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Conceived and designed the experiments: JCL. Performed the experiments: NB-P JCL KAF. Analyzed the data: JCL. Contributed reagents/materials/analysis tools: APD SSW JYB BDK. Wrote the paper: JCL.
The four dengue virus serotypes (DENV-1–DENV-4) have a large impact on global health, causing 50–100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4+ cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (p<0.0001 for all cytokines). No statistically significant changes were observed at any time-points for IL-10 (p=0.19), a regulatory cytokine, indicating that the response to DENV-1 was primarily proinflammatory in nature. We also observed little T cell cross-reactivity to the other 3 DENV serotypes. The percentage of multifunctional T cells (T cells making ≥2 cytokines simultaneously) increased with time post-DENV-1 exposure (p<0.0001). The presence of multifunctional T cells together with neutralizing antibody data suggest that the immune response generated to the vaccine may be protective. This work provides an initial framework for defining primary T cell responses to each DENV serotype and will enhance the evaluation of a tetravalent DENV vaccine.
40% of the world's population is at risk for developing dengue fever, an acute febrile illness caused by the 4 serotypes of dengue viruses (DENV). Though most of the 50–100 million annual DENV infections resolve without medical intervention, approximately 500,000 cases are severe and require hospitalization. Supportive care is currently the only available treatment for dengue disease. As a result, DENV infections cause strain on healthcare systems and economic burden in endemic countries. Much of the research in the dengue field has focused on understanding the mechanism of severe dengue disease. To better understand human adaptive immune responses to asymptomatic or mild DENV infections, we used longitudinal specimens collected following low dose vaccination with a live DENV-1 candidate vaccine. We found that CD4+ T cells made the proinflammatory cytokines, IFNγ, TNFα and IL-2, 3 weeks following exposure to DENV-1. IFNγ and TNFα production continued for 6 weeks post-vaccination, our final time-point. T cell responses were predominantly multifunctional: T cells produced ≥2 cytokines simultaneously. Lastly, we observed little cross-reactivity in T cell responses. This work helps establish the kinetics and characteristics of a primary adaptive immune response to DENV and aids in the development of a tetravalent vaccine against DENV.
Infections caused by the 4 serotypes of dengue virus (DENV-1- DENV-4) are significantly contributing to the increasing burden of vector-borne diseases globally [1]. Approximately 2.5 billion individuals living in >100 countries are currently at risk for acquiring DENV infection [1], [2]. The 4 DENV serotypes are endemic throughout the world's subtropical and tropical regions, especially in Asia and Latin America. DENV is also a re-emerging threat to areas in which it was previously controlled, as exemplified by recent 2009–2010 outbreaks in Florida [3]. Clinical disease ranges from the debilitating, but generally self-limited, dengue fever (DF) to life-threatening severe dengue disease which includes dengue hemorrhagic fever and dengue shock syndrome (DHF and DSS) [4].
Secondary infections, caused by a subsequent DENV infection in children and adults with a different DENV serotype than the primary infection, are associated with increased risk of severe disease (DHF/DSS) and are prevalent in endemic regions [5], [6]. Consequently, most studies have focused primarily on secondary DENV infections and comparisons of DF and severe disease rather than immune responses to primary infections. As safe candidate DENV vaccines are developed and introduced into naïve and endemic populations, a detailed understanding of the human immune response to both primary and secondary DENV infections is a necessary component of this effort. In particular, distinction is needed between post-infection immune responses which are beneficial or protective (including a correlate of protection) versus those leading to immunopathogenesis and DHF/DSS.
Two separate, but not mutually exclusive, hypotheses have been proposed to explain the immunopathogenesis of severe dengue disease: antibody-dependent enhancement (ADE) and cross-reactive memory T cells [7], [8], [9]. In the ADE hypothesis, cross-reactive antibodies from a prior infection increase the uptake of DENV by Fc-receptor-bearing cells but are incapable of fully neutralizing the virus [7]. Increased viral loads then trigger host inflammatory cascades and increased vascular permeability resulting in DHF/DSS. Notably, higher viral loads are correlated with severe dengue disease [10], [11]. The second hypothesis postulates that cross-reactive memory T cells from a previous DENV infection are preferentially activated over naïve T cells, producing an altered cytokine response that leads to a cytokine storm and ultimately ends in plasma leakage and DHF/DSS [12].
Complicating this picture is the observation that the 4 serotypes of DENV appear to be associated with distinct clinical and epidemiologic patterns, suggesting that each serotype varies in virulence and immunopathogenesis [13], [14], [15], [16], [17], [18], [19], [20]. The sequence of infection may also be important. In Cuba, for example, DENV-1 primary infections that were followed by DENV-2 or DENV-3 infections were highly associated with DSS [15], [16], [17]. Based on these observations, the primary immune responses may affect subsequent immune responses to different DENV serotypes. Thus, in order to understand DENV immunopathogenesis, we must understand the primary immune response to each DENV serotype.
Infection with live, attenuated monovalent DENV viruses offers a unique opportunity to define the precise kinetics of viral events for a known dose, serotype and genotype of DENV in a Flavivirus-naïve population that is not achievable in a natural setting. In natural DENV infections, evaluation of immune responses is complicated by an unknown date of infection and inoculum size. Prior DENV exposure history is often unknown or difficult to discern. Asymptomatic DENV infections are very difficult to capture. Furthermore, in endemic regions, multiple DENV infections are common making it impossible to study the precise effects of each serotype. Live monovalent DENV vaccines can be used as a model for asymptomatic or very mild DENV infection. Using longitudinal specimens from DENV-1 low dose vaccinees, we sought to better define the kinetics of the host cellular response and to evaluate the importance of serotype cross-reactivity. Our results suggest that multifunctional T cells that produce proinflammatory cytokines may facilitate control of DENV replication or clearance. Taken together, our data contribute to the understanding of the immune components that lead to recovery and protection from DENV infection.
Fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA); ACCUSPIN with Histopaque 1077 (A0561), Cell Freezing Medium (C6164), phorbol 12-myristate 13-acetate (PMA), β-mercaptoethanol and brefeldin A from Sigma-Aldrich, Inc. (St. Louis, MO); Hank's Balanced Salt Solution without Ca2+ or Mg2+ from HyClone (Logan, UT); all other reagents were purchased through Fisher unless specified. MEM GlutaMAX, phosphate buffered saline (PBS, pH 7.4), Staphylococcus enterotoxin B (SEB) [21], DNAase, RPMI 1640, and Live/Dead Blue were purchased from Gibco Invitrogen (Carlsbad, CA); and all antibodies and flow cytometry reagents were purchased from Becton Dickinson (Franklin Lakes, NJ).
Informed consent was obtained in writing from all subjects, and all work with human subjects was reviewed and approved by the University of Vermont Committees on Human Research prior to initiation of clinical studies.
Peripheral blood mononuclear cells (PBMCs) and sera were collected from healthy adults participating in a phase I trial of a live, attenuated DENV-1 candidate vaccine. All volunteers had been screened by plaque reduction neutralizing titer assay (PRNT) and were found to be naïve to DENV, West Nile, St. Louis encephalitis, yellow fever virus, and Japanese encephalitis virus exposure. Volunteers were vaccinated with a single, subcutaneous 10 plaque forming units (PFU) dose of the live, attenuated dengue serotype 1 vaccine (DEN1Δ30). Vaccine dosage of 10 PFU was confirmed by titering vaccine virus immediately following administration of vaccine. Vaccine construct, as well as standardized assays to measure neutralizing antibody responses and viremia post-vaccination were performed as described [22], [23]. Table 1 describes the clinical signs, symptoms and PRNT60 for the volunteers in this study as previously reported (Lindow, manuscript in preparation). PBMCs were separated within 2 h of blood draw using the ACCUSPIN System (Histopaque 1077). Following gradient separation, cells were washed three times with Hank's Balanced Salt Solution without Ca2+ or Mg2+, and cryopreserved in Cell Freezing Media (Sigma) in liquid nitrogen. Cells from a total of 12 vaccinated volunteers were used in this study.
Vero cells (WHO, a gift from S. Whitehead) were maintained using standard methods. Briefly, Vero cells were grown in MEM GlutaMAX (Minimum Essential Medium with glutamine, Invitrogen) plus 2% fetal bovine serum (FBS) at 37°C, 5% CO2. Inactivated DENV-infected Vero cell antigen was prepared as described previously [24]. Uninfected Vero cells were treated identically and used as negative controls for all experiments. Vero cells (85–90% confluent) were infected with DENV-1 (rDEN1 WP), DENV-2 (NCG Prototype), DENV-3 (Sleman/78) or DENV-4 (rDEN4) at an MOI=0.01–0.25 and cultured in MEM GlutaMAX+2% FBS until >50% of the Vero cells were visibly cytopathic. Monolayers were harvested and the supernatant cleared by 6 min of centrifugation (1350xg) at 4°C. Cells were washed 2 times with phosphate buffered saline (PBS) (pH 7.4). The pellet was resuspended in PBS, fixed for 15 min with 0.025% gluteraldehyde at 4°C, washed 3 times with PBS, and resuspended in RPMI 1640+L-glutamine+10% FBS at 1/40 the original volume. Antigen preps were then sonicated on ice using a Fisher Scientific Sonic Dismembrator Model 100 (6, 30 s cycles at 60% power). Preps were centrifuged for 10 min at 4°C at 1350xg, the supernatant was aliquotted, and frozen at −80°C. Antigen was thawed immediately before use. Antigen was titrated for reactivity on DEN1Δ30 vaccinee samples. Briefly, each DENV antigen and corresponding Vero prep were tested on PBMCs specific to each DENV serotype at 110, 120 and 140. DENV1 was also tested at 1100. The dilution resulting in the highest DENV-specific stimulation relative to Vero background was selected as the optimum dilution (data not shown). Time courses were also performed to determine the optimum stimulation conditions similar to those previously published [25]. Similar preparations were made for DENV-2 (NGC Prototype), DENV-3 (Sleman/78) and DENV-4 (rDEN4, S. Whitehead). A single preparation of antigen was used for all assays.
All assays were performed on a BD LSR II 4 laser flow cytometer. Thawed PBMCs were washed twice in 37°C cRPMI-10 (RPMI 1640+10% FBS)+2.7 U DNase/106 cells/ml. Cells were resuspended at 5–10×106/ml and rested for 6–18 h. Cells were washed once in warm c-RPMI-10. PBMCs were not used unless they had ≥86% viability following thaw and ≥90% viability following rest. The rest period allows dying cells to complete apoptosis prior to initiating the assay. The following was added to ~1×106 cells in wells containing 10 µg/ml brefeldin A (BFA) and anti-CD28/CD49d (1 µg/ml each) for 12 h: 140 DENV-1 whole cell antigen, 120 DENV-2 antigen, 140 DENV-3 antigen, 140 DENV-4 antigen, 140 Vero antigen (negative control), 120 Vero antigen (for DENV-2 only), or 3 µg/ml SEB (positive control). (As little antigen as possible was used as it autofluoresces in the FITC and APC channels, and had to be gated out using unstained cells treated with antigen.) After stimulation, cells were removed from culture plate for 10 min with 2 mM EDTA then washed twice with 1 ml PBS. Viability of cells was determined by staining with 11000 Live/Dead Blue surface stain (Invitrogen) for 30 min at 4°C in the dark. Cells were washed in 1 ml PBS, fixed for 10 min at room temperature (RT) using BD FACS Lysing Solution, then washed twice with wash buffer 1 (WB1=PBS+1% FBS+0.1% NaN3). Cells were then permeabilized for 10 min at RT using 250 µl BD FACS Permeabilization Solution 2. Cells were washed twice with WB2 (110 BD Perm/Wash Buffer) followed by staining with the following cell surface markers and cytokines: CD3+ PerCP Cy5.5 (UCHT1), CD4+ V500 (RPA-T4), CD8+ V450 (RPA-T8), IFNγ Cy5.5 (B27), TNFα FITC (6401.1111), IL-2 PE (5344.111) and IL-10 APC (JES3-19F1). Stained cells were washed twice with WB2 and run the same day on a BD LSR II. Compensation was performed using beads prepared according to manufacturer's protocol (Becton Dickinson). Fluorescence minus one (FMO) experiments were run on multiple individuals as gating controls. 300,000 events were collected for a majority of time-points, with a minimum of 175,000 events for any time-point analyzed, and all with lymphocyte viabilities ≥90%.
Data were analyzed using FlowJo version 9.3.1 (TreeStar) and SPICE v.5.22Beta software [26]. Boolean gating was used to determine simultaneous cytokine production in cells. For each of the cytokines, a positive response is defined as the difference between the DENV-antigen stimulated cells and the background (uninfected Vero control). The fold-increase of antigen-stimulated cells divided by Vero-stimulated cells (background) was statistically higher on days 21, 28, and 42 for IFNγ and TNFα (p<0.05) and days 21 and 28 for IL-2 (p<0.01). All wells produced ≥3 cytokines when stimulated with SEB. PMA-ionomycin stimulated cells produced all 4 of the analyzed cytokines. Increasing CD3+ and CD4+ gate sizes did not alter the results shown.
Cytokine data was analyzed following background subtraction (the identical cell population treated with uninfected Vero prep) from the DENV stimulated sample. The fold increase of signal over background was also analyzed for all cytokines at all time-points. A single-group repeated measures analysis of variance was used to examine cytokines or multifunctional T cells produced at each time-point relative to pre-vaccination. Additionally, differences in multifunctional T cell production between those who did and did not develop viremia were tested using a two-group repeated measures analysis of variance. Repeated measures analyses were performed using SAS, version 9.2; Wilcoxon rank sum tests were computed using SPICE v.5.22Beta. p values≤0.05 were considered significant.
To evaluate cellular immune responses following infection with DENV-1, we collected peripheral blood mononuclear cells (PBMCs) at pre-vaccination and at weekly time points post-vaccination (days 8,14,21,28,42) from the 15 Flavivirus-naïve volunteers vaccinated with 10 plaque forming units (PFU) of the live, attenuated DENV-1 vaccine, rDEN1Δ30, and 2 placebo recipients. A full description of clinical outcomes is described elsewhere (J Lindow, manuscript in preparation). We observed low-level DEN1Δ30 viremia (0.5 log10/ml serum), starting at study day 10 post-vaccination, in 8/15 (53%) vaccinees, as determined by a standardized viral amplification assay. Two individuals experienced mild rash, and 14/15 volunteers (93%) seroconverted to DENV-1, as defined as ≥4-fold increase in PRNT60 (60% of virus is neutralized by antibodies) to wild type DENV-1 on post-vaccination days 28 or 42 compared to the pre-vaccination titer (day 0). We did not detect any other clinical signs or symptoms such as leukopenia or elevated liver enzyme levels in any of the volunteers. 12/15 subjects, 6 viremic and 6 non-viremic, along with 2 placebo recipients were randomly selected for phenotypic responses of T cell responses (Table 1). All 12 subjects seroconverted to DENV-1.
To evaluate the kinetics of the T cell cytokine responses following DENV-1 primary infection, we stimulated PBMCs collected from a pre-vaccination time-point (day 0) and 5 post-vaccination time-points (days 8, 14, 21, 28, and 42) with either wild type DENV-1 antigen or a negative control antigen (Vero cells). The DENV-1 antigen contains structural and non-structural proteins and therefore allows an unbiased, global analysis of T cell responses. Using an 8-color flow cytometry panel, we then measured the number of CD3+ CD4+ or CD3+ CD8+ T cells producing cytokines that have been implicated in natural dengue disease: IFNγ, TNFα, IL2, and IL10 [27], [28], [29], [30]. Representative gating of CD4+ T cells from DENV-1-stimulated cells (Subject 03, day 28) is shown in Figure S1.
CD4+ T cell responses for pre- and post-vaccination time-points for DENV-1 vaccinees are summarized in Figure 1. All 3 proinflammatory cytokines, IFNγ, TNFα and IL-2, were produced by CD4+ T cells 3 weeks after DENV-1 infection (Figure 1A–C). More IFNγ+ CD4+ and TNFα+ CD4+ T cells were present on days 21, 28 and 42 relative to day 0 (pre-vaccination) (p<0.0001 for analysis of differences over time; p<0.05 and p<0.01 for days 21–42 versus day 0 for IFNγ and TNFα, respectively) (Figure 1A and B). Significantly more IL-2+ CD4+ T cells were also observed on days 21 and 28 relative to day 0 (p<0.0001 for differences over time; p<0.01 for days 21 and 28) (Figure 1C). The timing of these T cell responses occurred 7–11 days after the onset of detectable viremia based on the 6 viremic subjects tested. Of particular interest, we did not observe any significant trends in the production of IL-10, a regulatory cytokine (p=0.19) (Figure 1D).
We observed little or no stimulation of CD8+ T cells using this DENV-1 antigen (data not shown). This is similar to what other groups have observed using this type of DENV antigen [22], [24].
Multifunctional T cells (T cells making ≥2 cytokines simultaneously) have been correlated with both protection from disease and lessening of progression of disease for a variety of human pathogens including HIV, Leishmania major, and Mycobacterium tuberculosis [31], [32], [33], [34], [35]. We Boolean gated the CD4+ T cell cytokine responses to determine the breadth of the T cell response following DENV-1 infection. We observed that multifunctional T cells increased with time; on days 21–42 we observed a significant increase in the number of multifunctional T cells compared to days 0–14 (p<0.0001) (Figure 2A and B). To determine the abundance of multifunctional T cells relative to T cells producing only 1 of the 3 proinflammatory cytokines, we summed the total number of multifunctional T cells producing each of the cytokines for a particular day and divided the sum by the number of T cells making only that cytokine alone. We found that there were 3.8-fold, 5.1-fold, and 5.0-fold more multifunctional T cells making IFNγ+, TNFα+, and IL-2+, respectively, compared to single cytokine producing T cells (Figure 2A). We observed similar increases on days 28 and 42 (Figure 2A).
We next wanted to analyze each possible T cell phenotype (8 possible with 3 cytokines) to determine whether specific combinations of cytokines were produced at specific times post-DENV-1 infection. To this end, we plotted the percentage of CD4+ T cells producing a particular combination of cytokines for each of the 6 time-points (days 0, 8, 14, 21, 28, and 42) as shown in Figure 2B. This analysis revealed that there is a marked increase in IL-2+ IFNγ+ TNFα+ CD4+ T cells (triple producers) 3 to 6 weeks post-vaccination (days 21–42), with significant increases at days 21 and 28 relative to day 0 (p=0.005 and p=0.049, respectively) and day 42 approaching significant difference from day 0 (p=0.055) (Figure 2B). While T cells simultaneously producing all 3 cytokines were relatively abundant, we also found significant numbers of CD4+ T cells making different combinations of 2 cytokines(IFNγ+ TNFα+, TNFα+ IL-2+, and IL-2+ IFNγ+) on days 21–42 relative to pre-vaccination (day 0) (p≤0.023) (Figure 2B).
To better understand how each of the 8 different T cell subsets changed over time, we generated pie charts for each day, with each slice representing the proportion of a specific T cell subset compared to the entire T cell population. The pie charts show that multifunctional T cell subsets increase as a function of time, emerging as early as day 14 (Figure 2C). Further, the pie charts show that T cells making only 1 cytokine (red, green, or blue) represent a smaller fraction of the total T cell population, especially at later time-points (days 21–42) (Figure 2A and C). Together these data show that the proinflammatory CD4+ T cell response to DENV-1 is largely multifunctional and lasts a minimum of 6 weeks post-infection.
53% of DEN1Δ30 vaccinees developed low level viremia (0.5 log10 PFU/ml) 10–14 days post-infection. To test whether individuals who developed viremia had a delayed or less robust T cell response, we compared CD4+ T cell cytokine responses from the 6 individuals who developed viremia to 5 individuals who did not, at all time-points. We were not able to collect ≥250,000 events for 1 viremic subject past day 8 so we excluded the subject from this analysis. We found no significant differences in the pattern and/or magnitude of individual cytokines produced over time by CD4+ T cells between viremic and non-viremic subjects (p≥0.13 for each cytokine). However, we observed that on day 28, non-viremic subjects were trending toward having elevated multifunctional T cells relative to viremic subjects (p=0.064) (Figure 3A and B).
One possible mechanism for increased disease severity upon exposure to a new, heterologous DENV serotype in a secondary infection is that memory T cells from a primary DENV infection are activated and produce an altered, low affinity response to the infecting DENV serotype that is insufficient to clear the new infection. To determine whether cross-reactive T cells were generated following DENV-1 infection, we stimulated PBMCs from 2 subjects with each of the 4 DENV serotypes separately (Figure 4). Fewer CD4+ T cells responded to heterologous antigens compared to DENV-1, thus, the cytokines produced were of lesser magnitude than the DENV-1 response, were observed at later time-points (day 28 or day 42) (Figure 4). Neither subjects' T cells produced IFNγ significantly above the day 0 value in response to heterologous DENV stimulation (Figure 4 A, E). Both subjects showed a very minor response to DENV-4 with production of TNFα at day 42 (Subject 1) or day 28 (Subject 2), and 1 subject responded to DENV-2 (Subject 2) by making small amounts of TNFα. Again, all responses were well below those observed for the homologous DENV-1 antigen. IL-2 was produced on day 42 in Subject 1 when stimulated with DENV-4 while Subject 2 responded similarly, though to a lesser extent, to DENV-2 and DENV-3 on day 28. Lastly, a robust IL-10 response was observed in Subject 1 when stimulated with DENV-2 on days 21 and 42, but not day 28. Subject 2 produced minor amounts of IL-10 when stimulated with DENV-3 or DENV-4. Together, these results show that the level of cross-reactivity in the T cell responses is relatively low, at least in comparison to the response to homologous DENV-1 antigen in a small number of subjects.
We demonstrate that a DENV-1-specific CD4+ T cell response with specific kinetics was elicited in Flavivirus-naïve, healthy adults following vaccination with a low dose (10 PFU) of live DEN1Δ30 virus. The observed cytokine response was proinflammatory; IFNγ, TNFα and/or IL-2 production became evident 3 weeks following vaccination. We did not detect significant amounts of IL-10, a regulatory cytokine, at any particular time post-vaccination. We also noted no or low levels of DENV serotype T cell cross-reactivity in a small number of subjects. Lastly, we found that a majority of the cytokines were produced by multifunctional T cells (≥2 cytokines produced simultaneously). As has been observed in other infectious diseases, the presence of multifunctional T cells following DENV-1 vaccination suggests that live, attenuated DENV vaccines safely and effectively prompt immune responses associated with control of infection and protection from re-infection.
In natural primary DENV infections, the precise timing of infection cannot be determined, but is generally 3–14 days prior to disease onset, and disease symptoms last approximately 1 week [36]. DENV-specific T cells have been observed 0–5 days prior to defervescence, when viremia levels were dropping or undetectable [9], [37]. In our vaccine platform, low level viremia (0.5 log10 pfu/ml serum) occurred 10–16 days after vaccination in 6 subjects, but DEN1Δ30 virus was undetectable in the other 5 subjects, though all seroconverted to DENV-1. Statistically significant CD4+ T cell responses to DEN1Δ30 vaccination were measurable 21 days following vaccination and pro-inflammatory cytokines remained elevated for at least 6 weeks, the latest time-point samples were available. In the 6 subjects with viremia, which occurred about 2 weeks post-vaccination, the T cell responses occurred later than expected and were only measurable 5–11 days after the onset of viremia. Similar kinetics of T cell responses were observed in studies in macaques: T cell responses were evident ~20 days following the peak of viremia (days 3 to ≥11) in macaques inoculated with a high dose tetravalent DENV vaccine, DENVax [38]. Given these similarities, we suspect that the increased length of time required to elicit a measurable T cell response is a function of the attenuated DEN1Δ30 virus or inoculum size. The live-attenuated DEN1Δ30 vaccine candidate has decreased replication kinetics, which may slow the spread of the virus, resulting in lower antigenic loads and delayed or muted T cell responses.
The role of proinflammatory cytokines in the immunopathogenesis of DENV is a research area of intense interest. Proinflammatory cytokines such as TNFα, IFNγ, and IL-6 have been associated with severe secondary dengue disease in several studies [27], [28]. Far less is known about the role of these cytokines in primary disease, and in particular, mild or asymptomatic disease. In a study that included primary natural DENV infections, higher percentages of T cells (CD4+ and CD8+) produced IFNγ and/or TNFα in DHF versus DF patients during the acute phase of disease [39]. However, a recent study demonstrated that the pro-inflammatory cytokines, TNFα, IFNγ and IL-2, were produced by T cells (CD8+ and CD4+) at higher levels in individuals who subsequently developed subclinical secondary DENV infections versus symptomatic infections [40]. Human challenge studies conducted with under-attenuated DENV vaccines also correlated sustained levels of IFNγ produced by PBMCs with protection from re-challenge with the same viruses [41]. These studies indicate that the production of proinflammatory cytokines may be part of a continuum between asymptomatic infection and pathogenesis.
Understanding the characteristics of T cells, including multifunctional T cells, involved in protection from DENV is important for assessing vaccine immunogenicity, safety, and efficacy. In other disease models, extensive work has focused on the role of multifunctional T cells, both CD4+ and CD8+, and protection from disease or lessening of disease severity in a variety of infections: HIV, tuberculosis, malaria, leishmaniasis, influenza, and vaccinia [31], [32], [33], [34], [35], [42], [43], [44]. We observed significantly increased numbers of multifunctional CD4+ T cells by day 21 of infection, and these largely remained present through day 42. While we do not have data past 6 weeks to determine the duration of the multifunctional responses, our initial results parallel other models, and offer the first suggestion that multifunctional T cells may be associated with protective responses.
We observed a trend of more multifunctional T cells in non-viremic vaccinees relative to viremic vaccinees. While these data did not reach statistical significance because of the limited number of available subjects, they suggest that multifunctional CD4+ T cells may be indicators of individuals who are better able to control DENV infection, and therefore may have less severe clinical disease. This is consistent with findings from HIV studies: individuals with lower viral loads have larger percentages of multifunctional T cells, in particular IFNγ+ TNFα+ IL2+ (triple producers) or IFNγ+ IL-2+ (double producers) CD4+ T cells, while those who have higher viral loads and more severe disease progression produce greater numbers of IFNγ+ CD4+ T cells [45], [46], [47], [48]. However, HIV causes a chronic infection while DENV results in an acute infection. This difference might affect the nature of the T cell response. More work is clearly required to discover whether the correlation between levels of viremia for DENV and production of multifunctional T cells is a reason some individuals may develop more severe DENV disease. Of note, no significant differences in neutralizing antibody titers were observed between the viremic and nonviremic subjects or high (Subjects 2, 3, 5, 6, 8, 14) and low (Subjects 4, 7, 10, 12, 18) T cell responders; all groups generated robust antibody responses. Future work should also address whether memory T cell responses maintain specific multifunctional properties or reduce the number of cytokines produced, and whether memory T cell populations are correlated with neutralizing antibody outcome.
The role of IL-10 in DENV disease is still contradictory as it has been correlated with severe dengue disease, despite having multiple regulatory functions that aid in DENV clearance [30], [49]. In general, IL-10 is a regulatory cytokine, produced by CD4+ T cells, activated CD8+ T cells, and activated monocytes, that can reduce IFNγ-related antiviral effects [50], [51], [52], [53]. We detected little to no IL-10 production at each specific time-point suggesting that it is either not produced at the times sampled following DENV-1 inoculation or our flow assays did not capture the cell population producing it. The role of IL-10, if any, following natural DENV exposure or vaccination therefore requires further investigation.
Our work also contributes to the understanding of memory T cell cross-reactivity following DENV infections, which is thought to contribute to increased disease severity [8]. While we observed small increases in the number of cross-reactive CD4+ T cells producing TNFα, IL-2 or IL-10, especially on day 42, the magnitude of the responses were much lower than those observed for homologously stimulated T cells (stimulated with DENV-1 antigen). Similar to the rationale outlined above for delayed T cell responses, the low dose and/or attenuation of the infecting DENV-1 vaccine strain may decrease the antigenic load affecting the overall T cell response, including cross-reactivity. Higher viral loads, as seen in natural exposure, may be required to activate lower affinity T cells such as memory T cells, which cross-react to a different DENV serotype [8]. Alternatively, the difference between our data and other reports could be the result of the small subject numbers or differences in study populations. The issue of T cell cross-reactivity is important to clarify as it potentially affects dengue vaccine safety in Flavivirus-experienced populations. Future work will continue to address whether the dose of virus affects the development of cross-reactive T cell responses and whether cross-reactivity varies amongst populations.
Although CD4+ T cells play an important role in resolving of DENV infections, CD8+ T cells are active participants in clearing DENV infections [54], [55], [56], [57]. We likely did not observe robust CD8+ T cell responses because the inactivated DENV antigens we used are not ideal antigens for CD8+ stimulation from frozen PBMCs which are not able to process full-length antigens efficiently [40]. Low CD8+ responses have been observed using this type of antigen by other groups [24], [25]. Future work using specific DENV Class I peptides will help define the role of CD8+ in the overall T cell response to monovalent live vaccines.
Longitudinal DENV vaccine specimens provide a unique opportunity to determine T cell profiles in primary, mild DENV infections in a controlled, clinical setting. Further analysis using these and other specimens from DENV monovalent vaccine clinical trials will enable us to determine whether there are differences among serotypes, since each DENV serotype may stimulate a unique cytokine production pattern in T cells. We can also test the effect of antigenic load on the immune response using specimens from different doses of the same vaccines. These are nearly impossible goals to achieve in natural settings as the time of infection and infectious dose cannot be determined, and sometimes even the infecting serotype is difficult to determine due to cross-reactive immune responses. To best determine the role of T cell responses in protection, future work will need to compare specimens from controlled settings to field-setting specimens from individuals with known clinical outcomes. A detailed understanding of a protective versus aberrant immune response following DENV infection will greatly enhance the development and evaluation of an efficacious tetravalent DENV vaccine.
CD4+ T cells respond to DENV-1 antigen. Gating scheme and raw representative data of PBMCs stimulated with DENV-1 antigen for Subject 03 on post-vaccination day 28. Cells are initially gated on singlets followed by the lymphocyte population. CD3+ are gated from the lymphocyte population after selecting for live cells. CD4+ and CD8+ are separated from the CD3+ population. CD4+ CD8− cells are gated for IFNγ, TNFα, IL-2 and IL-10 based on negative controls (bottom panel) and FMO controls. The percent positives are determined by subtracting the negative control values from the corresponding DENV-1 stimulated cell. All DENV-1-specific cytokine signals were statistically higher than the negative control for days 21, 28, and 42 excepting IL-10 (p<0.05).
We thank Anuja Mathews for technical assistance and Iori Ueki for assistance with flow assays. We also express our gratitude to the many volunteers, nursing and clinical staff who have made this work possible.
The authors have declared that no competing interests exist.
This work was supported by contract HHSN272200900010C, the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Disease, and COBRE P20RR021905. The COBRE P20 award is described at: http://grants.nih.gov/grants/guide/pa-files/PAR-09-079.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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- Colored: No
- Brand Name: TOP-PRINT
- Model Number: TP-TN660
- Place of Origin: Guangdong, China (Mainland)
- Service: To Be Top of Your Printing Solutions!
- OEM/ODM: Available
- Payment terms: L/C,T/T,Western Union
- Warranty: 18 months for toner cartridges
- Page yield@5% coverage: 2600pages
- Sutable working humidity: 30-80
- Suitable working temperature: 10-35
- Certification: ISO9001/ ISO14001/RoHS/REACH/CE/STMC
- Defetive rate: <1% for Black, <2% for Color
- Quality: Grade A & customized standard
Packaging & Delivery
|Packaging Details
|1. Neutral Packaging (airbag, white or color box), Top-print Package, Customized Packaging and others
2. Packing Dimension: 53*33.5*31CM, 10pcs/CTN
3. Mode of transport: 1) 20GP: approx 4500 - 5000 pieces; 2) 40GP: approx 8000 pieces; 3) 40HQ: approx 10000-11000 pieces.
|Delivery Time
|7-15 working days upon receipt of payment.
SpecificationsCompatible Toner Cartridge for TN660
Environmental Protection
Fast delivery
Flexible payment
New arrival! New Compatible toner cartridge Brother TN660/2350/2360/2310/2320/630 Black Toner Cartridge
|Model Number:
|Compatible Brand:
|Suitable for:
Brother HL-L2300D/HL-L2320D/HL-L2340DW/HL-L2360DW
|Page yield(5% coverage):
|18 months after shipping date
|Defective rate:
|Black toner cartridge: < 1%, Color toner cartridge: <3%
|Payment terms:
|T/T,Western union, L/C, etc.
|Trade terms:
|EXW, FOB, CIF, etc.
|Packing Dimension:
|53*33.5*36CM, 10pcs/CTN
Factory show:
Quality Control:
All products must go through three checks before manufacturing process.
We have professional quality control and R&D department to strictly manufacture each product.
Shipment: By sea for bulk order, by air(airport to airport), express(door to door) for small order or samples.
1. We can guarantee 18 months after delivery.
2. Our products are 100% on line tested before shiping.
3. The defective rate is less than 1% and with long-term technical support. If more than 1%, test pages or samples will be needed to send us for engineer analysing in case the problem caused by Top-print, the defective one can be retured to factory or our abroad warehouse for replacement 1:1.
4. Any inquiry and claims will be reply in 12 hour since receiving your information.
5. Sample, OEM, ODM service is available.
6. Fast delivery and Flexible payment terms. We have flexible production line to meet customers' urgent demand.
7. Industry and trade integration business.
8. High quality toner powder and Top quality OPC drum.
WELCOME TO VISIT TOP-PRINT, A HIGHLY PROFESSIONAl AND RELIABLE MANUFACTURER OF PRINTER CONSUMABLES IN CHINA.
1. We are a professonal manufacturer of toner cartridges which is range from HP, Canon, Samsung, Xerox, Brother, Lexmark, Dell, Epson, Konica Minolta, Ricoh, OKI, etc.
2. With the production capactiy of over than 500 thousand pieces each month.
3. More than 15 years experience on OEM toner cartridge service. and covers an area of 30,000 square meters.
4. With about 1000 employees in the factory. We have excellent R&D ability and experienced R&D team, and professional quality control.
5. With warehouses in DUBAI and Los Angeles to save time for your order and offer better after-sale service.
6. We have to go through 3 times strict testing before delivery.
7. With ISO9001, ISO14001, EMS, STMC, CE Certification.
8. We can guarantee 18 months after shipping date.
Why choose TOP-PRINT?
1:1 Replacement Guarantee: Any defective products or quality problem which cause by factory can be compensated in the next order or refund.
High quality toner powder
Top quality OPC drum
You are our No. 1 priority!!! Customer satisfaction is our top priority.
What is the necessary we do is to provide best service for you!
Our prices are very competitive! Contact us to get your pricelist.
Basic knowledge
1. Please do not turn over the OPC freely after the new toner cartridge took out from the box .And to prevent it collide with hard objects.
2. Keep the toner cartridge away form water, moisture, radiation and long time exposed in the light.
3. When the laser printer can not operatenormally or the cross white line influence the prints seriously, it owe to the toner cartridge installed improbably. You can remove and reload it.
4. If there is a little print quality problems , please print a few more pages to solve it.
5. When laser printer alerts" Change toner Cartridge" orthe prints turn out vertical white unevenly, take out the toner cartridge and shake it for 2 to 3 times. It can make full use of the toner.
Warm Notice
If you are interested in our products, please Add us to Inquiry Cart or Add to My Favorites or share to Facebook, Twitter, which is on the top of the page to make it easily to find us. If you are in need it one day in the future, please feel free to contact with us. We do appreciate it for your kindness!
- Contact person: Ashley He
- Skype: ashleyhe19880713
- Mobile: 86-18675618435
- Tel: +86 756 6116333 EXT:1038
- Fax: +86 756 6116698

subset: commoncrawl


text:
Zoeken naar een online exemplaar
Links naar dit item
cdm15932.contentdm.oclc.org Digital reproductions available online.
Zoeken naar een in de bibliotheek beschikbaar exemplaar
Bibliotheken met dit item worden gezocht…
|Manuscript collection
Sound recordings
|Genoemd persoon:
|James Earl Chaney; Andrew Goodman; Jan Hillegas; Phil Lapsansky; Kenan Barrett Lawrence; Michael Henry Schwerner
|Soort document
|Archiefmateriaal, Internetbron
|Alle auteurs / medewerkers:
|Freedom Information Service.
|0.4 c.f. (1 archives box), 3 reels of microfilm (35 mm), and 1 tape recording; plus 0.1 c.f. of additions.
|Meer informatie
Included are materials written and printed by FIS, and items created by allied Mississippi civil rights groups, all of which were distributed by the Service. In the collection are freedom primers; political handbooks concerning county and municipal government; leaflets and flyers on a variety of political, economic, and agricultural subjects; news releases; and a small file of correspondence and printed form letters. There are also copies of the proposal for organizing FIS, and a prospectus for later expansion; with small files regarding the Mississippi Freedom Democratic Party and the Student Nonviolent Coordinating Committee (SNCC).
From the 1970s are records created and collected by Jan Hillegas and Ken Lawrence, some of which were produced under the aegis of FIS. These later documents concern women's rights and feminism, the Republic of New Afrika, farm workers and labor, and racism in the military. On microfilm are papers and clippings which pre-date the organization of FIS, but which were deposited for use by FIS by the Council of Federated Organizations. These materials include WATS reports; clippings, statements, and news releases concerning the disappearance and murder of Michael Schwerner, James Chaney, and Andrew Goodman in 1964; and similar documentation of incidents of brutality and harassment. These items are mounted on printed COFO news releases and other papers, which are also of interest.
Also in the collection is "A Visit to Sunflower County, Mississippi," which was the home of Senator James O. Eastland, a recording made by FIS head of news and tape programs, Phil Lapsansky, in August 1966.
The processed portion is summarized above and is described in the register. Additional accessions are described below.
Vergelijkbare items
- Chaney, James Earl, -- 1943-1964.
- Goodman, Andrew, -- 1943-1964.
- Hillegas, Jan.
- Lapsansky, Phil.
- Lawrence, Kenan Barrett, -- 1907-
- Schwerner, Michael Henry, -- 1939-1964.
- Council of Federated Organizations (U.S.)
- Mississippi Freedom Democratic Party.
- Student Nonviolent Coordinating Committee (U.S.)
- Feminism.
- African Americans -- Civil rights -- Mississippi.
- Agricultural laborers -- Mississippi.
- Community organization -- Mississippi.
- Mississippi -- Politics and government.
- Sunflower County (Miss.)
- United States -- Armed Forces.


text:
This item from Inspiration in Jewelry is now in the Shop Backroom.
More from Inspiration in Jewelry
Arabesque necklace, 14k and leather
This pendant was created using a photo of an Oakland Ballet dancer performing an especially beautiful arabesque in the Hansel and Gretel ballet. The border of the skirt is hand engraved and the face, hands, and pointe shoes are beautifully detailed. The dancer is slightly more than 1 1/8 inch high and is 1 1/4 inch long, and varies between 1/16 and 1/8 inch thick. It suspends from a 2mm thick leather neck cord 16 1/2 inches long with a barrel style clasp. "14K" and my hallmark are stamped on the back of the pendant and "14K" is stamped on the clasp.
Gender: Female, Age Group: Adult, Color: gold, black, Size: 1 1/8 inch high
If you were the pending buyer of this item, go to My Account to view, track and check payment for this item.
Inspiration in Jewelry
Pamela Lorence
Richmond, CA
Old World Craftsmanship For the 21st Century!
Spring Sale !!!! Ask me about my convenient layaway plan.


text:
Listen to free music played by justglenn
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station and start listening to @justglenn.
What other DJs are saying about Grounded Anode - Misfortune
sassikassi Not so bad being grounded!!!! @xSilkiex
Sep 29, 2010 at 4:14 am • reply to | AMAZON | ITUNES
happyhotelier For all those grounded at airports


text:
The Walled Gardens at Picton – An Appeal
When you visit Picton Castle gardens today you will find the walled garden brimming with an eclectic and colourful collection of plants informally arranged in large beds.
Look beyond this and you will become aware of some partially derelict buildings that included the Head Gardeners office and a fruit storage room, above which is a rare surviving example of a grape store.
Next door was the gardener's bothy which provided accommodation for young, single gardeners amongst whose duties would include stoking the furnace and boilers and providing ventilation to the glasshouses during the night. Adjacent to this were the potting sheds which now form Picton's indoor fernery. Here you will find a door with potting mixtures written on it in pencil dating from the 1890's.
Walled garden range of greenhouses before they were destroyed by storm in the 1950s
Our Grade II Listed Walled Garden is however in dire need of some extensive restoration work to save and restore this unusual, rare and historically significant group of buildings and bring them back into sustainable use whilst retaining their character and setting.
Impact assessments revealed that Picton's Walled Gardens are home to Greater and Lesser Horseshoe bats, Brown Long-eared bats and Soprano Pipistrelles
Of great importance will be the restoration of the walled garden walls and its wonderful gates, iron railings and gateposts which are topped with what we believe to be Coade stone pineapples. In addition it is hoped to create outdoor planted areas exploring the world of plants and their uses to mankind. It will also include a training and education room, an interpretation suite, improved volunteer and visitor facilities and in doing so, continue to allow and maintain access to bat roosts.
Assisting in the Walled Garden Project are a team of experts in their field including Conservation and Environmentalists, Architects, Surveyors, Interpretation and Educational Specialists. It is currently being part funded by the Picton Trust and the Heritage Lottery Fund.
The collapsed roof adjoining the bothy
A sketch of how the walled garden area is planned to look upon completion
We Need Your Help!
This is a £1.1 million project and in order to complete it we need to raise a further £40,000.00
You Can Make a Donation Today Here By Clicking This Link
because you really can make a difference!
Thankyou for your generosity
Together we can preserve this part of our heritage for generations to come
Donations & Sponsorship by Individuals
The Picton Castle Trust is a Charity (No.519693) and its objectives are to restore and preserve the Castle and Gardens for the pleasure of the public, and to safeguard its future. The generosity of our supporters and local businesses has been crucial to achieving these aims and securing our future. Long and short term plans and projects on our 'wish list' can be seen here, however if you wish to make a difference on our current project of restoring the Walled Garden, here's how you can help:
How To Make A Donation
You can make a donation at any time and pledge any amount by any of these means:
In Person
Online, Using the Form Below
By Scanning the QR code above
By Visiting our Special Donation Website
By Post : Cheques should be posted to
The Picton Castle Trust, The Rhos, Haverfordwest, Pembrokeshire, SA62 4AS
GIFT AID – How It Works
If you pay UK Tax, then the Gift Aid Scheme makes your gifts of money go further and increases the value of donations to charities. Gift Aid donations are regarded as having basic rate tax deducted by the donor. Charities such as ourselves take your donation, which is money you've already paid tax on, and reclaim the basic rate tax on its 'gross' equivalent – the amount before basic rate tax was deducted. Basic rate tax is 20 per cent, so this means that if you give £10 using Gift Aid, it could be worth £12.50 to us! Find out more on Gift Aid basics here. If you claim age-related allowances or tax credits, Gift Aid donations can sometimes increase your entitlement.
In order that we can claim this, you don't need to do anything other than let us know that you wish to do this (the online form will take you through the process) – we then reclaim the money from HMRC. Donations to charities under the Gift Aid Scheme are currently increased by 25p for every pound donated.
Higher-rate taxpayers can also claim back the difference between the 20 percent and 50 percent tax rates at year end when they fill out their tax return. If this relief is then donated to charity it too can be boosted by Gift Aid. For example, for each £100 donated by you paying 50% tax in the forthcoming tax year, approximately £37.50 can be reclaimed in relief. If this tax relief is donated as well, then we will receive, for example, a total of £176.28 for each £100 of your own money you donate.


text:
Hi All,
So I got a new fridge for my cake "studio" and it's amazing. It's a double door stainless steel Love it!!! only problem I have is that I can't put my fondant cakes in it becuase it's makes them all gummy! why is that? I've tried regulating the temp but I'm not sure if i should turn the temp up or down? I can put them in my regular fridge and they are fine, but this one is not doing what it should. It keeps all others good and fresh. Anyone else heard of anything like this? Can any one give me any tips on how to fix it?
it would be GREATLY Appreciated.
Thanks so much
Changing the temp in the fridge won't really help. The issue is the amount of moisture/humidity in the fridge.
Home refrigerators are usually very dry, so condensation isn't a problem. Commercial refrigerators are generally designed to be more humid, often if humidity controls.
First of all, are the cakes visibly "gummy" when you take them out and before they've been exposed to room air?
If so, and if you're new fridge is a residential model, I can think of several reasons for increased humidity.
Does it have a special produce drawer with humidity control? If so, I'd set that to the least humid as is possible.
Are you putting warm things into the fridge when cold, fondant covered cakes are in there? That will quickly cause condensation on the cold items.
If this is a side by side or french door fridge, if the doors and seals aren't working perfectly, then humid warm air is getting inside all of the time.
Now, if the cakes get gummy after being taken out of the fridge & sitting on the counter for awhile, it has NOTHING to do with the fridge. It's merely normal condensation because the air is warmer and more humid and it condenses on the cold cake.


text:
Huntsman School Global Opportunities Scholarship
The application deadline for scholarships awarded for 2017-18 has passed. If you have questions about your application, please contact Liz Allred, firstname.lastname@example.com.
To promote understanding of the global economy through participation in:
an Huntsman School Global Learning Experiences
an internship outside the US (with preference given to internships with academic credit)
an academic conference outside the US approved by a faculty member
- Undergraduate students admitted to the Huntsman School, with preference given to upperclassmen
- Minimum GPA of 3.0
Award Amount: Global opportunities scholarship awards are based on type of program and GPA
- For GPA 3.0-3.29
- City Break Program (London or Paris): $350
- Asia Summer Program: $700
- South America Summer Program: $550
- International Conference: $350
- For GPA 3.3-4.0
- City Break Program (London or Paris): $700
- Asia Summer Program: $1,400
- South America Summer Program: $1,100
- International Conference: $700
- For international internship or a semester exchange program: $1,250
Application Schedule:
Extended Application Deadline: Friday, March 17, 2017
The USU Office of Study Abroad provides a listing of various scholarship opportunities for students undertaking a study abroad experience. For more information, go to: http://globalengagement.usu.edu/htm/study-abroad/outgoing-students/study-abroad-scholarships.


text:
Producer Potluri Vara Prasad is extremely confident of his latest production venture 'Ghazi' starring Rana Daggubati. He is saying that Ghazi is a never before film in the 100 year old history of Indian Cinema. He said he is proud of producing a film like this. Even though the project started off as a small venture, it has become a crazy project now. He said people will know why he is talking about this film like this on February 17.
"Our Production 'Kshanam' was ready by this time last year. It was released in February and went on to become a super hit. We are coming up with Ghazi in the same month this year. Thousands of movies made in India till now. Mythological, historical, socio fantasies.. there
are several other genres. But you wouldn't have watched this kind of film on the Indian screen. I am proud to be part of this project. The movie started off as a small project. But the project has grown in to very big size now. You will come to know how we made the movie on February 17. It is going to remain as a special film in the history of Indian Cinema".
Ghazi is a first Indian film to be made in the backdrop of a submarine. Rana is playing a Naval officer role in the movie. Ghazi is directed by debutante Sankalp Reddy.


text:
Monthly Archives: September 2016
Ways To Stay Cool Outdoors In The Summer
When summer arrives, hot weather is everywhere. In some parts of the country, temperatures can reach a sweltering 100 degrees or more. Now, if you're anything like me, the longer days days are great but the hot, outdoor temperatures present a challenge. If I'm outside in the hot weather, a beach is absolutely necessary for me to enjoy it.
For those instances when a beach is not convenient, solutions are needed to keep cool. From low-key picnics to large backyard BBQ parties, the summer is made for outdoor fun. See cool ideas you can incorporate into your summer plans to ensure everyone is comfortable and enjoying the best of what summer has to offer.
1. Swimming Pool
A swimming pool can bring hours of fun with friends and family, with instant relief from the heat. Host a pool party with neighbors to bring together the community, or enjoy floating on a sunny afternoon. A swimming pool will prove its convenience on a hot summer day.
The keys to truly enjoy your pool this summer is with pool maintenance. The average cost to maintain a swimming pool is $254, but this can depend on the size of the pool. Maintenance is important for the overall health and the longevity of your pool. Here are some ways to regularly care for your pool this summer:
- Skim Debris
- Clean Out Baskets & Filters
- Vacuum
- Test pH Balance
- Maintain Water Level
2. Backyard Water Slide
Swimming pools are a large investment that take extra time to care for properly. If you're looking for a short-term option to keep the kids cool and entertained outdoors, look to the traditional backyard water slide. Many stores offer a wide variety of backyard water slides, with sprinklers attached to various areas and inflatable options for safety. These bring hours of fun to those of all ages and are a welcome activity on hot days. Always read safety instructions for each model so you can ensure a great time in the water.
Most homeowners are concerned about lawn damage when it comes to these water activities for children. To prevent harm to your grass, be sure to only have the water slide out when it's in use, to prevent burning or damaging the grass. Put it away immediately after use. It's likely the slide will flatten out your grass. If you're able to plan for it, let your lawn grow longer than you normally would before using the waterslide. Cut the grass once you have removed the water slide. Damaged grass may take up to two weeks to return to its green hue again.
3. Seek Shade
If water activities aren't for you, adding more shade can help to cool down. An arbor or pergola installation can help reduce the amount of sun you come into contact with. Not only will this help you keep cool, but it will also increase your home's ROI.
Pergolas are a stylish way to add partial shade to your yard and can be placed almost anywhere. For extra coverage, try draping a large canvas or fabric between the slats. This beautiful, airy look will have you loving summer in no time.
Arbors typically are attached to the home and will provide you with complete shade, as it is a solid frame. They are a great way to add a permanent, shady retreat to your yard.
Whatever you choose, be sure to include comfortable lawn furniture or a hammock to rest and relax in the shade.
4. Deck Out Your Deck
As nicer weather begins to become the norm for a few months, more homeowners begin to utilize their decks. Enjoy a dinner outdoors or find a cozy place to read, there are plenty of things you can do to stay cool on your deck. An awning or umbrella can add shade where you need it, when you need it. While awnings tend to be semi-permanent features, umbrellas can be moved from one area to another typically without an issue.
If your deck has a roof, a ceiling fan might be just the touch you need to get a little breeze going outside and keep the bugs away. The average cost to install a ceiling fan is $229. Even though your deck may provide some shelter from the elements, it's important to choose a fan that is weather-resistant.
5. Cool Food Ideas
If you need a quick fix for the heat, choose a cold food to provide some relief. First, remember to stay hydrated. Not drinking enough water when working or playing outside in hot weather can cause dizziness, cramps and in severe cases, heat stroke. Keep a water bottle on hand when you're out, regardless of activity.
Fruits like watermelon and pineapple are naturally cooling and nutritious. Though ice cream may be tempting, dairy and hot weather don't always mix. Popsicles are a great way to enjoy a cold treat on a hot day and are even better if you make them yourself. Purchase a popsicle mold and fill with your favorite fruit juices and fresh fruit. This is a great treat for both kids and adults on a hot summer day! Here are a few great recipes from PopSugar.
There are so many activities to enjoy during the summer months and keeping cool is key. Whether it's diving in the pool or reading in the shade, you're sure to enjoy everything the season has to offer by utilizing these ideas.
Deck Railing Design Ideas For Outdoor Living Space
A deck is a wonderful addition to your home. More and more homeowners are seeing the value of this useable outdoor space as a place to gather and relax. As beautiful as they are, having a deck railing is a must for the safety of anyone who is using the deck.
But deck railings don't have to be an eyesore. All you need is a little inspiration to find the right deck railing design that matches your style.
Are you ready to install or replace a deck railing? Contact a pro today for up to four free quotes from local decking pros in your area.
Deck Railing Height
If you're building a new or updating an old deck, a railing is a must have. Even if your deck is low to the ground, it may still need a railing. Deck railings help prevent friends, family and pets from falling. International Residential Code requires that decks 30" above the ground must have a railing and it must be at least 36" in height. However, be sure to check with local and city codes so you can ensure you have the proper railing height.
Cable Deck Railing
If you're looking to create a chic, modern look in your outdoor decking design, cable deck railings are a must. Cables are arranged either vertically or horizontally to achieve the look you'd like. It's also great to keep the view from your deck without sacrificing safety.
Metal Deck Railing
If you'd like to change up the look of your deck, metal railings can be the answer. With different types of metal and the ability to create intricate designs, you have plenty of options to choose from.
An Aluminum deck railing is an affordable choice for anyone looking for flexibility and low maintenance decking. They come in a variety of colors, so it's easy to find the perfect one that matches your outdoor look.
For a classic look, a wrought iron deck railing is a great fit. The classic ornate spiral spindle is a look that will never go out of style. In addition, iron railings are durable and long lasting.
Vinyl Deck Railing
For decks that require a perfect style and shape, vinyl railings may help. They are low cost and come in a variety of colors and shapes. Many vinyl railings are offered as kits, so it's possible to DIY your deck railing.
Composite Deck Railing
A popular option among homeowners, composite railings are versatile, low maintenance and long lasting. They come in many styles from natural looking wood to intricate spindle designs. Additionally, they are environmentally friendly.
Glass Deck Railing
Some homeowners love their decks because it provides a view that is unmatched. If you are one of these homeowners, don't delay on installing your deck railing because it may ruin the natural beauty. A glass deck railing can provide an uninterrupted viewpoint. These, however, do take some maintenance and upkeep to stay in great shape, so be aware before installation.
Wood Deck Railings
Traditional wooden decks are sometimes best accompanied by a traditional wooden railing. The cost of the railing will depend on the type of wood you use. Many times, wood railings come with a straight line design, but there are some styles that allow for more intricate details. Remember, this type of railing may take specific upkeep, depending on the wood you choose. While many choose to match the wood they have already used for their deck, you can mix it up. Here are some wood deck railing types:
- Cedar
- Mahogany
- Pressure-Treated Lumber
- Redwood
- Teak
Deck Railing Designs
The design of a deck railing does not stop simply with the railing itself. There are plenty of ways you can add your unique style. Here are a few ideas to include in your deck railing design:
Deck Railing Planters
If you love gardening and want to bring that element to your outdoor oasis, a railing planter is what you're looking for. Many are easily attached over the railings, with brackets, so it sits securely and can hold your favorite plants for added décor.
Deck Lighting
Not only will your deck railing help keep you safe, but deck lighting can also have additional safety benefits as well. Deck lights help you enjoy the outdoors well into the evening and sometimes, add a bit of style to your desk. You can find many options of deck lighting to suit your style. Read Deck Lighting Ideas to see how you can make lighting a decorative addition to your railings.
Built-In Seating
Finding the right seating and furniture for your deck can be a challenge. However, by building in permanent deck seating to your railing upgrade, you'll always have a place to relax and entertain guests on your deck.
Finding and installing a deck railing is not only important for the safety of all who use the space but can also add to the outdoor décor. Thankfully, you have many options of deck railing design to help you create the outdoor oasis you've always dreamed of.
10 Bathroom Amenities
Regardless of how large or small your bathroom is, you can still find ways to upgrade it to the bathroom you desire. After all, it's a room that requires function to get a fresh start on the day ahead, but it needs features to help you relax at night.
If you're looking for inspiration, look no further than these luxury bathroom amenities. They will have you dreaming of all the changes you can make to create the relaxing bathroom you've always wanted.
Are you ready to add a little luxury to your bathroom? Contact a bathroom remodeling pro today for up to four quotes from contractors in your area for free.
Bathroom Remodeling Costs
Besides being a space to rest and relax, if you're selling your home, the bathroom could be a large factor in those looking to buy. The look and feel of a bathroom can impact a buyer's decision. If you're considering selling your home, now is the perfect time to consider a bathroom remodel. Much of the cost depends on what luxurious amenities you decide to add to your new bathroom. The average cost to remodel a bathroom is $8,748, with most homeowners spending between $6,535 and $9,788.
1. Luxury Bathroom Vanities
If you're looking for a bathroom amenity that can change how you function, look no further than a double bathroom vanity. Instead of fighting over a single sink in the morning, this gives you and your significant other the space you need to prepare for the day. Choose a countertop material like marble to really give the look of luxury to your bathroom vanity.
2. Luxury Bathroom Faucets
Simply changing out your faucet with a newer model can sometimes give your bathroom the look you desire without the price tag of a complete remodel. Wall-mount sink faucets will give your bathroom a sleek look and not get in the way of your vanity display. The average cost to install a faucet is $235, but if you're installing a wall-mount faucet, you may have a few additional costs to consider.
3. Luxury Bathroom Sinks
Once you update your faucet, you must update your bathroom sink to complete the look. Nothing quite says extravagance like a vessel sink. Not only is it a modern upgrade, but it's also a décor focal point of the bathroom. The average cost of a ceramic vessel sink is between $80 to $120 and glass vessel sinks between $150 to $300.
4. Luxury Bathroom Shower
Your shower experience can be instantly changed with an update to your shower head. Having dual shower heads or including a rainfall shower head can be just what you need to relax at the end of the day.
If you're remodeling your bathroom, consider expanding the size of your shower for the look of luxury. Glass or frosted glass doors will make all the difference here. This is an expansion that will never go out of style.
5. Luxury Bathroom Tub
If you're remodeling your bathroom to the best, you must include a soaker tub. Not only do they look beautiful, they are the remedy to a long and busy day, providing a place to unwind and relax. Soaker tubs come in many varieties from freestanding to drop in, so it's easy to match with the look you want. The good news is that if you'd love a soaker tub, but not sure you have the space available, there are small bathroom tub options to choose from.
6. Luxury Bathroom Lighting
The function of your bathroom can largely be enhanced by the lighting of your bathroom. One of the first ways you should upgrade your bathroom lighting is by adding a dimmer switch. This allows you to control how much light you'd like in the room. Keep it bright in the morning as you're getting ready for the day and turn it down low at night to relax. Dimmer switches can be added to any bathroom, regardless of size. The cost of a dimmer switch largely depends on the manufacturer, but expect to pay between $13.50 minimum and $40.50 maximum per unit.
If you're looking to really make a statement of extravagance in the bathroom, a chandelier will give the look you desire. Bathroom chandeliers come in various options; some are ceiling-mounted and some can be mounted on the wall above your vanity. Whatever you choose, you're bound to get the look of luxury.
7. Luxury Bathroom Rugs
Don't forget about your feet when you're upgrading your bathroom. Regardless of size, you can add a few plush rugs to the room to warm it up. Consider memory foam options that make it feel like you're walking on a cloud! Keep in mind, you'll want something that's mildew-resistant, as there tends to be a lot of moisture in the bathroom.
8. Luxury Bathroom Accessories
When you're remodeling for luxury, don't forget to include accessories that can improve the way you use the space. Adding a towel warmer to your bathroom can ensure you have a cozy way to dry off every time you step out of the shower.
Don't forget the smaller items like wastebasket, toothbrush holder and soap dispenser. Look for trendy options that will last to give your bathroom the elegance it needs.
9. Luxury Bathroom Technology
The latest advances in technology are now available to integrate into your bathroom. Consider upgrading your shower to include digital shower control. You can easily set your desired temperature every time, without worrying about an unexpected splash of cold water.
If your shower is where you sing your best, you may want to consider adding a way to stream your favorite music. Kohler's Moxie Shower Head includes a speaker so you never have to worry about damaging your sound system.
Finally, consider technology upgrades in the bathroom that allow you to conserve water and energy. Eco-friendly sensor faucets allow family members to only use the water they need with motion-detecting technology.
10. Luxury Bathroom Fireplace
One of the most relaxing bathroom amenities you could add is a fireplace. Not only does it exude elegance, it can enhance your bathroom experience with the ambiance it provides. For the bathroom, an electric fireplace is the way to go. The average cost to install a fireplace is $2,072, with most homeowners spending between $1,244 and $2,484.
You can create the bathroom you've always wanted. While you may not be able to include all these dreamy amenities to your bathroom, you can still draw inspiration from these ideas to upgrade your bathroom. It's time to fall in love with your bathroom again!
Tips for Selecting Options and Upgrades from Your Builder
Now that you've signed a contract to purchase your new home, how do you customize it to make it your own?
One of the advantages of purchasing a new home is the opportunity to select features and finishes that reflect your own personal tastes and lifestyle. But for many homebuyers, particularly first timers, the array of choices can be dizzying.
Cabinets, countertops, flooring, fixtures, appliances — and even structural changes such as additional bathrooms or garages — are just the beginning of a potentially endless choice of options or upgrades that your builder may allow you to select.
So what's a buyer to do?
1. Structural Changes
Select structural additions or changes when you sign the contract, or immediately thereafter. "Buyers are a little overwhelmed when they sign the contract because of all the legal documents," says Sue Goodrich, vice president of sales and marketing for Cachet Homes in Scottsdale, Ariz. "The only thing we talk about then is structural options."
Buyers who want to add rooms or garages, move doors or add a fireplace or recessed lighting, for example, need to make their structural changes early in the process because those changes may impact the building permit pulled by the builder. These types of changes — as well as any electrical or plumbing changes that would require walls to be opened — would also be costly to make after the home is completed.
2. Post-Purchase Upgrades
Remember that it might make sense to make certain changes after you purchase your home. Cosmetic features in particular, such as paint, landscaping, lighting and plumbing fixtures, epoxy garage flooring, crown molding, chair rails, window treatments and even certain appliance upgrades can often be made after the closing, particularly by homeowners who have a budget.
Grenadier Homes in Dallas, for example, doesn't include refrigerators in the base price of their homes, says Kathy Costa, a Grenadier Homes design consultant. That way buyers might be able to get a good deal on their own. Still, by purchasing these upgrades through the builder, you might be able to roll the cost into your mortgage, as opposed to paying out of pocket.
In addition, upgrades completed after the closing will not be covered by the builder's home warranty — and may void it, Costa warns.
And, of course, there is the hassle factor as well: are you willing to spend time after the closing to work on your home — or would you rather move in knowing that your home is exactly the way you want it to be?
3. Builder Timeline
Follow your builder's timeline to select other options or upgrades. About two to three weeks after the contract is signed and approved, your builder will arrange a meeting at its design center. Depending on the builder, you may or may not be able to make changes after this meeting, so be prepared with a list of the items you want. Consider bringing photos of kitchens and baths you like to help guide the designer.
4. The Model Look
Remember that the model home you fell in love with may have thousands of dollars of options and that the base home may look very different. While many builders include a number of standard features in the base price of their homes, others don't. That thick carpeting or granite countertop may cost extra.
5. Prepare a Budget
Prepare a budget — and stick to it. Walking into a builder's design center is a little like being a kid in a candy store. You'll see cabinets and granite and top-of-the-line flooring. To avoid overspending — and overextending yourself — prepare a budget before your design meeting.
Goodrich, of Cachet Homes, says that a good rule of thumb is to expect to pay about 12 percent of the base price of a home on interior upgrades. Make sure that the price of your home, including any upgrades, falls within the pre-qualification guidelines for your mortgage.
6. Needs Versus Wants
Be flexible. Remember that you have a budget and that you may not be able to afford all of the extras you'd like, so prepare a list of must-haves and want-to-haves. For example, Joel Whitley and his wife Taylor purchased a three-bedroom, two-bath home from Cachet Homes at the Santa Rita community in Phoenix for $346,900.
The couple budgeted an additional $30,000 for options, of which they spent $26,000 to upgrade cabinets and flooring and to add additional ceiling fan outlets and a soft water loop for a water softening system.
"We would have loved to do a backsplash throughout the entire kitchen, but we knew we didn't have the budget for that," says Whitley. "So we came up with a compromise where we have a backsplash just behind the oven in a fancy stone design."
7. Resale Value
Don't over-customize. Of course, new-home buyers want their homes to reflect their personal style and taste. But, it's important to consider the resale value, as well. "Those lavender granite countertops in the kitchen that you're thinking about may make your home difficult to resell in a few years," says Andy Weiser, a real estate agent with Coldwell Banker in Fort Lauderdale, Fla.
"A better choice might be to paint the walls a great shade of lavender, but to go with more neutral countertops. That way, when you decide to move, you will appeal to the most buyers possible."
8. Builder Trust
Deal with a builder you trust. Its design team will guide you through the process and offer expert advice on not only design choices, but also how best to apply your budget.
"It's their job to make your home everything you want it to be," says homebuyer Whitley. "Trust your home builder for their expertise. After all, this is what they do every day."


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Pre-Occupational Therapy Concentration
Please note that occupational therapy programs vary for their requirements. Be sure to check all programs you are considering applying to early during your undergraduate career for specific pre-requisites needed to apply to that school's program. The information for this list was compiled from the occupational therapy programs in North Carolina.
Be sure to check programs of interest to see if online courses are accepted, especially Anatomy and Physiology courses.
Standardized Exam required for admission: GRE Resource for national standards: American Occupational Therapy Association
Required Courses:
BIO 213 Anatomy & Physiology I
BIO 214 Anatomy & Physiology II
MAT 130 Introductory Statistics for Social Sciences or ENV Data Analysis for Natural Sciences or other statistics course or other statistics class
PSY 101 General Psychology (this is pre-requisite for upper level psychology courses)
PSY 271 Abnormal Psychology
PSY 210 Developmental Psychology
ATH 101 Cultural Anthropology and/or SOC 101 Introduction to Sociology (check programs) In addition to the above, the following courses are recommended depending on the programs you are applying to:
NUR 105 Medical Terminology (required for some programs)
PHL 310 Biomedical Ethics or other philosophy course including logic as an option
Upper level Sociology courses (required for some programs)
Consider starting Spanish or continuing it from high school to increase your ability to work with Spanish-speaking patients as this skill will be valuable with demographic trends in the United States.


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Magento and Facebook Open Graph: Introduction.
The main difference of version 1.1.0 of the Spread The Word extension is Facebook Open Graph technology support. This guide will tell you how to set up Spread The Word and how to befriend Magento and Facebook Open Graph.
First of all, we assume that you've already registered your Facebook application before and you have OAuth keys for this social network saved in your Spread The Word configuration. Otherwise, please follow the steps described in the "Setting up OAuth authorization" - "Facebook" section of the User Guide.
Creating Facebook Open Graph objects and actions.
By following the steps below we will create Facebook Open Graph Objects and Actions for Spread The Word invitations. We will also create Aggregations - little reports that will provide a resume of social activity concerning these objects and actions.
First of all, log in to Facebook go to your "Apps" page at https://developers.facebook.com/apps.
1. Select the app you'll work with in the left menu, click "Edit App".
2. Use the "Setting" menu on the left to go to the "Basic" page. Make sure that the "Sandbox Mode" option is disabled on the "Basic Info" tab. Otherwise, select the "Disabled" box and save changes.
3. Copy the value of the "Namespace" field and save it in the "Facebook App Namespace" field on the "Facebook Open Graph Object Settings" tab in Spread The Word extension configuration (Magento Backend - System - Configuration - Add In Mage:: Extensions - Spread The Word).
4. Use the "Setting" menu on the left to go to the "Permissions" page. Make sure that your app permissions correspond to those on the screenshot below:
User & Friend Permissions: "email"
Extended Permissions: "publish_stream", "read_friendlists"
Auth Token Parameter: "Query String (?code=...)"
5. Use the menu on the left to go to the "App Details" page. Make sure that you have filled in all the required fields. Please refer to the screenshot below for an example. After you set up Facebook Open Graph and Spread The Word extension in Magento Backend according to the steps below, your application should be submitted for approval by Facebook. Click "Submit App Detail Page" to do this.


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???Almost synonymous with being a student is drinking, living off no money and reading though large amounts of old textbooks, but it is the last of these that ForeMind's latest app for the iPhone is hoping to ease.
The ArticleSearch iPhone App essentially allows you perform simultaneous searches for academic articles across a range of sources, which not only saves you the time of having to search on a range of different engines, but also the irritating hassle of having to type all your criteria in every time.
The concept is fairly simple: you start by filling out one field (or a form for a more advanced search) and tap search which will prompt the app to gather results from the libraries, search-engines and databases that you have selected. It then displays the number of results in a nice little 'test-tube' graphic so you can easily see where the bulk of the results lie; to then access the specific content — which really is what you want to do — you just tap the relevant tube and the built-in web browser let's you look at the results list and then on selection the raw text.
So the premise is promising: you can quickly search across a whole range of academic sources whenever and wherever you like, but there are a few other nice little features that make the app well worthwhile. For starters sharing the articles that you find through the search couldn't be easier with simple buttons to both add it to favourites and send it to your friends/class mates through SMS or email. Also for those who like a nice visual touch the app has an accelerometer feature which means that when you shake the phone previous searches are cleared and the coloured test tubes empty.
So not only does this help students by making articles dead easy to find but it is also very easy on the pocket: the basic ad-free version which allows you to search on two standard and one premium search engines at a time is completely free. However if you are likely to be doing a lot of specialist searches it may be worth investing in the upgraded versions which allows you to access 3, 7 or an unlimited number (i.e. whatever your phone can cope with) of 'premium' libraries at a time.
Having only been released at the end of January the current source list isn't all that expansive (there's just over 40 different premium libraries) and boasts an odd mix of specialist and general engines — like 'Google Scholar' and 'Astrophysics Data System' – so you may want to check to see if the selection is useful for you. If it is then you are in luck and you will probably have found your next best friend when it comes to researching writing essays, theses and dissertations, but don't be too disheartened if not as future updates with greatly increased numbers of libraries are promised for the future.
Admittedly the ArticleSearch iPhone App is fairly niche – you are unlikely to ever use this unless you happen to be studying in the subject that the search engines cover – but for those that do fall within that small group it could quickly end up being an invaluable tool, especially for those last minute additions on the bus!


text: Anyone out there doing Halloween autograph pages for the characters? We are going in October and I thought I would like to create my book but instead of the standard pages use pages with a Halloween theme for each character. I like using the 12x12 pages but can't seem to find many with Halloween themes for each character. Any help/suggestions?


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2960 Christmas Way, Coquitlam, BC V3C 4E6
+ taxes & licensing
Accident Free, Cherokee North loaded with power options, beautiful alloy wheels, blue tooth and much much more! Looking for a smaller SUV with off road capabilities with added fuel economy, this is the Jeep for you! Comfort and style that wont break the bank! Call Click or visit today Only at Coquitlam Chrysler Dodge Jeep.
Please contact the seller to confirm pricing, features, odometer, and availability of this vehicle.
See the Carpages.ca Terms & Conditions for more details.


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The iWalk Tempered Glass Anti-Scratch Invincible Screen Protector provides superior screen protection for your Galaxy S5 with its special chemically-tempered glass that not only offers scratch prevention but also shock and shatter resistance. Its fingerprint-free film means you won't have to clean your screen very often. This anti-glare screen protector is also ultra-sensitive for safety and premium optical clarity.
Unlocked Cells offers a 90 day warranty for all accessories. This warranty excludes normal wear and tear or incidental damages caused by abuse. The client is responsible for return shipping fees on any claims that are not warranted.
For all warranty claims visit our warranties center.


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When the GUN strikes, it generates SMOKE. GUNS n SMOKE tells different genre of Guns and its sounds and make.
The best Guns app that supports Modern Warfare Guns supported to all resolutions of devices.
*Best and Hit Guns and weapons like Riffle , Shot Guns, Hand Guns, Machine Guns, Bombs, Assault Riffles,Snipers for Free*
*Super hit bullet Action for free*
*Tap the screen to shake, vibrate and fire the Guns & weapons for free in this addictive app*
*Buy Pro version to shoot birds*
* Cow Boy & weapons theme*
You Can select any model of
Rocket launchers with actual voices.
Get details of the different Guns and its background and History.Tap the targets to shoot. RICH GRAPhics of insanely powerful Armor and other explosive power-ups to get your hands on with real simulation.
Comments & Reviews
It's cool!!!
by jennifer– May 29, 2011
Amazing graphics, one of the most intense shooters ever!
by crystal– April 22, 2011
This is awsome
by jak– May 14, 2011
Disclaimer: This free app is ad supported and may contain ads in the notification tray and/or home screen. The required permissions are just for ads.
Download PRO version completely Free of Ads.
Uploaded: Jun 22, 2017 at 13:48 CDT
File size:45.39MB
Uploaded: Aug 24, 2016 at 17:21 CDT
File size:49.15MB
Uploaded: Oct 23, 2014 at 2:15 CDT
File size:41.82MB
Uploaded: Nov 16, 2016 at 8:11 CST
File size:40.64MB
Uploaded: Sep 10, 2016 at 16:01 CDT
File size:44.1MB


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Follow Satch in lead up to the release of Black Swans & Wormhole Wizards on his cool youtube vlog. The series gives you insight to the writing of the songs, the studio, the band members and very importantly the origins of the crazy album title!
Learn guitar with the ultimate step by step system.
Follow Satriani Behind the Scenes
Follow Satch in lead up to the release of Black Swans & Wormhole Wizards on his cool youtube vlog. The series gives you insight to the writing of the songs, the studio, the band members and very importantly the origins of the crazy album title!
Labels: cool, drums, interview, song writing, trivia, video, vlog
Steve Lukather & Marty Friedman Jamming!
Hearing a well known guitarist cover another well known guitarists song is one thing but hearing 2 well known guitarists cover each others song while sitting in the same room is another thing all together. Confused? Then watch the video and see what I yaking on about :)
If you are easily offended skip the potentially offensive lead in banter and jump straight to the guitar action at 20 seconds.
Learn guitar with the ultimate step by step system.
Labels: cool, cover song, funny, guitar solo, metal, riff, trivia, video, weird
Rock Stars Kidnapped By Ninjas!
So I recently got into this whole video blogging thing as an outlet for airing my thoughts on guitar, new music, bands etc. Subscribe to the channel at http://www.youtube.com/user/nailguitarvlog
Leave your comment below sharing... if kidnapped by ninjas which sexy star you would want to save you? And why? The best answer will feature in my next vlog.
Learn guitar with the ultimate step by step system.
Labels: cool, favourite music, trivia, video, vlog
Mario played with a Theremin
Remember that weird instrument called a theremin that was used for making spooky sounds in old school horror movies? (If not click here) Well it appears that with the right know how you can hook one up to play Mario!
Learn guitar with the ultimate step by step system HERE
Lessons Of A Slide Blues Pro
Jack Pearson, quietly spoken but a devil of a slide player. You can hear the years of experience in every note, so intentional, so mature. This video is part 1 of five pieced together by Gibson.
Learn guitar with the ultimate step by step system
Labels: acoustic, guitar solo, inspiring, interview, lesson, preview, trivia, video, wisdom
Approaching Warp Speed, Hold On To Your Butts!
so I heard a few mentions of Andy James in the same sentence as Paul Gilbert who Im a huge fan of so decided to give his videos some investigating... 3;30mins later Im here trying to pull my eyebrows down my forehead!
Occasionally you find a player who absolutely flaws you from the word go, Andy James is one of those players. There are a zillion shredders out there but Andy James is one of the few who manages to maintain incredible note clarity even at warp speed.
Speed aside he also has some great phrasing and menacing vibrato. Enough of my yakking, press play and prepare to feel the need to for a couple of decades of practice :)
Learn To Shred
Labels: guitar solo, metal, super skills, video
First 3D Music Video
Awwwwweful music, but the 3D is great if you have red/blue glasses! Apparently its the first 3d film clip on youtube, if not its probably the first well produced one.
(at time of publishing more than 50% of the 3D movies on youtube aren't very affective.)
Need glasses try Ebay, I got mine for under $5 delivered and their actual plastic glasses not just the old paper ones. Enjoy, even if that requires turning the sound down :)
Nail Your Guitar Chords For Good
Engaging The Lighter Side Of Metal
I had a good laugh while watching this Killswitch Engage video clip... a great reminder that music is all about enjoyment, and big part of that being not taking things tooo seriously. A good lesson we can all be reminded of from time to time.
Get the guitar course designed to help you nail all your essential guitar skills as quick as possible. Click Here
Jam To The Ninja Guitar Beat!
Mr Fast Finger is the magical meeting of shred guitar, animation and humour. What's more he's a very nice guy because he's now provided us with a blog of wicked shred licks to learn + and a very groovy ninja backing track loop.
Hear Fast Finger fan Ugur Dariveren ripping up over the loop with his slippery licks then try it yourself HERE!
Still stuck at the beginner's stage? Get the course that's teaches you all the essentials, step by step click here
Labels: cool, free stuff, guitar solo, inspiring, lesson, riff, video


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Listen to free music by Bill Geldard
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station.
Bill Geldard – Hot Line
Bill Geldard
Hot Line
Bill Geldard songs played by Blip.fm DJs
Bill Geldard – Flute Salad [1975]| play
Nov 26, 2011 at 6:37 am via YouTube | AMAZON | ITUNES
Artists Like Bill Geldard
Blip.fm DJs who play Bill Geldard
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"The team at Irwin Mitchell has a 'pre-eminent reputation' for travel personal injury work"
Legal 500 (2016)
The International Personal Injury team at Irwin Mitchell is concerned to learn that guests became ill with gastric symptoms after staying at the Melia Caribe Tropical in the Dominican Republic this year.
Despite some positive reviews, Irwin Mitchell has discovered reports relating to the food at the resort. Holidaymakers have expressed that the food was unappealing and that "the milk tasted spoilt". Furthermore, guests have said that the food was sometimes covered in flies.
A visitor has also highlighted that their room was dirty with a dusty closet. Other holidaymakers described that the air smelt of sewage and have reported falling ill after eating at the hotel, suffering with gastric symptoms.
If you have also travelled to the Melia Caribe Tropical in the Dominican Republic and subsequently suffered from symptoms of illness, we would like to hear from you.
For more advice on making a holiday illness claim, contact us on 0800 056 0066 or complete our enquiry form and we will call you back.
« Back
Call us free on
"We were very happy with the outcome of our case, especially as we were initially offered nothing from the Tour Operator. Irwin Mitchell were very professional throughout and we are very happy with the outcome."
Chris and Michael, Worksop


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MV - West Grand Blvd Now We Will (w daphne khoo) MP3
An early music video we produced when we were a young company for the Singapore band West Grand Boulevard. Prod. Co: Monochromatic Pictures Director: ...
Delightful 'Grand Blvd Residence' in California Exuding Freshness Throughout MP3
Designed by Robert Thibodeau of Du Architects, the Grand Blvd Residence in Venice, California exudes freshness throughout. With a living surface of over 3000 ...
Sandestin Grand Blvd Tour MP3
Sandestin Grand Blvd Tour give you a drive buy look at the main shops of Grand Boulevard in Sandestin, Miramar Beach, Florida. Download a printable ...
1036 East Grand Blvd, Corona, CA 92879 - Presented by Realtor, Kym Talbert of Keller Williams Realty MP3
Realtor Kym Talbert of Keller Williams Realty Serving Corona, Orange County, Riverside County, California. Specializing in Homes for Sale, Home Buying, Short ...
FLIPnw - 2514 Grand Blvd [COMPLETE RENOVATION] MP3
Music ~ The Intro - The XX Photos courtesy of Jeremy Jones, The Jones Art Channel Currently for sale in Vancouver, WA, USA 2514 Grand Blvd Vancouver, WA ...
3040 Oasis Grand Blvd # 1103 - Fort Myers, Florida (Furnished) MP3
This pristine unit has been professionally decorated and complete with over $30000 in designer upgrades, this unit show cases hardwood floors, custom ...
San Bruno City Council Meeting September 10, 2013 10a. Grand Blvd Case Study MP3
San Bruno City Council Meeting September 10, 2013 10a. Grand Blvd. Initiative's Complete Streets Case Study.
Morph Man at Grand Blvd MP3
Morph Man hits SLU.
Ain't No Sunshine - West Grand Blvd MP3
Ain't No Sunshine - West Grand Boulevard performing at the Summer Concert Series in Pleasanton, CA on June 6, 2014.
Grand Blvd Walleye Fishing..Spring 2014, Day 2 MP3
Day 2 of Walleye Fishing on the Detroit River , 10 Walleye in 2 hours, Good day for fishing!! Let's get em'
San Bruno City Council Meeting April 8, 2014 10f. Grand Blvd. MP3
San Bruno City Council Meeting April 8, 2014 10f.Grnd Blvd. Initiative's Complete Streets Case Study.
Driving Near the Motown Museum "Hitsville U.S.A." W. Grand Blvd Detroit, MI MP3
Driving on W. Grand Blvd near the Motown Museum in Detroit, MI. Filmed on a RoadLens dash camera http://www.RoadLens.com.
Grand Blvd Alviso CA MP3
View From main door.
Adamo Group - Demolition of I-94 bridge @ W Grand Blvd - Detroit Drone Aerial Video MP3
Aerial Video shot of the demolition of the West Grand Blvd bridge over I-94 in Detroit.
Bobby Sixkiller - 2648 West Grand Blvd MP3
Available from http://www.redstar73.com/tienda.
Ain't Too Proud to Beg - West Grand Blvd MP3
Ain't Too Proud to Beg - West Grand Boulevard at the Pleasanton Summer Concert Series at Lions Wayside Park, June 6, 2014.
602 Grand Blvd. Mishawka, IN 46544 MP3
Visit our website at www.renthomeworks.com to view an up to date listing of our homes, our rental requirements and our rental application.
Nicole Curtis Grand Blvd house MP3
Subscribe! Thanks for watching! I post rants and other videos for entertainment and you might even learn a thing or two.
425 Grand Blvd San Mateo, 94401 MP3
http://425grandblvd.2seeit.com/ 425 Grand Blvd San Mateo, 94401 For Rent $1000 Lovely 2 Bedroom 1 Bath House Call for information: (804) 571-1917 Lovely ...
GRAND BLVD at Riverhouse Grille Sweet Home Alabama.
140 Grand Blvd Benton Harbor, Michigan Harbor Shores MP3
A unique custom home nestled along Harbor Shores Development! Enjoy views of Lake Michigan and the premier Jack Nicklaus Signature Golf Course.
Commercial Property For Sale: 8220 Grand Blvd Hobart, Indiana 46342 MP3
For more information visit http://commercial.century21.com/in/hobart/8220-grand-blvd-46342/listing-C21LHQ9PY 8220 Grand Blvd Hobart Indiana 46342 MLS# ...
1134 Grand Blvd, Cedar Falls, Iowa 50613 MP3
Contact VirtualOpenHouseHD at 866-599-3560 - http://www.virtualopenhouseHD.com - Located in the beautiful Grand Boulevard Neighborhood - perfect for ...
425 Grand Blvd San Mateo, 94401 MP3
http://425grandblvd.2seeit.com/ 425 Grand Blvd San Mateo, 94401 For Rent $1000 Lovely 2 Bedroom 1 Bath House Call for information: (804) 571-1917 Lovely ...
Falling down grand blvd by Big Drew Grafix MP3
Homes for sale - 7302 Grand Blvd, Hobart, IN 46342 MP3
1845 Bayou Grand Blvd NE Saint Petersburg FL 33703 - Carolyn Kopco MP3
http://homesite.obeo.com/viewer/default.aspx?tourid=826337&refURL=youtube Contact: Carolyn Kopco Coldwell Banker St Petersburg Central 727-365-0534 ...
For Rent 11/181-203 Grand Blvd Craigieburn Vic 3064 - English MP3
For Rent - 11/181-203 Grand Blvd Craigieburn Vic 3064 Raine & Horne Roxburgh Park 2 Bedrooms 1 Bathroom 1Garage Clean, Neat & Tidy!!! This seamless ...
10788 Grand Blvd West Palm Beach FL 33401 MP3
232 grand blvd MP3
This video is about 232 grand blvd.


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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
Added 20/04/2017
Small repair to cracked tile (we will supply) on garage roof plus plastic covering over existing...
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Gardener / Garden Designer
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Services required: lawn mowing and edging, hedge trimming Approximate length of garden: more...
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Wiring up an outside shed (not yet erected) as a workroom. We need to connect to the mains...
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Regular house clean small 3 bed cottage
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new house on my house for my house because my house is not big enough for my house
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Managing your job is easier with the Rated People Homes App, download it today.