URL
Searching ParenthesesIf * is not used in the search, all parentheses data including the keyword will be searched. All you need to do is putting the parentheses.
- All entries with the text John in parentheses : '(John)'
- All entries involving the Item John, with the text boy in parentheses : 'John(boy)'
- All entries involving Items of which their names start with Jo, with the text boy in parentheses : 'Jo*(boy)'
Copy URL
Related reportsRelated report means the reports that show up only under the specified search. It may not come up or might appear many other reports. This includes the following.
- Changes in (Entry/Item/Customer account): Any changes during the certain period will be appeared in line graph. Assets and Liabilities start up from the accumulated amount.
- Items(Customer accounts) in Entry: Total sum of items (or customer accounts) in searched entry and Accumulated amount for customer accounts.
- Entries in Account: This includes every entry in the account. Assets and Liabilities are appeared in accumulated amount.
- Flow in (Account/ Entry): The flow of every account/entry in account or entry unit. It is a graph that appears like folders so you can fold and open to grasp the point more specifically.
Copy URL
Transfer of related contentA breakdown shown on the screen includes links based on the relevant letter. If you click the item ''Snack', it will refer the breakdown of 'Snack' for a certain period. Click the 'Back' button, if you wish to go back to the previous page.
Copy URL
Graph toolsThere are graph tools that you can use to outline the relevant figures on the right upper side of the page.
- Pop-up: To open a new window for a graph that you are looking at. It would be used to analyze related graphs on several pages.
- Printing: Open a new window to print the only related data. It could be used to see the data in a simple graph.
- Excel storage: You can download data in Excel. Data in Excel is arranged mainly in rows which is suitable for using in filter or pivot table.


text:
Seed Swap
Join us on the 1st of February for the 2nd Annual Seed swap at Oakkham Castle.
Bring your seeds and see what we have for an exchange.
Kits for beginners also available.
Join the Sunflower Challenge - seed kits available from last years winner which was 3.9 metres tall - can you do better?
Free entry, refreshments available and children welcome
When: 10am-1pm on Saturday 1st February
Where: Oakham Castle


text:
09 April 2019
SD Card & e-stant software: find out the benefits!
A few simple steps to PROGRAM, UPDATE THE FIRMWARE and manage the VOICE PROMPTS of the 2W autodiallers.
In Esse-ti the evolution anticipates the needs of the user: thanks to the e-stant software with the SD Card, it becomes extremely easy to program, manage and customize the voice messages of the 2W autodiallers.
The e-stant software is available as free download for:
- local and remote programming
- creation of a QR-code containig the cofiguration profile
- reading/sending all the autodiallers' settings
- alarms texting
- events' log storage and query
- customizing/playing the audio files
- firmware update
- configuration, speech customization and firmware update of the 2W-serie autodiallers via SD CARD.
Watch the new tutorial VIDEO!


text:
Oakland, Berkeley, And East Bay News, Events, Restaurants, Music, & Arts
What is the sound of one corporate culture falling? Matthew Herbert is on the warpath to find out -- and make it happen. Since 1995, the acclaimed left-field dance producer (who's released numerous records under the guises of Herbert, Doctor Rockit, and Wishmountain) has closed the gap between groove culture and daily life by building his house, electro, and mutant jazz tunes with digitally manipulated sounds of everyday objects. His live appearances have found him tearing potato chip bags, clinking bottles, and crushing soda cans into compelling rhythmscapes. Now, under the guise of Radio Boy, Herbert comments on multinational companies and their wasteful behavior by thrashing their products into beats.
The results are refreshing and provocative. The album's fifteen percussion-centered house-tempo tunes don't serve up any dance music clichés beyond hypnotic rhythms, and the atmosphere of each is positively kinetic. Looking at the credited "sole sound sources" brings up several questions: How the hell did Herbert get all that funk out of the sound of a straw being plunged into the lid of a McDonald's soft drink cup? And can we really discern that he made music with "one caramel latte and one Frappuccino" from Starbucks? But does it really matter if the point's made via the producer's own lucid liner notes, and if the music makes you cut loose? Especially on "The Whisper of Friction," made from field recordings from May Day worker protests in Europe. Though he's obviously not the first to expand on found sound or sampling, Herbert's explorations are qualitatively unique. Mechanics' tech-noise neither clangs with the postwar metallic scrapings of industrial pioneers like Einstürzende Neubauten nor guffaws with the mediated irreverence of Negativland. Instead, it sensually squishes with the "soft" rips and pours of late-century convenience culture, which -- in conjunction with its lucid liner notes -- opens intimate and vulnerable spots in our conscience. Of course, like any piece of media that denounces corporations, Mechanics evokes those nagging questions of hypocrisy -- after all, at least a few monoliths are inevitably involved in its production, manufacture, and distribution. But to be fair, Herbert has put his politics where his wallet is: Mechanics will reportedly retail for two dollars in stores. Viva!
Contact the author of this piece, send a letter to the editor, like us on Facebook, or follow us on Twitter.
News - October 26, 4:00 AM


text:
Course Outline
This course is designed for those who are required to respond to fire incidents as a first responder. This course has theoretical and practical components that participants must successfully complete.
On completion of training, participants will be able to:
- Evaluate and communicate the location, nature and extent of the fire emergency in a timely and appropriate manner.
- Maintain personal safety at all times in accordance with WHS guidelines.
- Put on appropriate protective clothing in accordance with organisation procedures.
- Select appropriate extinguishing agents based on knowledge of fire and fuel types.
- Operate basic fighting equipment safely, according to manufacturer specifications and organisation procedure, in order to contain the fire emergency.
- Follow emergency reporting procedures.
- Identify appropriate authorities and notify.
- Clearly and unambiguously communicate information concerning the emergency.
- Evacuate area in a safe and controlled manner when first response has failed to control the fire emergency, or has proven inappropriate.
- Secure immediate area of the emergency to ensure no further loss occurs to people, equipment, process and environment.
Students who attend and successfully meet all assessment requirements of the course will receive a nationally recognised statement of attainment with the following unit of competency: MSMWHS212 Undertake first response to fire incidents.
There is a maximum number of 12 participants per program.
There are no pre-requisites for this course.


text:
CNP Wrinkle Repair Eye Cream 15ml
The anti-wrinkle eye cream contains Adenosine in nano-capsule to provide elasticity and reduce creases around delicate eye area.
Nano-Capsule Adenosine cares for fine lines around the eyes for skin firming effects.
The ingredients formulated in the product has no colorant, no mineral oil added and is safe to use around the eye area.
The Vitamin P creates moisture barrier for firmer and smoother skin around the eyes.
The EGF ingredient and Peptide compound strengthens the skin barrier, adding vitality to the eyes.
Before the last step of skincare regime, apply Wrinkle Repair Eye Cream sparingly under the eyes and dab lightly using the fourth finger. Finish off with moisturizer and cream.
Water, Butylene Glycol, Dimethicone, Glycerin, Neopentyl Glycol Diheptanoate, Dipentaerythrityl Hexa C5-9 Acid Esters, Butyrospermum Parkii (Shea Butter) Extract, Cetearyl Alcohol, Squalane, 1,2-Hexanediol, Cyclopentasiloxane, Malachite Extract, Cetyl Alcohol, Polyethylene, Polyglyceryl-10 Distearate, Argania Spinosa Kernel Oil, Beeswax, Glyceryl Stearate, Polyacrylamide, Hematite Extract, PEG-40 Stearate, PEG-100 Stearate, C13-14 Isoparaffin, Spent Grain Wax, Cetearyl Glucoside, Betaine, Dimethicone Crosspolymer, Saccharomyces Cerevisiae Extract, Pentylene Glycol, Laureth-7, Tocopheryl Acetate, Stearyl Glycyrrhetinate, Lecithin, Sclerotium Gum, Disodium EDTA, Hesperidin Methyl Chalcone, Fragrance, Arginine, Adenosine, Acrylates/C10-30 Alkyl Acrylate Crosspolymer, Steareth-20, Phenoxyethanol, Chlorhexidine Digluconate, Rhodiola Rosea Root Extract, Tocopherol, Dipeptide-2, Potassium Sorbate, Sodium Oleate, Hydrogenated Lecithin, Palmitoyl Tetrapeptide-7, Copper Tripeptide-1, rh-Polypeptide-1, rh-Oligopeptide-1.


text:
Speedy 28mm Lined Passing Rings Chrome
Speedy 28mm Lined Passing Rings Chrome
Save 22% RRP: £11.49
Save 22% RRP: £11.49
- Finish: Chrome
- Diameter: 28mm
- Pack Quantity: 8
- Delivery: 2 to 3 working days
The 28mm lined bay pole passing rings antique brass are ideal for that extra support. The plastic lining inside the ring offers a smooth and silent glide across the curtain pole. The lined feature gives the rings a luxurious characteristic. Purchased in a pack of 8. Also available in antique brass, satin silver and polished graphite.
This product can be found in:
Standard delivery: £7.95
FREE delivery on orders over £100
Delivery is usually within 2 to 3 working days.
Prices above are for mainland UK only
Scottish Highlands and Islands £19.50
Isle of Man: £24.50
Northern Ireland: £24.50
Channel Islands: £24.50