text:
Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1β, IL-8, IL-10, TLR-3 and TLR-9. IL-1β and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1β, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1β. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1β, IL-8 and TGF-β already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells.
Citation: Ingerslev H-C, Ossum CG, Lindenstrøm T, Engelbrecht Nielsen M (2010) Fibroblasts Express Immune Relevant Genes and Are Important Sentinel Cells during Tissue Damage in Rainbow Trout (Oncorhynchus mykiss). PLoS ONE 5(2): e9304. https://doi.org/10.1371/journal.pone.0009304
Editor: Jacques Zimmer, Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg
Received: January 20, 2010; Accepted: January 29, 2010; Published: February 18, 2010
Copyright: © 2010 Ingerslev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by The Directorate for Food, Fisheries and Agri Business Grant 3310-06-00116 and the Strategic Research Council grant 2101-08-0017. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Throughout the distant evolutionary lineage from the very primitive invertebrates to the phylogenetically more 'modern' vertebrates, an inflammatory reaction is established when tissue is damaged or infection is initiated [1]–[4]. The types of cells and the signalling molecules taking part in this process are dependent on the evolutionary position of the organism [5]. Among the vertebrates, the inflammatory response is in general characterised by recruitment of cells like polymorphnuclear neutrophilic leucocytes, monocytes and macrophages to the tissue of injury and/or infection [6], [7]. Invasion of these cell types mediate enhanced phagocytosis and expression of pro-inflammatory cytokines as IL-1β, TNF-α, and of numerous chemokines like IL-8 and other CXC chemokines [8]-[10]. The inflammatory immune reaction may not only be elicited by 'traditional' immune cells like macrophages and neutrophils that are recruited or already present in the tissue. Several reports suggests that in humans, the conventional non-immune cell-type fibroblast, also have immune regulating capabilities [11]–[13] and can thus serve as highly important sentinel cells [14]. Fibroblasts are the least specialised member of the connective tissue family, whose main function is to maintain the structural integrity of connective tissue by secreting precursors of extracellular matrix rich of type I and/or type-III collagen [15]. At the same time, human fibroblasts are known to express a wide range of cytokines. These include, among others, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-γ, CCL20 and macrophage colony stimulating factor [12], [16]. These cytokines are also known in lower vertebrates like the fish species rainbow trout and zebrafish (Danio rerio) [17]-[26]. In addition, human fibroblasts also respond to the secretion of cytokines like IL-1β, TNF-α e.g. by enhancing the production of CCL20 and hyaluronan [12], [27]. Besides expressing various Toll-like receptors (TLRs) [28] these characteristics indicate the presence of several other immune receptors on fibroblasts [12] and subsequent downstream pathways to convey signalling through these [29]–[31]. Further, some human fibroblasts have also been found to be phagocytic and capable of generating respiratory burst reactions as well as being responsive to lipopolysaccharide (LPS) from E. coli. [32]–[35]. The sensitivity to LPS most likely relates to the presence of CD14/TLR2/TLR4 receptors on the fibroblast surface, which further indicate that fibroblast serve an important sentinel function through pathogen-associated molecular pattern (PAMP) recognition [11], [32], [36]–[38]. It then seems reasonable that fibroblasts also respond to damage-associated molecular patterns (DAMPs) like necrotic cells and other hydrophobic portions (hyppos) from 'self' parts exposed through tissue damage [39].
Whether fibroblasts in lower vertebrates also exhibit phagocytic capacity and/or serve as sentinel cells is currently not known, but previous results from carp (Cyprinus carpio) could indicate this. Thirty minutes following injury of the skin, significant up-regulation of CXC receptors, IL-10 and TNF-α was reported [40]. This could indicate the presence of local, responsive cells that were already present in the tissue prior to injury. In lower vertebrates such as fish from the carp family, presence of TLR2 and TLR4 have been shown [41]. However, in evolutionary older fish species like the salmonids, they do not seem to be present although other TLRs have been reported [42]–[44]. Due to the phylogenetic position of fish, they are by choice considered to be an excellent animal to study immune mechanisms since it is the earliest divergent vertebrate, which have both an innate and adaptive immune system [1], [3]. Conversely, the scarce availability of antibodies in teleost research to date often hampers the opportunity for functional experiments. However, the ongoing sequencing of immune genes in fish makes real-time RT-PCR for measuring gene expression a good alternative. As in humans, the adaptive immune system in fish is characterised by the presence of antigen-specific receptors (T cell receptors), immunoglobulin on lymphocytes and MHC class I and II surface molecules [1], [45]. However, the immunoglobulin isotype repertoire in teleost fish is more limited than in e.g. mouse and humans and consists of only IgM, IgD and a unique fish isotype termed IgT [46], [47]. The specific immune cell-repertoire consists of T- and B-cell subpopulations, whereas the non-specific cells consist of phagocytic cells like monocytes/macrophages, granulocytes and natural killer cells [1], [48]. Several different fibroblast cell lines have been characterised from fish. In rainbow trout (Oncorhynchus mykiss) at least three fibroblast cell lines termed RTG-2, RTG-P1 and RTHDF exist of where the RTG-2 cell line is expressing the immune genes Mx, iNOS, IL-1β and IL-18 [49]-[55]. In order to examine the immune capabilities of fibroblasts in fish, an in vitro assay was setup using the rainbow trout RTHDF cell-line [53]. The cells were subjected to either LPS from E. coli, debris or supernatant from sonicated RTHDF cells in order to introduce fibroblast receptors to both PAMP and DAMP stimuli. Further, in order to examine the role of fibroblasts in vivo, a model of sterile, mechanical tissue damage of muscle tissue in rainbow trout was established. From both experiments, the muscle tissue and the fibroblasts were subsequently subject to real-time RT-PCR. Expression of the pro-inflammatory cytokines IL-1β and IL-8 as well as the anti-inflammatory cytokine IL-10 was measured since these are key molecules in the initial, inflammatory reaction [56]–[59]. The multifunctional gene transforming growth factor-β (TGF-β) was also included in order to show whether regenerative and proliferative responses were activated following tissue damage. Finally, we examined the expression of the four different TLRs; 3, 5, 9 and 22 in order to show if these were sensitive to PAMPs and/or DAMPs [42], [60], [61]. Together, this study provides new and important information about the role of fibroblasts in lower vertebrates in relation to inflammation, tissue damage and immune competence.
Materials and Methods
Culturing of RTHDF Fibroblasts
Rainbow trout hypodermal fibroblasts (RTHDF [53]) were cultured in Leibovitz L-15, supplemented with 15% (w/v) foetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 µg/ml) at 21°C and atmospheric air, as described previously [53]. Trypsin solution for cell detachment was made by dissolving 0.1% (w/v) trypsin and 1 mM disodium EDTA in phosphate-buffered saline, PBS [137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4] [62]. All cell culture reagents were used cold from the refrigerator. Cells were subcultured in 25 cm2 tissue culture flasks 3 days prior to the experiments and were confluent when harvested. The amount of cells per culture flask when confluent was estimated to 4.5×105 as earlier described [53]. All cell culture reagents were purchased from Life Technologies Inc. (Naperville, IL, U.S.A.) and cell culture plastic wear were purchased from TPR (Trasadingen, Switzerland). Chemicals were from Sigma Aldrich, unless otherwise stated.
Stimulation of RTHDF Fibroblasts with LPS, Cell-Debris and Supernatant
Confluent 25 cm2 cell culture flasks of RTHDF cells containing 3 ml of cell-culture medium were either incubated with sonicated RTHDF fibroblasts, the supernatant from the sonicate or E.coli 0111:B4 LPS (Sigma-Aldrich). The sonicate was prepared by an initial trypsination of confluent cell culture flasks followed by sonication for 30 s (amplitude 10 microns) using a MSE Soniprep 150 sonicator (Sanyo). The sonicate was then centrifuged for 2 min at 14.000× g and the clear supernatant was transferred to a new tube. The pellet containing necrotic cells and cell debris was thereafter resuspended in L-15 medium and used for stimulation. The RTHDF fibroblasts were then stimulated in triplicate of cell culture flasks per sampling point. The amount of sonicate added per flask of stimulated cells originated from one flask of RTHDF fibroblasts. The LPS stimulation was performed using 20 µg ml−1 of LPS and the amount of supernatant used was 100 µl per cell culture flask. Non-stimulated cells were used as controls. Harvesting of cells for isolation of total RNA was then performed 1 hour, 4 hours and 24 hours post stimulation. This was accomplished by removal of the cell culture medium from the flasks followed by addition of 500 µl of lysis buffer plus 5 µl of β-mercaptoethanol from the GenElute Mammalian™ Total RNA Miniprep Kit (Sigma-Aldrich). The cells were then scraped off the bottom of the flasks with a cell scraper (Greiner Bio-one) and RNA was isolated according to the manufacturers instructions.
Phagocytic Assay
Phagocytosis of the RTHDF fibroblasts was studied using carboxylate-modified polystyrene fluorescent latex beads with a diameter of 1.0 µm (Sigma-Aldrich) as described by Ganassin et al. [63]. A suspension of beads was prepared by adding 2 µl of the commercial latex suspension (2.5% solids latex) to 5 ml of growth medium, which replaced the regular growth medium. The cell-cultures were then observed at 1 hour, 4 hours and 24 hours after incubation and observations were made with a Nikon fluorescence microscope. Prior to observation, the cells were washed four times with PBS in order to wash away adhered, non-ingested beads.
Rearing Conditions of Mechanically Damaged Fish
Unvaccinated, healthy rainbow trout (Oncorhynchus mykiss) reared at Agerskov Dambrug (Bording, Jutland), where delivered to the experimental facilities at the National Institute of Aquatic Resources, Technical University of Denmark at 21 March 2007. The fish were then acclimatised one month prior to mechanical injury and maintained in 200 l plastic tanks with aerated local tap water at 14°C and exposed to a light regime of 16 hours of light followed by 8 hours of dark. Average weight (g ± SD) and length (cm ± SD) at the samplings were 9.5±2.6 and 9.5±0.9, respectively (n = 40). At each sampling point five injured fish and five control fish were collected. All procedures were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate permit 2007/561-1302 and in compliance with European Community Directive 86/609.
Procedures for Mechanical Injury and Tissue Sampling
Thirty fish were mechanically injured just below the caudal end of the dorsal fin at April 26 using a home made device containing twenty five sterile needles made from 19G syringes (Becton Dickinson) with a thickness of 1.1 mm equally distributed on an area of 6 mm×6 mm. The needles had a depth of 6 mm to ensure penetration of both the skin and underlying muscle tissue of the fish. Prior to mechanical injury, fish were anaesthetised in MS-222 (50 mg/l) (Sigma-Aldrich). The injury was performed posterior to the dorsal fin above the lateral line and the device was penetrated twice through the skin, giving rise to fifty holes per fish. Prior to sampling of muscle tissue, fish were killed in an overdose of MS-222. Sampling occurred at 4, 8 and 24 hours post injury using a sterile disposable scalpel. The tissue collected was 1) muscle tissue from the site of injury and 2) non-injured muscle tissue from the opposite side of the fish relative to the injury tissue and 3) muscle tissue from non-injured control fish. By sampling the internal control it was possible to show whether the responses in the injured fish were local or systemic. Tissue was collected from five injured and five control fish per sampling point and was transferred to cryo tubes containing RNA later® (Sigma-Aldrich) and stored at −20°C until isolation of RNA. For this, fifty mg of the sampled tissues were homogenised by sonication for 30 s (amplitude 10 microns) using a MSE Soniprep 150 sonicator (Sanyo) and RNA was further isolated using a GenElute Mammalian™ Total RNA Miniprep Kit (Sigma).
CDNA Synthesis
The RNA quality and quantity from the RTHDF cells and the muscle tissue was checked by OD260/280 measurements on a GeneQuant II Spectrophotometer (Pharmacia Biotech) and the RNA was finally treated with DNase-I (Sigma-Aldrich) to remove any genomic DNA. Random hexamer primed reverse transcription reactions were performed from 400 ng of total RNA in a 20 µl setup using TaqMan® Reverse Transcription reaction (Applied Biosystems). The synthesised cDNA samples were diluted 1:10 in MilliQ H2O and stored at −20°C.
Quantitative RT-PCR
Quantitative RT-PCR was performed using a Stratagene MX3000P™ real-time PCR system, dual-labelled TaqMan® probes conjugated with either a 5' HEX or a 5' FAM fluorophor, a 3' BHQ1 quencher and desalted primers (Sigma-Genosys). The assays for TLR-3, TLR-9 and TLR-22 examined were run using SYBR® Green (Sigma-Aldrich) instead of a probe. The genes chosen for investigation were IL-1β, IL-8, IL-10, TGF-β, TLR-3, TLR-5m (membrane bound form), TLR-9 and TLR-22. For use as an internal control and for normalisation of the results the reference gene ribosomal protein S20 (RPS20) and elongation factor-1α (ELF-1α) were validated for their transcriptional stability in muscle tissue and the RTHDF cells (data not shown). RPS20 was used within muscle tissue since it was more stably expressed between the different individuals and injured versus non-injured fish compared to ELF-1α. For the RTHDF cells, the elongation factor-1α gene was found more suitable [64]. The sequence for the primers and probes, amplicon length and GenBank accession numbers are shown in Table 1. The primers were optimised according to MgCl2 and primer concentrations. The cycling conditions for the TaqMan® assay were 94°C for 2 min followed by 40 cycles of 94°C for 15 s and 60°C for 1 min. The cycling conditions for the SYBR® Green assays were the same but the run was terminated by a melting curve analysis where the fluorescence was continually measured during a temperature increase from 60°C to 95°C. Wells for the TaqMan® assays contained 12.5 µl of JumpStart™ Taq ReadyMix™ (Sigma–Aldrich), 0.25 µl ROX (Sigma–Aldrich; diluted 10x), 2.5–5.5 mM MgCl2 (Sigma–Aldrich), 0.75–1.25 µl forward and reverse primer (10 mM), 1 µl TaqMan® probe (200 nM), 5 µl of diluted cDNA and autoclaved MilliQ water to a volume of 25 µl. Wells for the SYBR® Green assays contained 12.5 µl of SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich), 0.25 µl ROX (Sigma–Aldrich; diluted 10x), 3.5–5.5 mM MgCl2 (Sigma-Aldrich), 1 µl forward and reverse primer (10 mM), 5 µl of diluted cDNA and autoclaved MilliQ water to a volume of 25 µl. The expression results were analysed using the 2−ΔΔCt method after verification that the primers amplified with an efficiency of approximately 100% (doubling of the product between every cycle in the log-linear phase) and data were shown as −ΔΔCt-values and fold expression relative to non-injured control fish or non-stimulated RTHDF cells [65]. The threshold cycle (Ct) was determined manually and set to 0.01 in the lower level of the log-linear area. The statistical analysis was performed on the ΔCt values by a two-tailed T-test in cases of normally distributed data and Mann-Whitney U-test in cases when they were not normally distributed. The statistical software GraphPad Prism version 4.03 was used to calculate the statistics and create graphs.
Constitutive Expression of Selected Genes
All genes except for TLR-22 in the RTHDF cells were expressed within 40 cycles of PCR in non-injured tissue and non-stimulated cells (Figure 1). Except for TLR-9, the constitutive level of expression of the immune genes was higher in the RTHDF cells than in muscle tissue. The highest difference was seen for IL-1β and IL-8 that were approximately 20 and 330 folds higher expressed in the RTHDF cells compared to muscle tissue, respectively. The constitutive expression level of the housekeeping genes was lower for the RPS20 in the muscle tissue compared to the ELF-1α in the RTHDF cells and both genes showed a lower transcriptional variance between individuals/cell replicates than the immune genes.
The Ct-values for RPS20, ELF-1α, IL-1β, IL-8, IL-10, TGF-β, TLR-3, TLR-5m, TLR-9 and TLR-22 are shown for muscle tissue (black bars) and for RTHDF cells (white bars). The data are presented as mean expression of the control fish from all samples points and mean expression of control RTHDF cells from all sample points. The Ct value is defined as the threshold cycle number of PCR at which the sample fluorescent signal passes a fixed threshold above the baseline.
Expression in RTHDF Cells following Stimulation with LPS, Debris and Supernatant
Figure 2 A-E shows the effect of E. coli LPS (0111:B4), debris or supernatant from sonicated RTHDF cells on the expression of IL-1β, IL-8, IL-10, TLR-3 and TLR-9 in the RTHDF cells after 1, 4 and 24 hours of stimulation. No influence on the expression of TGF-β and TLR5m was seen and TLR-22 was not expressed in the RTHDF cells after 40 cycles of PCR (data not shown). Overall the variation in expression between cell replicates was much lower compared to individual fish. The highest effect on all genes was seen for IL-1β and IL-8 after stimulation with LPS. The expression increased significantly after 1 hour of stimulation for both genes peaked after 4 hours to 42.5 and 22 folds, respectively, followed by a decrease in expression to 9.4 and 9.9 folds after 24 hours, respectively (P<0.05). The pattern for IL-10 was different and the response was slower than for IL-1β and IL-8. IL-10 was only significantly expressed at 24 hours after stimulation by 2.7 folds relative to non-stimulated cells (P<0.05). Stimulation by debris revealed significant impact on only IL-1β, but the response was weak and only significantly elevated at 4 hours post stimulation by 2.2 folds (P<0.05). Supernatant from the sonicated RTHDF cells increased the expression of both IL-1β and IL-10, but weakly compared to the effects of LPS. The IL-1β expression increased to 1.9 folds after 4 hours of stimulation, while for IL-10 it was 2 folds after 24 hours relative to non-stimulated cells (P<0.05). TLR-3 and TLR-9 showed similar expression patterns. Both LPS and supernatant increased the expression significantly after 24 hours; LPS to approximately 4 fold for both genes and supernatant to 1.6 and 2.5 folds for TLR-3 and TLR-9, respectively (P<0.05).
Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) IL-10, (D) TLR-3 and (E) TLR-9. Black bars represent expression in E. coli LPS stimulated cells relative to control cells; white bars indicate expression in cells stimulated with debris from sonicated RTHDF cells relative to control cells and striped bars denotes expression in cells stimulated with supernatant from sonicated RTHDF cells relative to control cells. The data are normalised relative to the expression of elongation factor-1α and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from three cell replicates. * Depicts statistical significance between stimulated cells and control cells (*P<0.05; **P<0.01; ***P<0.001). A −ΔΔCt value of 0 means no regulation relative to control cells.
Phagocytic Assay
The addition of latex beads to the RTHDF cells did not indicate that the cells were able to phagocytose (data not shown). After incubation with beads and washing, a few beads were still attached to the glass slide surrounding the cells, and there was no indication that any beads were taken up and positioned inside the RTHDF cells.
Mechanical Injury of Muscle Tissue and the Effects on Gene Expression
The device used for mechanical tissue damage is shown in figure 3 A–B. The following effects of mechanical injury of the muscle tissue on the expression of the genes IL-1β, IL-8, TGF-β and TLR-3, 5 m, 9 and 22 at the time points 4, 8 and 24 hours post injury (p.i.) is shown in figure 4 A-F. No significant changes were seen for IL-10 (P>0.05) (not illustrated). The study showed a strong induction of immune related genes, especially of the cytokines IL-1β, IL-8 and TGF-β in the injured muscle tissue at all samplings. The average up-regulation in the injured fish for the three sampling points was between 5.2 to 21.8 folds for IL-1β; 20.0 to 59.3 folds for IL-8 and 4.9 to 13.9 folds for TGF- β. For all three genes the mean expression level was increasing from 4 hours to 24 hours post injury and the expression at the site of injury was significantly higher at all sampling points compared to the non-injured samples from the same fish (P<0.05).
The fish were injured on the left side behind the dorsal fin using the damage instrument. Muscle tissue samples were taken in the injured area while the internal control samples were taken from the same place relative to the dorsal fin on the right side of the fish (A). The vertical position of the site of injury/sampling site is shown in (B). Sampling of muscle tissue from non-injured control fish was performed in the same area as shown for injured fish (not shown).
Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) TGF-β, (D) TLR-3, (E) TLR-9 and (F) TLR-22. Black bars represent expression at the site of injury relative to control fish and white bars indicate expression at the non-damaged internal control site relative to control fish. The data are normalised relative to the expression of ribosomal protein S20 and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from five individuals. * Depicts statistical significance between injured fish and control fish (P<0.05); Δ denotes statistical significant difference between site of injury and internal control site (P<0.05). A −ΔΔCt value of 0 means no regulation relative to control fish.
The expression of the TLRs in muscle tissue showed significant changes in the expression for TLR-3, 9 and 22, but not for TLR-5m. TLR-3 was significantly up-regulated at the site of injury 24 hours post damage relative to the control fish corresponding to 4.6 folds while no changes was seen for the internal control samples (P<0.05). The expression of TLR-9, however, was significantly up-regulated at the injury site 4 hours and 24 hours by 2.5 and 7.2 folds, respectively (P<0.05). TLR-22 showed significant differences between injured and non-injured site 4 hours and 24 hours p.i. indicated by small down-regulations at the non-injured site of 3.2 and 2.8 folds, respectively (P<0.05). Twenty-four hours post injury, the expression of TLR-22 was significantly up-regulated by 5 folds at the site of injury relative to control fish (P<0.05).
Previous studies have shown that mammalian fibroblasts have immune regulatory capabilities due to their ability to express cytokines and immune receptors on their surface and their reactivity to pathogen associated molecular patterns (PAMPs) [12], [13]. In the present study it is demonstrated, by using the rainbow trout as a model, that these characteristics of the fibroblasts are evolutionary old and that Toll-like receptor and cytokine gene orthologoues to the mammalian counterparts are induced following stimulation in fibroblasts from this fish species. Further, it was shown that rainbow trout fibroblasts are reactive to damage associated molecular patterns (DAMPs). Seong and Matzinger (2004) proposed the DAMP model and according to this, any molecule that is normally not exposed can be a DAMP if it is revealed during, after or because of injury or damage [39]. Hence, in order to show if 'foreign' molecules were more potent inducers than 'self' in the fibroblasts, the sonicated fibroblasts were separated into debris containing cell-surface fractions and supernatant containing endogenous molecules like mitochondrial DNA and intracellular proteins [39], [66]. Interestingly, both debris and supernatant induced the expression of IL-1β, whereas only the supernatant stimulated IL-10, but IL-8 was not induced at all. However, debris might also contain 'foreign' parts of molecules and structures since the interior side of the cell wall is normally not exposed and could explain why debris stimulated the IL-1β expression. Together, this shows that fibroblasts potentially are able to take part in the inflammation in the body and react to signals released from injured cells. Worthy of note, the results also showed that only LPS induced IL-8 expression in fibroblasts in vitro, whereas neither fraction of the injured cells or supernatant could do so. Hence, subsequent neutrophil recruitment via IL-8 chemoattraction most likely relies on stimulation by PAMPs rather than by DAMPs. Alternatively, other cell types in the injured tissue could account for the increase in IL-8 expression observed in vivo. However, the constitutive expression of IL-8 was higher in the RTHDF cells than in muscle tissue, indicating that the contribution of IL-8 from fibroblasts is not without significance. In this study, the idea of looking into the immune regulating capabilities of fibroblasts in vivo was derived from the results that were obtained from the in vitro study using the RTHDF cell-line. From this, we hypothesized that local cells in the musculature of the rainbow trout were able to express cytokine genes following tissue damage and hence were able to react to DAMPs. Previously, it has been shown that skeletal muscle tissue in humans produce IL-6, IL-8 and IL-15, and it could be suggested that local fibroblasts contributes to this [67]. By applying the damage model in the rainbow trout, internal controls in muscle tissue from the injured fish opposite to the damaged side could be sampled. This gave a unique opportunity to show whether the responses were local or systemic. An activation of local cells was initiated at the injury site due to a rapid induction four hours post sampling of IL-1β, IL-8 and TGF-β. This did not occur within the internal control site. It seems realistic that an up-regulation earlier than four hours took place since the level of especially IL-8 was high four hours post injury. The in vitro experiment supports this idea, since LPS stimulated RTHDF cells expressed IL-1β and IL-8 significantly higher than control cells already one hour after stimulation. Further, in zebrafish an induction of H2O2 following tail cutting has been observed in the wound a few minutes after cutting, indicating a very rapid activation of local cells [68]. During the evolution from the early vertebrates (fish) to mammals a number of different TLRs have evolved. Fishes comprise a large heterogeneous group with a high degree of evolutionary distance between species, which is also reflected in the numbers and specific types of TLRs present. In the rainbow trout, five different TLRs named 3, 5, 9 and 22 are known, where TLR-22 is only found in fish [61]. In the evolutionary more 'advanced' pufferfish Fugu rubripes and the zebrafish Danio rerio, a complete set of TLRs orthologous to the ten mammalian TLRs is described [69]. In addition, they also express TLR-21 and 22, where TLR-21 is unique to fish [61]. Both the damage model and the fibroblast in vitro experiment showed that the TLR-3 and 9 genes were inducible by DAMPs at the site of injury and by the supernatant from the sonicated fibroblasts, but not by the debris. Hence, this was in accordance with the DAMP model proposed by Seong and Matzinger since endogenous proteins and molecules released following tissue damage were able to trigger the expression of immune genes [39]. Further, LPS induced TLR-3 and 9 expression in the fibroblasts showing that PAMPs could also stimulate these cells. In contrast, TLR-5 was not affected either in mechanically damaged tissue or in stimulated fibroblasts, indicating that this receptor is triggered by other ligands. TLR-22 was induced by mechanical damage in the rainbow trouts, but not expressed in the non-stimulated RTHDF cells. In the mammals, TLR-3 binds dsRNA, but is also a sensor of tissue necrosis during acute inflammation [70]. Our findings in the rainbow trout fibroblasts suggest that this characteristic is evolutionary old. It is not known whether TLR-3 and 9 are endogenous positioned in cells from rainbow trout, as generally seen in mammalian cells. However, although mostly expressed endosomally, TLR-3 have been shown to be surface expressed in human skin and lung fibroblasts, in certain human fibroblast cell lines and on epithelial cells. Whatever might be the case in fish; our results show that these receptors are inducible in the rainbow trout fibroblasts without the cells being able to phagocytise latex beads. This is in contrast to e.g. macrophages and granulocytes in the rainbow trout, which are phagocytic cell-types [48], [71]. Future studies using nano particles could reveal if the RTHDF cells are able to take up smaller particles [72]. Further, the ability of supernatant to induce TLR-9 expression in the fibroblasts showed that this receptor also functions as a sensor of necrosis. In the closely related species Atlantic salmon (Salmo salar), an increase in TLR-9 expression after CpG stimulation have been shown in lymphoid tissue indicating a homologue function of this receptor to the mammalian TLR-9 [73]. Since the salmonids lack TLR-2, it might therefore be suggested that TLR-3 and 9 in these species could have a similar role as TLR-2 in the mammals, where this receptor is also a sensor of necrotic cells, while TLR-9 in the mammals recognises bacterial and viral DNA, unmethylated CpG and DNA without CpG motifs [39], [74]. Together, these results indicate that evolutionary fibroblasts are primitive cells, which have maintained the ability to express genes of immune functions, when stimulated. Their function as being positioned in the tissue, as 'already there' before the more potent immune cells are recruited, seems advantageous when damage, injury or infection occurs. However, due to their lack of phagocytosing capability it seems that their immune capacity is not fully developed like traditional immune cells such as macrophages and granulocytes.
Author Contributions
Conceived and designed the experiments: HCI TL MEN. Performed the experiments: HCI CGO. Analyzed the data: HCI MEN. Contributed reagents/materials/analysis tools: HCI CGO MEN. Wrote the paper: HCI MEN.
- 1. Kaiser P, Rothwell L, Avery S, Balu S (2004) Evolution of the interleukins. Developmental and Comparative Immunology 28: 375–394.
- 2. Martin P, Leibovich SJ (2005) Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 15: 599–607.
- 3. Magor BG, Magor KE (2001) Evolution of effectors and receptors of innate immunity. Developmental and Comparative Immunology 25: 651–682.
- 4. Sepulcre MP, Alcaraz-Perez F, Lopez-Munoz A, Roca FJ, Meseguer J, et al. (2009) Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-kB Activation. J Immunol 182: 1836–1845.
- 5. Litman GW, Cannon JP, Dishaw LJ (2005) Reconstructing immune phylogeny: new perspectives. Nat Rev Immunol 5: 866–879.
- 6. Diegelmann RF, Evans MC (2004) Wound healing: an overview of acute, fibrotic and delayed healing. Front Biosci 9: 283–289.
- 7.
Murphy KP, Travers P, Walport M, Janeway C (2008) Janeway's immunobiology. New York: Garland Science.
- 8. Dinarello CA (1997) Interleukin-1. Cytokine Growth Factor Reviews 8: 253–265.
- 9. Kobayashi Y (2008) The role of chemokines in neutrophil biology. Front Biosci 13: 2400–2407.
- 10. Zhang W, Chen H (2002) [The study on the interleukin-8 (IL-8)]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 19: 697–702.
- 11. Hatakeyama J, Tamai R, Sugiyama A, Akashi S, Sugawara S, et al. (2003) Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system. Oral Microbiol Immunol 18: 14–23.
- 12. Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T (2005) Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin. Clin Exp Immunol 142: 285–291.
- 13. Chen B, Tsui S, Smith TJ (2005) IL-1 beta induces IL-6 expression in human orbital fibroblasts: identification of an anatomic-site specific phenotypic attribute relevant to thyroid-associated ophthalmopathy. J Immunol 175: 1310–1319.
- 14. Smith RS, Smith TJ, Blieden TM, Phipps RP (1997) Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation. American Journal of Pathology 151: 317–322.
- 15.
Alberts B (1989) Molecular biology of the cell. Garland Pub . New York:
- 16. Ruiz C, Perez E, Garcia-Martinez O, Diaz-Rodriguez L, Arroyo-Morales M, et al. (2007) Expression of cytokines IL-4, IL-12, IL-15, IL-18, and IFNgamma and modulation by different growth factors in cultured human osteoblast-like cells. J Bone Miner Metab 25: 286–292.
- 17. Yoshiura Y, Kiryu I, Fujiwara A, Suetake H, Suzuki Y, et al. (2003) Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits. Immunogenetics 55: 296–306.
- 18. Zou J, Grabowski PS, Cunningham C, Secombes CJ (1999) Molecular cloning of interleukin 1 beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site. Cytokine 11: 552–560.
- 19. Ohtani M, Hayashi N, Hashimoto K, Nakanishi T, Dijkstra JM (2008) Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish. Immunogenetics 60: 383–397.
- 20. Iliev DB, Castellana B, MacKenzie S, Planas JV, Goetz FW (2007) Cloning and expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus mykiss). Molecular Immunology 44: 1803–1807.
- 21. Sangrador-Vegas A, Lennington JB, Smith TJ (2002) Molecular cloning of an IL-8-like CXC chemokine and tissue factor in Rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization. Cytokine 17: 66–70.
- 22. Zhang DC, Shao YQ, Huang YQ, Jiang SG (2005) Cloning, characterization and expression analysis of interleukin-10 from the zebrarish (Danio rerion). Journal of Biochemistry and Molecular Biology 38: 571–576.
- 23. Laing KJ, Wang TH, Zou J, Holland J, Hong SH, et al. (2001) Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha. European Journal of Biochemistry 268: 1315–1322.
- 24. Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, et al. (2005) Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: The first Th1-type cytokine characterized functionally in fish. Journal of Immunology 175: 2484–2494.
- 25. Peatman E, Liu ZJ (2007) Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity. Immunogenetics 59: 613–623.
- 26. Wang TH, Hanington PC, Belosevic M, Secombes CJ (2008) Two macrophage colony-stimulating factor genes exist in fish that differ in gene organization and are differentially expressed. Journal of Immunology 181: 3310–3322.
- 27. Campo GM, Avenoso A, Campo S, Angela D, Ferlazzo AM, et al. (2006) TNF-alpha, IFN-gamma, and IL-1beta modulate hyaluronan synthase expression in human skin fibroblasts: synergistic effect by concomital treatment with FeSO4 plus ascorbate. Mol Cell Biochem 292: 169–178.
- 28. Kurt-Jones EA, Sandor F, Ortiz Y, Bowen GN, Counter SL, et al. (2004) Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J Endotoxin Res 10: 419–424.
- 29. Dziarski R, Gupta D (2000) Role of MD-2 in TLR2- and TLR4-mediated recognition of Gram-negative and Gram-positive bacteria and activation of chemokine genes. J Endotoxin Res 6: 401–405.
- 30. Takeuchi O, Akira S (2001) Toll-like receptors; their physiological role and signal transduction system. Int Immunopharmacol 1: 625–635.
- 31. Medzhitov R, Janeway C Jr (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173: 89–97.
- 32. Nemoto E, Sugawara S, Tada H, Takada H, Shimauchi H, et al. (2000) Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production. J Immunol 165: 5807–5813.
- 33. Skaleric U, Manthey CM, Mergenhagen SE, Gaspirc B, Wahl SM (2000) Superoxide release and superoxide dismutase expression by human gingival fibroblasts. Eur J Oral Sci 108: 130–135.
- 34. Abraham LC, Dice JF, Lee K, Kaplan DL (2007) Phagocytosis and remodeling of collagen matrices. Exp Cell Res 313: 1045–1055.
- 35. Arora S, Jain J, Rajwade JM, Paknikar KM (2009) Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol Appl Pharmacol 236: 310–318.
- 36. Uehara A, Takada H (2007) Functional TLRs and NODs in Human Gingival Fibroblasts. J Dent Res 86: 249–254.
- 37. Sugawara S, Sugiyama A, Nemoto E, Rikiishi H, Takada H (1998) Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide. Infect Immun 66: 3043–3049.
- 38. Kyburz D, Rethage J, Seibl R, Lauener R, Gay RE, et al. (2003) Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling. Arthritis Rheum 48: 642–650.
- 39. Seong SY, Matzinger P (2004) Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 4: 469–478.
- 40. Gonzalez SF, Huising MO, Stakauskas R, Forlenza M, Lidy Verburg-van Kemenade BM, et al. (2007) Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses to injury mimicking infection with ectoparasites. Developmental and Comparative Immunology 31: 244–254.
- 41. Jault C, Pichon L, Chluba J (2004) Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Molecular Immunology 40: 759–771.
- 42. Rodriguez MF, Wiens GD, Purcell MK, Palti Y (2005) Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics 57: 510–519.
- 43. Skjaeveland I, Iliev DB, Zou J, Jorgensen T, Jorgensen JB (2008) A TLR9 homolog that is up-regulated by IFN-gamma in Atlantic salmon (Salmo salar). Developmental and Comparative Immunology 32: 603–607.
- 44. Purcell MK, Smith KD, Aderem A, Hood L, Winton JR, et al. (2006) Conservation of Toll-like receptor signaling pathways in teleost fish. Comparative Biochemistry and Physiology D-Genomics & Proteomics 1: 77–88.
- 45. Hordvik I, Grimholt U, Fosse VM, Lie O, Endresen C (1993) Cloning and Sequence-Analysis of cDNAs Encoding the MHC Class-II Beta-Chain in Atlantic Salmon (Salmo salar). Immunogenetics 37: 437–441.
- 46. Hansen JD, Landis ED, Phillips RB (2005) Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proceedings of the National Academy of Sciences of the United States of America 102: 6919–6924.
- 47. Warr GW (1995) The Immunoglobulin Genes of Fish. Developmental and Comparative Immunology 19: 1–12.
- 48.
Secombes CJ (1996) The nonspecific immune system: Cellular defences. In: Iwama GaN T, editor. The fish immune system: Organism, pathogen, and environment. New York: Academic Press. pp. 63–103.
- 49. Collet B, Secombes CJ (2001) The rainbow trout (Oncorhynchus mykiss) Mx1 promoter - Structural and functional characterization. European Journal of Biochemistry 268: 1577–1584.
- 50. Trobridge GD, Leong JAC (1995) Characterization of a Rainbow-Trout Mx-Gene. Journal of Interferon and Cytokine Research 15: 691–702.
- 51. Collet B, Boudinot P, Benmansour A, Secombes CJ (2004) An Mx1 promoter-reporter system to study interferon pathways in rainbow trout. Developmental and Comparative Immunology 28: 793–801.
- 52. Wolf K, Quimby MC (1962) Established Eurythermic Line of Fish Cells in Vitro. Science 135: 1065–&.
- 53. Ossum CG, Hoffmann EK, Vijayan MM, Holt SE, Bols NC (2004) Characterization of a novel fibroblast-like cell line from rainbow trout and responses to sublethal anoxia. Journal of Fish Biology 64: 1103–1116.
- 54. Zou J, Bird S, Truckle J, Bols N, Horne M, et al. (2004) Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss). European Journal of Biochemistry 271: 1913–1923.
- 55. Wang TH, Zou J, Cunningham C, Secombes CJ (2002) Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss). Biochimica Et Biophysica Acta-Gene Structure and Expression 1575: 108–116.
- 56. Bowie A, O'Neill LAJ (2000) The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. Journal of Leukocyte Biology 67: 508–514.
- 57. Stylianou E, Saklatvala J (1998) Interleukin-1. International Journal of Biochemistry & Cell Biology 30: 1075–1079.
- 58. Secombes CJ, Bird S, Cunningham C, Zou J (1999) Interleukin-1 in fish. Fish & Shellfish Immunology 9: 335–343.
- 59. Commins S, Steinke JW, Borish L (2008) The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J Allergy Clin Immunol 121: 1108–1111.
- 60. Rebl A, Siegl E, Kollner B, Fischer U, Seyfert HM (2007) Characterization of twin toll-like receptors from rainbow trout (Oncorhynchus mykiss): evolutionary relationship and induced expression by Aeromonas salmonicida salmonicida. Dev Comp Immunol 31: 499–510.
- 61. Oshiumi H, Tsujita T, Shida K, Matsumoto M, Ikeo K, et al. (2003) Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome. Immunogenetics 54: 791–800.
- 62. Ma C, Collodi P (1999) Preparation of primary cell cultures from lamprey. Methods Cell Sci 21: 39–46.
- 63. Ganassin RC, Bols NC (1996) Development of long-term rainbow trout spleen cultures that are haemopoietic and produce dendritic cells. Fish & Shellfish Immunology 6: 17–34.
- 64. Ingerslev HC, Pettersen EF, Jakobsen RA, Petersen CB, Wergeland HI (2006) Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol 43: 1194–1201.
- 65. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25: 402–408.
- 66.
Alberts B (2008) Molecular biology of the cell. New York: Garland Science. 1 v. (various pagings) p.
- 67. Nielsen AR, Pedersen BK (2007) The biological roles of exercise-induced cytokines: IL-6, IL-8, and IL-15. Appl Physiol Nutr Metab 32: 833–839.
- 68. Niethammer P, Grabher C, Look AT, Mitchison TJ (2009) A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459: 996–999.
- 69. Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, et al. (2004) Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish. Mol Immunol 40: 773–783.
- 70. Cavassani KA, Ishii M, Wen H, Schaller MA, Lincoln PM, et al. (2008) TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events. J Exp Med 205: 2609–2621.
- 71. Ganassin RC, Bols NC (1998) Development of a monocyte/macrophage-like cell line, RTS11, from rainbow trout spleen. Fish & Shellfish Immunology 8: 457–476.
- 72. Shan X, Liu C, Yuan Y, Xu F, Tao X, et al. (2009) In vitro macrophage uptake and in vivo biodistribution of long-circulation nanoparticles with poly(ethylene-glycol)-modified PLA (BAB type) triblock copolymer. Colloids Surf B Biointerfaces.
- 73. Strandskog G, Skjaeveland I, Ellingsen T, Jorgensen JB (2008) Double-stranded RNA- and CpG DNA-induced immune responses in Atlantic salmon: comparison and synergies. Vaccine 26: 4704–4715.
- 74. Kumagai Y, Takeuchi O, Akira S (2008) TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev 60: 795–804.


text:
Veterinary Science & Medical Diagnosis Journal is an international, Online Open Access, peer reviewed journal that mainly focus on latest research in the fields of veterinary and animal husbandry. It covers all clinical, medical, scientific and technological aspects of veterinary sciences, anatomy, physiology, biochemistry, pharmacology, microbiology, pathology, parasitology, infectious diseases, alternative veterinary medicine and other biomedical aspects.
Aim and Scope
Our aim is to serve the scientific knowledge to the readers with unlimited free access. We consider research manuscripts, review manuscripts, editorials, commentaries, opinion pieces, case studies, case reports, clinical images, letters and perspectives. Editorials, perspectives and opinions are submitted by authors and editorial board members on topics pertaining to Veterinary Science.
All published articles are assigned to Digital Object Identifier (DOI) - Crossref.
All published articles are permanently archived and available at Gavin Publishers website in HTML and PDF formats.
· Veterinary Physiology
· Veterinary Pathology
· Microbiology and Medical Ethics
· Animal Nutrition and Veterinary Pharmacology
· Animal Husbandry
· Veterinary Diagnosis & Treatment of animal diseases
· Animal Vaccination & Veterinary immunization
· Translational/Pre-clinical Studies in Animal Models
· Animal Anatomy
· Animal Nutrition
· Animal Surgery
· Animal Antibiotic resistance
· Animal Parasitology
· Animal Infectious diseases
· Animal transmitted diseases
· Alternative veterinary medicines and biomedical fields
· Animal Breeding and experimental animals
· Environmental impacts of Veterinary practices
· Veterinary dentistry
· Veterinary Ophthalmology
· Animal Health
· Large/small animal medicine
· Animal Production and Management
· Wildlife diseases
· Wildlife management
· Alternative Veterinary Medicine
· Current Research in Diagnosis
· Veterinary hygiene
· Animal behaviour/ management
· Nutritional deficiency diseases
· Zoonosis

subset: commoncrawl


text:
565 Plus XLO Contact Insecticide
565 Plus XLO Contact Insecticide Details:
Prescription Treatment 565 Plus XLO Contact Insecticide is a non-residual contact insecticide that is specially formulated to provide instant knockdown of a long list of insects including ants, bed bugs, flies and mosquitoes. 565 Plus XLO Contact Insecticide's active ingredient is pyrethrins (.50%) and is derived from the Chrysanthemum flower so insect resistance is not a concern.
- Offers broad spectrum killing power to help combat a wide range of flying and crawling insects
- Use as a space spray for flying insects, pantry pests, stored product pests
- Provides quick action that attacks an insect's central nervous system
- Low odor formula
Target Pests
Ants, Bed Bugs, Booklice, Carpet Beetles, Centipedes, Chocolate Moths, Cigarette Beetles, Clothes Moths, Clover Mites, Granary Weevils, Horn Flies, House Flies, Indianmeal Moths, Mediterranean Flour Moths, Millipedes, Mosquitoes, Mud Daubers, Red Flour Beetles, Rice Weevils, Sawtoothed Grain Beetles, Silverfish, Small Flying Moths, Sowbugs, Spiders, Stable Flies, Ticks, Wasps, Angoumois Grain Moths, Cluster Flies, Cockroaches, Confused Flour Beetles, Crickets, Drugstore Beetles, Fleas, Flies, Fruit Flies, Gnats, Grain Mites
Active Ingredient:
Pyrethrin .50%
565 Plus XLO Contact Insecticide Product Label
565 Plus XLO Contact Insecticide SDS
Always read and follow label directions when using any product.
Shipping Information
All orders are shipped the same or next business day. We can only ship to addresses in the USA due to Customs restrictions in other countries.
Free shipping: Standard Free Shipping is UPS Ground or USPS First-Class (5-7 business days).
Aerosols must ship Ground. There is no Expedited shipping available for this product.


text:
We will likely talk about the best way to submit the pieces of your software, together with the timing associated with every bit. Knowledge the parts of your respective software, together with the admission prerequisites, will let you assemble a more robust application All round.
The Listening and Framework sections ended up Laptop or computer-adaptive, meaning that the difficulty volume of Each individual question depended on the correctness of previous responses. Considering that the computer have to rating each query to be able to choose the next one, the student had to reply Each individual problem as it had been introduced.??portion. If it is necessary for you to go ahead and take revised paper-sent test, which happens to be scored from 0-30 on Every of three sections, we commonly require a minimum amount complete rating of seventy five.|Its reason is to arrange Intercontinental college students for studying within an English-speaking region or program. Our Exercise TOEFL is right here to provide you with a simulation of your articles and is a superb primer whether you go ahead and take paper-primarily based (PBT) or Net-primarily based exam (iBT). Just about every testing style is damaged up into four sections.|When you've got taken the take a look at and want to post suggestions regarding your knowledge, be sure to fill out and post our feed-back sort}
Here is the official TOEFL guide penned by ETS, the Group that makes and administers the Test, so it is among the most exact source on the format on the Test along with the written content it handles. Right after introducing the overall structure from the TOEFL, the book devotes a chapter to every portion in the Test.
After getting your TOEFL vocabulary flashcards, you?�ll experience those cards one by one. For every card whose definition you are aware of easily, you?�ll put it in a ??Know It??pile. In the event you don?�t know the definition of a particular TOEFL word, put it in a ?�Struggled??pile.|IELTS enables you to: start your journey into work or analyze Out of the country verify your English language skills choose a module to match your aim ??Academic or Common Training.| For each TOEFL dilemma style, more Highly developed tactics are furnished, and also the section finishes with final learning techniques for undertaking properly on that A part of the TOEFL.|Lương Xuân Trường t�?tin "bắn" tiếng Anh tr�?lời phỏng vấn của AFC - Duration: 98 seconds.| These could make finding out far more productive and quicker than It will be without having a prep e book because you?�ll learn The simplest way to answer concerns without having to do demo-and-mistake strategies all on your own.|After the Examination day by itself, The main time to get a test-taker is the ideal ahead of the Examination. It may seem counterproductive, although the working day prior to the take a look at really should not be employed for researching or refreshing. Students should really try out to not Consider an excessive amount with regard to the TOEFL. Take it easy and check out to clear your intellect as a substitute. Pupils frequently stress by themselves out unnecessarily suitable before an Test. Make sure your whole tests components are Prepared and you've got your Instructions towards the testing Centre along with your testing registration printed out the night time ahead of. Be sure to get a great night time?�s rest the night time ahead of, but try out never to deviate out of your every day routine too much.|It contains six responsibilities, two unbiased duties and 4 integrated jobs. In The 2 independent responsibilities, college students must response viewpoint questions on some aspect of tutorial daily life. In two built-in looking at, listening, and speaking responsibilities, college students will have to examine a passage, hear a passage, and discuss regarding how the Tips in The 2 passages are relevant.|In untimed mode, people can choose solutions in Studying and Listening that will enable you to Examine the correctness within your solutions, and suggest no matter whether you might have guessed or not. It is possible to reply to as numerous queries as you desire for every on the internet session.|DO discuss in English around you maybe can when talking to family and friends users who're fluent by themselves.|Speaking - learners read passages and response thoughts on a number of subjects. These replies are digitally recorded and despatched to exam judges to generally be tested.|Pursuing Every phrase could be the definition, synonyms, and instance sentences. Following the text are launched, the chapter includes sample queries to check your knowledge of the terms.|Purchase analyze materials and just take as quite a few practice examinations as you could, especially in the previous couple of months in advance of check day.|While the numerous observe components are sufficient for some pupils, lots of Other people do most effective getting acquired one-on-a single coaching from a native English speaker. From the fluent mentor a student might get individualized help, and target parts where by they are having difficulties most. Quite a few pupils attest that using a TOEFL mentor who was fluent within the language assisted their scores to substantially improve.|DON'T suppose immersion is the one answer. A lot of what you may come upon on this test is academic in character, so examine formal English about you do the pop culture stuff.|Will not get disappointed if you are not pretty much as good at English on test working day as you wish to be. You may still pass the TOEFL no matter whether you happen to be a specialist or not.|Exercise assessments are the most effective means to organize to the TOEFL. We?�ve gathered information on the very best-top quality TOEFL follow assessments into one particular position so you may have usage of the most beneficial analyze materials.|Caution: Be sure to study your test Centre?�s information completely. There are numerous prohibited objects even though during the tests home like electronic devices and digital watches. Scoring|This site also consists of vocabulary exams, such as apply tests for the academic wordlist, as well as grammar tests which can be related to IELTS. Level and scores|Some customers have complained of complex difficulties with the web and cellular options. Most commonly, consumers have claimed that some take a look at thoughts have incorrect answers and that the cellular sources are tough to see and navigate.|Because the exam is new to a lot of scholars, the textbooks do a superb occupation of giving potential exam takers a good evaluate the things they can assume to find out on check working day. TOEFL Programs|Once you?�ve gone through each with the playing cards after, pick up your ?�Struggled??pile. You?�ll now go through this pile again. For words and phrases in which you know the definition, area them within a second ??Know It|Realize it|Understand it|Comprehend it}??pile beside (although not combined with) the very first ??Know It|Realize it|Understand it|Comprehend it}??pile.|Once you have your TOEFL word record that you must examine, you?�ll want to operate on memorizing their definitions. You don?�t should memorize the sample sentences, but it is best to critique them to make sure you understand more info how the phrase is applied.| You can also make your very own TOEFL flashcards or use a web site such as Quizlet which helps you to make on line flashcards without cost out of your TOEFL vocabulary list.|eight entire-size practice tests, to simulate the complete check working experience Producing and speaking samples evaluated by Kaplan industry experts Added Sources:|The PBT checks fundamentally the identical skills since the CBT, albeit with some discrepancies, significantly the amount of thoughts (that's greater during the PBT) and also the rating scales.|For terms you don?�t know, come up with a new ?�Struggled??pile. Repeat this method, placing new ??Know It|Realize it|Understand it|Comprehend it}??piles in the row from still left to ideal. This generates your waterfall. Repeat this process right until the ?�Struggled??pile has just a few words and phrases still left.|as common a tourist attraction than as preferred a tourist attraction as as much more preferred a tourist attraction as Suitable Erroneous Make use of the comparative phrase 'as + adjective + as' to indicate equality. eight. Although most New Yorkers have confidence in ghosts, ______ usually do not.}
The exam may perhaps mandate that you simply fill out tables full summaries. The good news is, you don't have to have read more to come back in the TOEFL with a previous idea of The subject at hand to be able to get the best respond to.
Conveys and understands only typical that means in really common situations. Recurrent breakdowns in communication arise.
Products and here solutions can be use in timed or untimed manner; timed method simulates the true TOEFL iBT; untimed is better for observe
Segment 3 can be a conversation between two major speakers (for example, a dialogue between two College students, Most likely click here guided by a tutor)
Your Test payment need to be designed within just three times of registration. The simplest way to pay for is through the use of our safe on the web bank card payment technique:
Along with substantial-excellent follow exams, these TOEFL preparation books also consist of in-depth respond to explanations to help you realize why a certain response is proper. extended passages and questions on the passages. The passages are on educational subjects; they are The type of fabric That may read more be found in an undergraduate College textbook. Pupils remedy questions on major Strategies, facts, inferences, sentence restatements, sentence insertion, vocabulary, operate and All round ideas.|When you have completed your exercise, Examine these best TOEIC tips to help improve your score.|Just more than 50 percent (51%) of take a look at takers get IELTS to enter greater education in a very international nation.[32] The IELTS minimum scores required by educational institutions and by course differ.|There was a least cut-off date of 90 times just before which anyone wasn't allowed to retake the test. Even so this restriction is withdrawn and presently there is not any Restrict for applicants to retake the exam.|For this follow session, any words which you see in red are terms you should hear over a recording in the real check. Any words and phrases the thing is in blue are phrases you would probably read within your test booklet.|Your Test payment has to be made inside of five days of registration. The easiest way to pay is through the use of our protected on the internet charge card payment method:|Pupils applying for grasp's stage CDM courses who get an English proficiency rating under the minimum prerequisite and who fulfill all other necessities for admission to an instructional software may be granted conditional admission to your blended CDM/ELA program.|Regardless of whether English was the language of instruction at your college, for those who did not analyze in one of those nations around the world You aren't exempt within the prerequisite. It's essential to submit a transcript that demonstrates you attended school in among the list of approved areas, and that the academic application was a minimum of two several years in duration.|New kinds of inquiries in the iBT call for paraphrasing, filling out tables, or finishing summaries. Generally prior understanding of the topic less than discussion just isn't important to come to the proper answer, though a priori information may possibly support.|The essay was study by two screening evaluators. Each gave the essay a rating. The two scores are averaged to generate the final Composing scores. In case the evaluators ended up much more than one place various within their assessment, a third evaluator scored the essay.|If you still have concerns right after talking to your graduate discipline, electronic mail firstname.lastname@example.com For more info.}
Want more info on how the TOEFL is scored? Consider our information to understand specifically how the TOEFL is scored so you can maximize the volume of factors you receive.
Just after you choose to sign-up for the take a look at at our locale, Listed below are methods for filling in the web registration:
The jobs here are damaged up into built-in and impartial. With the integrated, you might read a passage and hear a speaker discuss it. Then, you may generate a summary that specifics the entire crucial details within the listening passage and displays how it connects to the main takeaways with the studying passage. From the independent activity, you can merely create an essay on a well-recognized subject matter..|A replica within your ID needs to be submitted within just three times of registration. The next kinds of ID will likely be acknowledged for registration and identification:|ILSC Toronto proudly announces that from February onwards it's going to supply large-high quality wireless headsets for listening tests at all its venues. So hurry up and sign up for virtually any with the impending assessments at to benefit from the condition-of-the-art technology for a better listening working experience!|When you've got taken the exam and want to submit feed-back regarding your knowledge, remember to fill out and submit our responses sort}}
The 2-Minute Rule for toeic
We will likely talk about the best way to submit the pieces of your software, together with the timing associated with every bit. Knowledge the parts of your respective software, together with the admission prerequisites, will let you assemble a more robust application All round.


text:
This package is another great option to dive into the deep end of Council Tool premium axe goodness! This axe package contains 1 of each axe we carry that are within the Council Tool Co. "Wood Craft" premium axe line. When you purchase this package you receive two axes that have stolen my heart. This package is everything you'll need around the camp and on the hike to your camp!
**This package qualifies for free shipping!**
Package includes one of each:
1) 24" Pack Axe w/leather mask - 2lbs
2) 19" Pack Axe w/leather mask - 2lbs
3) 16" Camp Carver with leather mask
4) American Pioneer Sharpening Puck - just for fun!
A $437 total value that can be yours for $405!


text:
The Town Planning Board today (February 20) announced amendments to the approved Kwai Chung Outline Zoning Plan (OZP).
The amendments mainly involve the rezoning of a site at Kwai Yi Road from "Government, Institution or Community" and 'Road' to "Residential (Group E)1" for public rental housing development; rezoning of Tai Wo Hau Road Park from "Residential (Group A)" and 'Road' to "Open Space" and a site at 33 Wo Yip Road from "Comprehensive Development Area" to "Commercial(2)" to reflect their current uses; and revisions to the Notes to incorporate the latest refinements to the Revised Master Schedule of Notes to Statutory Plans.
The draft Kwai Chung OZP No S/KC/22 incorporating the amendments is now available for public inspection during normal office hours at the Secretariat of the Town Planning Board, the Planning Enquiry Counters in North Point and Sha Tin, the Tsuen Wan and West Kowloon District Planning Office and the Kwai Tsing District Office.
Any person can make written representation in respect of the amendments to the Secretary of the Town Planning Board on or before April 20. Representations made to the Town Planning Board will be available for public inspection.
Copies of the draft OZP are available for sale at the Map Publications Centres in North Point and Yau Ma Tei. The electronic version of the OZP can be viewed on the Town Planning Board's website at (http://www.info.gov.hk/tpb).
Ends/Friday, February 20, 2009
Issued at HKT 16:00


text:
The Festivals Company is proud to announce that the Purple Poppadom from Cardiff has been crowned Welsh Curry House of the Year 2012. The announcement was made by Rt Hon Carwyn Jones AM, First Minister of Wales at a glittering Awards Gala Dinner at The Red Dragon Centre, Cardiff Bay on Monday 23 April 2012.
"We knew the National Award would eventually go to South Wales but we didn't think it would take six years. The Purple Poppadom is a restaurant that represents a vision of the future but is a reality we can enjoy today. Imagination, flair and confident execution were all in evidence at this fine Indian restaurant which we believe can happily compete on the world stage. This is a restaurant that Cardiff and Wales can be proud of. The Purple Poppadom is a new restaurant that lives up to the hype. The attention to detail is quite spectacular and at the heart of the dining experience is one of the best chefs in the world, Anand George," said Niaz Taj, the Jury Chair.
"The competition is committed to celebrating the fine work of the 280 Indian restaurants we have in Wales. We are also committed to encourage the sector to use more locally sourced Welsh ingredients like Welsh Lamb. The response to the Welsh Lamb Curry dish which was offered in the top 30 restaurants confirmed a demand by the public for food using locally sourced ingredients. We would like to see such ingredients become a permanent part of menus in restaurants all over Wales," said Berwyn Rowlands from the Festivals Company.
The judges who were tasked with choosing the National Winner were:
• Niaz Taj, Jury Chair and Manager of the Spice Route.
• Shuel Ahmed, winner of the competition in 2008 and 2011 from the Sheesh Mahal in Llanelli.
• Bonny Young from London returns as a judge having travelled extensively in India.
• Sirajul Islam returns as a judge having spent 40 years in the Indian Restaurant sector.
A total of five awards were presented during the evening and the winners were:
• Purple Poppadom, 185 Cowbridge Road East, Cardiff. CF11 9AJ (NATIONAL WINNER)
• The Suhail, 12 Bastion Road, Prestatyn. LL19 7ES (NORTH WALES WINNER)
• Bengal Spices, Ashfield House, Llanymynech. SY22 6ER (MID WALES WINNER)
• Purple Poppadom, 185 Cowbridge Road East, Cardiff. CF11 9AJ (SOUTH WALES WINNER)
• Bombay Blue, 27a Cardiff Road, Caerphilly. CF15 7SS (BEST DESIGN)
The Welsh Curry House of the Year competition will return in 2013!


text:
Healthy Joyful Living
France Rentals
Books and E-Books
Our Paintings
Contact Us
Recipes from a Novice
France Rentals
Books and E-Books
Healthy Joyful Living
Our Paintings
Recipes from a Novice
Contact Us
January 2010
If you enjoy this newsletter, we would love it if you would forward it to one or two friends and have them sign-up by clicking
January 2010
SF Examiner.com
Subscribe to the Entrepreneurial Lifestyle
column and receive it directly to your mailbox.
Register for the Free Teleseminar
February 25th at 10AM PST
Two Page Integrated Business
and Lifestyle Plan
Help us with the subtitle for our new book.
Stay tuned for our new Book,
Courage and Croissants
We are still playing with the best subtitle, so help us out.
Go to
Survey Monkey
and vote on the subtitle of your choice.
Lifestyle Corner
One Easy Step to Enjoying a Meal with Friends
As many of you know, the French are well known for their two hour lunches. What you might not know is they are also known for their five hour dinners. In contrast, my personal research in California has shown me that most dinner parties with friends last an average of three hours (7-10pm). So what's the difference I ask?
The difference is the intimacy that occurs in those additional 2 hours. The depth of conversation that takes place. The laughter that occurs. The satiation of eating a long slow dinner.
As Julia Child said, "
It's fun to get together and have something good to eat at least once a day. That's what human life is all about--enjoying things."
Three tips to making a dinner with friends memorable:
Focus on simple and beautiful
. Add a sprig of rosemary or mint on each plate for decoration or a dabble of goat cheese and dried cranberries on your salad. It doesn't take much to make it taste good and serve as a conversation piece.
Spread the word that it will be a long relaxing evening.
In the states there is a feeling that we must be in bed before it is late or the next day will be wasted. The day is scheduled and there is much to do (even if it is Sunday). Let your guests know that you want this to be a special evening when no one rushes and they can kick up their feet. Plan places for kids to fall asleep or watch a stack of DVDs.
Plan some conversation starters.
The key to a great evening (besides the food and wine) is the conversation, sharing, and bantering. The joy is in the debate, the philosophizing, and learning from each other. It is not a conversation just about what you are doing, but about what you are feeling, thinking, and wondering about.
If you are concerned about what you are going to talk about all evening, have everyone send in or write a question that is on their mind. Throughout the evening choose questions out of the hat and share what everyone thinks.
Take a night and tell everyone to plan on staying until midnight. To know it is a night of conversation, of pleasure, of laughter, of good food. And then create the environment and see what happens! You will be surprised!
Read More at my
Entrepreneurial Corner
Make the First Hour Each Day Your Hour for Creating your Dreams
Starting your day with a great cup of coffee or tea is, of course, a must, but then what do you do once you settle into your desk at work? For most people, the answer is that they comb through their emails. They respond to what is important, click on a few items of interest and then spend another 15 minutes checking out a few blog posts, twitter discussions, and other social networking sites.
For the next month I urge you to try something different and see if it increases your productivity, creativity, visibility, and helps you to achieve some major marketing goals you have established for yourself.
Use the First Hour of Each Day to
Spend the first hour on making yourself visible to your customers. A few suggestions include:
Write a blog post and then tweet about it. Make sure it is linked to Facebook, LinkedIn, and other sites.
Research, write, or edit an article. Use the article for a free giveaway, a blog post, an ezine. After one year you will have 52 articles if you do one a week.
Comment on a blog post, a tweet discussion, or articles. Your focus is to get your name out there as someone who has something important to add to the conversation. Comment on blogs who are authored by people you admire, can help you in some way, or are your target market.
Yes, you can do this any time of the day and yet we never seem to find the time to consistently make ourselves as visible as we would like.
Try it for a week or a month, if you can, and let me know the results as you will surprise yourself.
Read More on
Entrepreneurial Life Coaching
for building a successful service business and enjoying life.
Book Review
The Right Questions
by Debbie Ford
I recently ran across an older book by Debbie Ford entitled, The Right Questions- Ten Essential Questions to Guide You in an Extraordinary Life.
A quick but fun read that has huge impact on how you make small and large decisions in your life. As she so elegantly says, "We are where we are because of the decisions we make every day. If we want to better understand how we have created our present-day reality, all we need to do is look at the choices we made and continue to make along the way".
Asking yourself and others the simple question such as, "Is this decision leading you towards what you want in life or away from it?" makes you stop, think and make more conscious choices.
Each day we are making thousands of small decisions that will lead us towards the lives we love living. The journey itself is fantastic if we see ourselves making these empowered choices. No matter how long it takes to achieve our goals or dreams, if we can feel empowered every day that we are on our way; we begin to realize how wonderful the journey itself is as well as achieving and living our dreams.
For More
Book Recommendations
please visit my blog.
The Road Not Taken
The Road Not Taken by Robert Frost is a poem, my 10 year old daughter has to memorize in school this week. Wow, at 10 she truly understands what it means to make choices that make a difference in her life.
In practicing it with her, I recognized how this poem is as relevant today as it was when Frost wrote it (1874-1963). Take a few minutes and read this poem and realize how we all have to choose different roads in different times in our lives and hopefully our choices have made all the difference.
The Road Not Taken
by Robert Frost
Two roads diverged in a yellow wood
And sorry I could not travel both
And be one traveler, long I stood
And looked down one as far as I could
To where it bent in the undergrowth;
Then took the other, as just as fair,
And having perhaps the better claim
Because it was grassy and wanted wear,
Though as for that the passing there
Had worn them really about the same,
And both that morning equally lay
In leaves no step had trodden black.
Oh, I marked the first for another day!
Yet knowing how way leads on to way
I doubted if I should ever come back.
I shall be telling this with a sigh
Somewhere ages and ages hence:
Two roads diverged in a wood, and I,
I took the one less traveled by,
And that has made all the difference.
We want to hear from you!
Our goal is to support men and women between 40-60 who are going through transitions, redefining their lives, and often wanting to step out to become solopreneurs, consultants, and creative professionals.
Let us know how we can help you or what you want to hear more about! We are trying to build our list and reach people just like you. Please forward this newsletter on to someone you think might enjoy it and ask them to
We thank you in advance!
Contact us for a 30 minute complimentary consultation.
Bon Courage – Go with Courage
Suzanne and Jean-Pierre
Follow me on:
To unsubscribe from this list visit
this link
To update your preferences visit
this link


text:
Gloria Trevi and Alejandra Guzman Kiss on Lips on Stage
Alejandra Guzman and Gloria Trevi surprised fans by kissing on stage.
Everyone is talking about their kiss at their concert…but, truth be told, it was a simple peck on the lips.
The two singers were talking about their differences and similarities on stage and how much they admire each other when fans started asking for a kiss.
They simply obliged.
Related posts:
Previous: OV7 Show Plan B Canceled
Next: Kabah in Boat Accident During Video Shoot


text:
The equation of a straight line takes the form y=mx+c, where m is the gradient (slope) of the line and c the y-intercept (The value of y where the line crosses the y-axis). The values of m and c can be calculated if two points on the line are known. If these points are denoted by (x1,y1) and (x2,y2), then y1=mx1+c and y2=mx2+c. From this, it follows that y2-y1=m(x2-x1). The value of m can now be found. To find c, insert the known values into the equation for y1 or y2 and rearrange.
A quadratic equation is one of the form y=ax²+bx+c, where a, b and c denote constants. The resulting curve is a parabola. Equations of the form y=f(x) in which f(x) consists only of powers of x and a pure number are termed polynomials. Polynomial equations in which the highest power of x is 3, 4 or 5 are cubic, quartic and quintic equations respectively.
Consider a circle of radius r and centre (0,0). If the radius is the hypotenuse of a right-angled triangle (The side not touching the right angle), and (x,y) denotes a point on the circumference, Pythagoras' Rule states that x²+y²=r². Now move the centre to (a,b). The x coordinate, to take an example, of the point (x,y) is now a units greater than the length of the horizontal side of the triangle. To return to the dimensions of the triangle, therefore, the equation of the circle must be written as (x-a)²+(y-b)²=r².
Imagine that the equation is written in the form x²+ax+y²+by=c. This can be rewritten as (x+0.5a)²-0.25a²+(y+0.5b)²-0.25b²=c, then as (x+0.5a)²+(y+0.5b)²=0.25a²+0.25b²+c, where 0.25a²+0.25b²+c=r². This technique is called completing the square.
The equation of an ellipse is of the form x²/a²+y²/b²=1. The centre of this ellipse is at (0,0), so the foci can be denoted by (-α,0) and (α,0). The lines d1 and d2 connect the foci to a point on the ellipse. The sum of the lengths of these lines is constant, so let us consider the points where the ellipse crosses the positive axes. At (a,0), d1=a+α and d2=a-α. Therefore, d1+d2=2a. At (0,b), d1=d2=√(α²+b²). Therefore, d1+d2=2√(α²+b²). Combining the equations gives 2a=2√(α²+b²), and rearranging gives α=√(a²-b²). Another equation is α=a∊, where ∊ denotes the eccentricity.
The equation of a hyperbola is of the form x²/a²-y²/b²=1. Rearranging this gives y=(b/a)√(x²-a²). The hyperbola gets closer to the asymptotes (The lines it does not cross) as x→∞ or -∞. There, √(x²-a²)≈√(x²), which in turn equals x or -x. As our hyperbola is centred at (0,0), the gradients of the asymptotes are therefore b/a and -b/a. A hyperbola is rectangular if a=b.
To find the intersection(s) of two lines, one can start by writing both equations in terms of the same variable, for example x. This gives equations of the form x=f1(y) and x=f2(y). If two quantities both equal x (or the same function of x), they must also be equal to each other. Therefore, f1(y)=f2(y). Now write this equation in terms of y. This technique only works where both the x and y values are the same for both lines, so one now has the y coordinate(s) of the intersection(s). Finally, substitute for y in either of the original equations to find the x coordinate(s).
[Contents][Previous Page][Next Page]


text:
Yesterday I played in a VIP tourney on Chess Cube. It was a 4 player round robin. I am please to have placed 1st. I decided to post the final game which had a pretty tactic (discovered check) finishing the game.
I am starting to feel more comfortable playing these tournaments. When I first started playing I did not play to my potential and my tournament rating was significantly lower then my regular rating. Now it is getting much closer.


text:
Whether on behalf of an absent business owner, receiver or financial institution, our directors will work with the team in place to achieve your goals and provide part or all of the following services……Read More
Feasibility Studies
Whether you are making a decision on whether to close a business, reduce or increase opening hours or invest cash into a business, it is vital to assess the potential gains or losses from your actions.Read More
Are you not receiving the financial information needed to keep your business or managed unit on track? Keystone can provide full outsourced financial functions, allowing you and your team to focus on the day to day of the business.Read More
Head Hunting
Does your business require new senior staff at middle to senior management level? From our years of experience in the business, we have an extensive network of experienced, professional and dedicated industry leaders who may be suitable for your role.Read More
Staff Training
Our training and personnel development courses are designed exclusively with the licensed trade in mind and are geared towards generating increases sales, improved customer satisfaction and greater return business.Read More
Asset Management
We can provide you with comprehensive short, medium or long term management of distressed units including operations, financial reporting and business development strategies, always working to add value to the asset during our tenureRead More
Independent Business Reviews
Whether on behalf of an insolvency firm, financial institution or a business owner, our IBR's will provide a comprehensive insight into the business' current and potential financial and operational performancesRead More
Sales and Marketing
Business development will always be the key driver of any business as cutting costs will only go so far. At Keystone we can provide a range of Sales and Marketing support including but not limited to….Read More


text:
Sunday February 4 at 9:00pm - Victoria - News of the horrific famine in Ireland has finally reached the queen. She is adamant that her government should be doing more to help, but meets with surprising opposition from her prime minister.
Monday February 5 at 10:00pm - Independent Lens - Explore the life of Winnie Mandela and her struggle to bring down apartheid, with intimate insights from those closest to her and testimony from the enemies who sought to extinguish her radical capacity to shake up the order of things.
Tuesday February 6 at 9:00pm - American Experience - The Gilded Age Meet the titans and barons of the glittering late 19th century, whose materialistic extravagance contrasted harshly with the poverty of the struggling workers who challenged them. The vast disparities between them sparked debates still raging today.
Wednesday February 7 at 10:00pm - Impossible Builds - Follow the construction of one of the most complex skyscrapers ever to make it off the drawing board. Its design is so radical that construction experts have turned to a building material never before used in skyscrapers.
Thursday February 8 at 9:00pm - Dickensian - Compeyson is riled by the arrival of Matthew Pocket and quizzes Arthur over his cousin. Later, a drunken night in the pub ends up with Compeyson challenging Matthew to some dares.
Friday February 9 at 9:00pm - African American Oral History Project - This documentary highlights the fascinating story of former slave and later United States Congressman Robert Smalls of South Carolina. In addition to the national story of Robert Smalls, the film also brings to light the personal struggles and triumphs of many others in the African American community located in south central Pennsylvania.
Saturday February 10 at 9:00pm - A Place to Call Home - With a religious occasion approaching, Sarah performs a spiritual cleansing in the local creek that's unfortunately witnessed by Doris Collins. But while Sarah finds it hard to deal with Doris's reaction to her nudity, it is Sarah's own, personal demons with which she most struggles.
Support for WITF is provided by:
Support for WITF is provided by:


text:
4 Tips When Starting a Car BusinessPosted on March 14th, 2015 No comments
Starting a new car business can be rewarding and provide you with a significant amount of income. However, you will need to invest a lot of money and effort into the business to ensure its success, especially at the beginning. Here are some tips when starting a car business.
1. Find capital
You will need to invest a significant amount of capital into your business to get started. Spend time working out how much your new car business will cost you, taking into account the funds you will need to rent a building, purchase vehicles, hire staff, spend on marketing, and cover other costs. You will need to contact different financial institutions about obtaining a loan for your new business, so shop around in order to find the best deals and lowest interest rates. A price comparison website will let you compare different business loans, and you will be able to find out more information about the customer service offered by a particular bank, and the terms and conditions of the loan.
2. Find a good location
Finding a good location for your new business is imperative, especially if you want to attract passing trade. Review the best places in your local area to set up your dealership, and compare the costs involved in renting different premises. You may want to set up your business in a location that is close to other car dealerships, but not any businesses that sell the same car brands as you do. This can be useful when attracting customers in the vicinity who are looking to purchase a vehicle.
3. Hiring staff
If you plan on opening your business throughout the week, and for long hours every day, you will want to hire staff. For example, you may need to hire salespeople who have experience in selling vehicles to the general public. Other people that you may need to employ can include finance staff, a sales manager, and cleaning staff.
4. Marketing your new business
If you are opening a new business, you will want the world to know about it. Come up with a marketing budget, and try to stick to it as closely as you can. There are a number of different ways you can advertise the new business, including via local radio, print media, and the internet. Social media can also be useful; set up Twitter, Google +, and Facebook pages for your new business, and ask your followers to spread the word about your business. Keeping customers informed about any offers, promotions, or new cars that you have can increase sales and interest in your company.
Three Succesful Car Dealers in ScandanaviaPosted on February 15th, 2015 No comments
Scandinavia is the name given to a broad group of countries within Northern Europe. These countries are Norway, Denmark and Sweden. With so many different car dealerships located throughout the region it can be tough to know who is the best. We reveal three of the most successful car dealers within Scandinavia.
BCA combines physical auctions with a secure online auction systems to provide one of the most popular car auction websites not only within Scandinavia but within Europe. Based in Sweden, this company holds over 800 auctions every year, with over 1.2 million vehicle sales a year. The website is easy to use and lists all of the available cars by their makes and models. You can check out the BCA auction website here.
Daytona is one of the oldest and most respected car dealers within Denmark. They specialize in dealing classic and vintage cars including exclusive brands like Ferrari, Bugatti, Facel and Maserati. You can get their contact information or view all of the cars that they have up for auction on their website.
Mollergruppen is Norway's largest car dealership with a whopping 63 dealers and 3,600 employees. As well as serving Norway they also sell cars to Sweden, Estonia, Latvia and Lithuania. They supply some of the biggest names in the car industry including Skoda, Volkswagen and Audi, although they only specialize in selling new cars. They are also able to provide a range of maintenance services that are fully guaranteed.
These are considered to be amongst the top car dealers in Scandinavia. You may want to browse several of the websites to see who offers the best deals around. If you are looking for a specific type of car then you can also try large online auctions sites such as eBay, as they will often have some good bargains. Good luck!
4 of the Best Pieces of Equipment for Car DealershipsPosted on January 27th, 2015 No comments
If you own a car dealership, there are a plethora of different products that keep things running as smoothly as possible, so you can spend more time concentrating on other aspects of your business. This equipment can include vehicle washing systems, and car displays – a useful way to advertise different cars to potential customers. Here is some of the best equipment you can purchase for your car dealership.
1. Car wash equipment
If you keep vehicles on display on a dealership forecourt, you will want to keep them clean to keep these cars to look their very best. Investing in car washing equipment can be a cost-effective way to do this, and will enable you to clean a wide range of different makes and models. Car washing equipment can include gutter cleaners, solar panel cleaners, and commercial vehicle washing systems.
2. Vehicle mover
There will be times when you will need to move vehicles from the dealership forecourt to inside your premises. Stringo car movers are a useful piece of equipment when you want to move a vehicle quickly and easily. The product can help you transport a car if you want to avoid exhaust fumes, if the car is damaged or has been freshly painted, or if there is a functional problem with the car. Instead of using several members of staff to move the car, this product can make the process easier, so you don't have to worry about injuries or other problems.
3. Car displays
Car displays are a great way to attract attention from passing trade and customers who visit your dealership forecourt. There are different products available, including windscreen displays, car top displays, and vehicle pricing units. You will be able to advertise prices, promotions, and other valuable information in order to drive revenue and increase sales. Car display ramps and stands can also be positioned outside your premises, or on the forecourt of your dealership. These products can easily be moved at the end of the day, and can include information about a particular vehicle, such as its price and specifications.
4. Vehicle lift
A vehicle lift provides you with instant access to the underside of a vehicle – ideal if you need to change a tire or install a piece of equipment. There are two different types of car lift – electric lifts and hydraulic lifts. Both of these make it safer when examining and installing equipment to the bottom of a car. However, you will need to adhere to safety when using one of these products to prevent a serious injury or accident from occurring. Vehicle lifts are available in different sizes, and can handle different amounts of weight, so always do your research before choosing a product.
6 Tips When Importing a Car into the UKPosted on January 19th, 2015 No comments
Purchasing an import car can save you money, and provide you with greater choice when buying a vehicle. However, you will need to complete a number of documents before you are able to drive the car in the UK. Here are some tips when buying import cars.
1. Shop around for the best deals
There are a number of websites that specialise in import cars from a particular country. Use these websites to find the best deals, and browse for different manufacturers, makes and models. Price comparison websites will let you compare and contrast different vehicles quickly and easily, providing you with a wealth of information before you make a purchase. You can also read review websites, which will have further information about a particular car, such as the specifications of the vehicle, and its performance and safety standards.
2. Make sure the car meets environmental and safety standards
When importing a car into the UK, the vehicle will need to be approved to ensure it meets the relevant environmental and safety standards. The company that is importing the vehicle for you will usually take care of this. Contact them directly to ask for more information before you make a purchase.
3. Know the rules
There are different rules when it comes to importing cars into the country. For example, you will need to obtain a European Certificate of Conformity if the car you are importing has a right hand drive, which you can get directly from the manufacturer. If the car has a left hand drive, you will need to obtain a European Certificate of Conformity, as well as a certificate of Mutual Recognition. Some companies may charge a fee for these documents, so you will need to factor in this cost when purchasing an import vehicle.
4. IVA certificates
If the car has no European approval, you will need to obtain an Individual Vehicle Approval, or IVA, certificate. For more information about these documents, and the process of importing a vehicle into the UK, contact the DVLA. You should also contact DVLA if the car is more than 10 years old, dating from the first registration of the vehicle, or the date of its manufacture.
5. Paying tax
You will also need to pay duty and vehicle tax, as well as VAT on a car you want to import into the UK. When you import a car from outside the EU, you will usually pay duty and VAT through customs.
6. Obtaining UK license plates
You will need to obtain UK license plates for your vehicle once it has been imported into the UK. For more information, contact DVLA, or refer to their website. However, there are exemptions for cars that are deemed as 'temporary imports'.


text:
My grandma came in and woke me before, hahah, not in a cute way with breakfast in bed. No, she yelled. I heard her from downstairs. She wants us to be up early so we can do something with our day. I'm so hungry, I'm just going to write this blogpost then I' going to eat.
So, today the plans are to go out with grandmas boat, I hope anyways. I really miss to be out with the boat. I don't think it's that hot out today… and I only have skirts with me. What is wrong with me?? Trusting Swedens weather like that?!?
I opened the balcony door last night, and I've really missed to fall asleep to seagull yells. And my grandmas sheets are so crispy and fine. I sleep like a princess in this room.
We biked down to this boat yesterday, and we watched the sunset. It was beautiful.
Later darlings!


text:
We will be in attendance at the 15th annual Learning Disability Today conference in London. You can come and visit our book store where you will be able to browse a selection of our titles, as well as take a sneak peek at the new Beyond Words Story App, due 2016. We will also be running a pop up book club at 12pm, led by our book club co-ordinator Sue Carmichael.
Back to All Events
Earlier Event: September 16Mental Wealth Festival
Later Event: April 28Picture This Launch


text:
The biggest free porn tube sites list on the net! Welcome to Linknurse.com's sex tube listings. From this list you will find every single remarkable free adult movie site. Each website is also reviewed, and checked that they are reasonable safe to use. So, say good bye for your old bookmarks, you need just only this resource, and you have everything you can imagine and beyond at your fingertips. Ready to get your dick hard or pussy wet? Just choose the tube and go!
In total the amount of content these sites offer collectively is so huge that I don't even try to calculate it. It's millions and millions of clips, and sure there are also full length movies. Categories range from amateurs, teens, lesbians to more specific such as Japanese bondage, German swingers, facial cumshots, real hidden cams and so on. Even if you are a real super consumer watching free xxx vids all night long (including days), you just can't watch this all – it is mission impossible. There comes constant updates on all of these sites, and each day they add collectively thousands of new movies daily. Does that sound good? Free porn is not going to end, amount of it only increases hour by hour and quality gets better and better.
Have you already got enough? I don't believe that. You never know from which tube you will find the most exciting videos. If you are masturbating only watching one or two old tubes, then you might have already lost your ticket to the mind blowing sexual pleasures – if you don't immediately go through all the adult movie websites listed below. There might be the one you have missed! The best one for your private (or public) pleasures. Don't be shy, these clicks doesn't cost you anything and you can really find the best porn tube below. It is there! Those all are good, but we have so many different expectations and tastes what it comes to rubbing cock or pussy that it is simple impossible to say which one is universally the best one. So, do you cock (or pussy) a favor, go on, and check those out by yourself!


text:
The Open-End Japanese Mutual Fund Puzzle
44 Pages Posted: 23 Apr 1998
Date Written: November 1997
Recent empirical evidence has suggested that the Japanese mutual fund industry has underperformed dramatically in the past two decades. Conjectured reasons for under performance range from tax-dilution effect to high fees, high turnover and poor asset management. In this paper, we show that this underperformance is largely due to tax-dilution effects and not necessarily due to poor management. Using a broad database of funds which includes investment trusts closed to new investment we show that once an instrument for the time-varying tax-dilution exposure is included in a factor model, there is little evidence of poor risk-adjusted performance. A style analysis of the industry demonstrates that managers appear to pursue tax-driven dynamic strategies.
JEL Classification: G2, F3
Suggested Citation: Suggested Citation