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The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4+CD25+T cells
DOI: 10.1186/ar1484
© Frey et al., licensee BioMed Central Ltd. 2005
Received: 1 November 2004
Accepted: 24 November 2004
Published: 11 January 2005
It is now generally accepted that CD4+CD25+ Treg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4+CD25+ cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4+CD25+ cells in the inflamed joint as compared with CD4+CD25- cells. These data imply a significant role for CD4+CD25+ Treg cells in the control of chronic arthritis. However, transferred Treg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of Treg cell transfer in potential therapeutic applications.
Keywordsarthritis regulatory T cells
Rheumatoid arthritis (RA) is the most common autoimmune disease in humans, affecting 1% of the population in western countries. Histologically, RA is characterized by hyperplasia and infiltration of the synovial membrane with mononuclear cells, development of an aggressive tissue called pannus and secretion of proteases, which are responsible for the destruction of articular cartilage and adjacent bone. It is well established that macrophages and synovial fibroblasts are effector cells of joint destruction, and it is presumed that autoreactive CD4+ T cells are involved in their activation [1]. There is now a large body of evidence that, in rodents, regulatory T cells (Treg) actively control the activation of autoreactive T cells and thus maintain immunological self-tolerance. Apart from adaptive Treg cells, which can be induced by antigen-specific stimulation of conventional peripheral T cells under tolerogenic conditions (for review [2]), there is no doubt that naturally occurring Treg cells exist in healthy mice as well as in humans and rats, and these are characterized by constitutive expression of CD25 [3–5]. Absence of these cells in vivo results in a multi-organ autoimmune syndrome [3, 6]. These CD4+CD25+ Treg cells leave the thymus as committed 'professional' suppressor T cells [7–9], proliferate in the periphery, and acquire an effector/memory-like phenotype [10]. In unmanipulated mice, Treg cells can also be found in the CD25- compartment, based on the expression of the integrin αEβ7 [10, 11], possibly reflecting differences in developmental stages of these cells.
The exact role played by naturally occurring CD4+CD25+ Treg cells in the pathogenesis of arthritis remains controversial. Arthritis is part of the autoimmune syndrome induced by transfer of CD25-depleted splenocytes into lymphopenic hosts [3], and CD4+CD25+ cells are protective in collagen-induced arthritis [12]. However, Bardos and coworkers [13] ruled out a role for naturally occurring CD4+CD25+ Treg cells in proteoglycan-induced arthritis.
To clarify this issue, we used the antigen-induced arthritis (AIA) model. AIA is a Tcell-dependent experimental arthritis that is induced by intra-articular injection of antigen (methylated bovine serum albumin [mBSA]) into knee joints of preimmunized mice [14, 15]. This results in an acute inflammatory reaction, which is characterized by exudation of neutrophils and fibrin, which later proceeds to a chronic arthritis with synovial hyperplasia, infiltration of mononuclear cells, and cartilage and bone destruction – histopathological changes similar to those that occur in RA. Autoimmune responses against cartilage constituents such as collagen types I and II and proteoglycans are involved in rendering the disease chronic [16, 17]. Beyond the 100% incidence of arthritis, another major advantage of the AIA model is that the time point of induction of arthritis is known, allowing manipulation of CD4+CD25+ Treg cell number in vivo at defined stages in the disease. Using depletion of CD25-expressing cells or transfer of CD4+CD25+ cells, in the present study we demonstrated that Treg cells modulate the onset of AIA but are ineffective at later stages, calling into question their value as a new therapeutic approach to established chronic arthritis.
Animals, arthritis induction and assessment
For all animal experiments, female C57Bl/6 mice (Charles River, Sulzfeld, Germany; age range 6–10 weeks) were used. Animals were kept under standard conditions, fed a standard diet and given free access to water. All animal studies were approved by the government commission for animal protection.
At 21 and 14 days before arthritis induction, mice were subcutaneously injected with 100 μg mBSA (Sigma, Deisenhofen, Germany), emulsified in complete Freund's adjuvant (Sigma) supplemented to 2 mg/ml heat-killed Mycobacterium tuberculosis (strain H37RA; Becton Dickinson [BD], Heidelberg, Germany). Simultaneously, mice received 5 × 108 heat-inactivated Bordetella pertussis (Chiron-Behring, Marburg, Germany) intraperitoneally. Arthritis was induced by intra-articular injection of 100 μg mBSA in 25 μl phosphate-buffered saline (PBS) into the right knee joint cavity.
Arthritis severity was monitored by measurement of lateral joint diameter using a vernier caliper (Oditest, Kroeplin Längenmesstechnik, Schlüchtern, Germany). Histological severity of arthritis was scored in a blinded manner by two investigators (PKP and MG) in frontal knee joint sections, stained with haematoxylin and eosin and prepared as described previously [14]. Briefly, at least four sections per knee joint were semiquantitatively examined on a 0–3 point scale for each of the following: extent of synovial hyperplasia, mononuclear infiltration, cartilage destruction and pannus formation.
Antibodies and reagents
The following antibodies were grown and purified from the culture supernatants in our laboratory: anti-CD25 (PC61), anti-CD3 (145 2C11), anti-CD4-FITC and FITC-labelled anti-CD4-F(ab) (GK1.5), anti-CD8 (TIB105), anti-CD28 (37.51) and anti-Mac-1 (M1/70). The following antibodies and secondary reagents were purchased from BD Pharmingen (Heidelberg, Germany): PE-Cy5-labelled anti-CD4 (H129.9), biotinylated anti-αEβ7 (M290), biotinylated anti-CD25 (7D4), allophycocyanine or FITC-conjugated anti-CD25 (PC61), streptavidin-allophycocyanine and streptavidin-PE, and matched antibody pairs for ELISPOT analysis of IFN-γ (R4-6A2 and biotinylated XMG1.2) and IL-4 (BVD4 1D11 and biotinylated BVD6-24G2) production.
In vivodepletion
Mice were injected with 0.5 mg purified anti-CD25 antibody (PC61) 4 and 2 days before intra-articular antigen injection. Polyclonal rat IgG, purified from normal rat serum, was used as control. The degree of depletion was determined by fluorescence-activated cell sorting, using a non-cross-reactive biotin-labelled anti-CD25, FITC-labelled anti-CD4 and streptavidin-conjugated allophycocyanine. Measurement was performed using FACSCalibur® (BD) and data were analyzed using WinMDI http://www.scripps.edu.
Preparation, pre-activation and transfer of regulatory T cells
Pooled spleen and lymph node cells from naive C57Bl/6 donors or, if indicated, from immunized mice were incubated with anti-CD4-FITC (clone GK1.5) and anti-CD25-biotin (clone 7D4; BD). CD4+ T cells were isolated using an anti-FITC-Multisort-Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) in accordance with the manufacturer's instructions. CD4+ T cells were sorted into CD25- and CD25+ cells using anti-biotin MicroBeads (Miltenyi Biotech). Purity was greater than 92% for CD4+CD25- and greater than 80% for CD4+CD25+ cells.
CD25-expressing and αEβ7-expressing subsets were sorted by FACS. Briefly, pooled spleen and lymph node cells from naive mice were stained with anti-CD25-FITC, anti-αEβ7-biotin and streptavidin-PE. The stained cells were enriched with anti-FITC and anti-PE MicroBeads, using the AutoMACS separation unit (Miltenyi Biotech). Thereafter, the cells were sorted into subsets according to their expression of CD25 or αEβ7 using a FACSDiVa cell sorter (BD). The purity was 90–95%, as determined by FACS.
For activation, cells were cultured for 24–72 hours in the presence of plate-bound anti-CD3 (3 μg/ml), anti-CD28 (10 μg/ml) and rhIL-2 (100 U/ml; Chiron, Ratingen, Germany) in RPMI 1640 containing 10% fetal calf serum (FCS; Gibco, Karlsruhe, Germany). Thereafter, cells were washed with PBS and transferred intravenously via lateral tail vein into mice at the time point of AIA induction or at later time points when indicated.
Delayed-type hypersensitivity reaction
Seven days after arthritis induction, mice were challenged by intradermal injection into their ears of 5 μg mBSA in 10 μl PBS. Ear thickness was measured before injection and 24 and 48 hours later using a vernier caliper (Kroeplin).
Proliferation assay and ELISPOT analysis
Single cell suspension from spleens and lymph nodes (inguinal, popliteal, axillary) were cultured at a density of 1 × 106/ml in RPMI 1640, containing 10% FCS, 2 mmol/l L-glutamine, 10 mmol/l Hepes, 1 mmol/l sodium pyruvate, 0.5 μmol/l 2-mercaptoethanol and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin; all from Gibco) in the presence of medium alone or 25 μg/ml mBSA for 72 hours in 96-well tissue culture plates (Greiner Bio One, Nürtingen, Germany). Cells were pulsed with 0.5 μCi [3H]thymidine (Amersham-Buchler, Braunschweig, Germany) for the last 18 hours of culture. Thereafter, cells were harvested onto 96-well glass fibre filters (Packard Bioscience, Groningen, The Netherlands), and [3H]thymidine incorporation was measured with a scintillation counter (Top-Count; Packard Bioscience).
For ELISPOT analysis, PVDF-membrane 96-well microplates (Millipore, Eschborn, Germany) were coated overnight at 4°C with the primary antibody diluted in sterile PBS. After washing, plates were blocked for 2 hours with RPMI 1640 containing 10% FCS. Thereafter 2 × 105 (IL-2 and IFN-γ) or 1 × 106 (IL-4) cells were cultured in duplicate wells for 24 (IL-2 and IFN-γ) or 48 hours (IL-4). After washing again plates were incubated overnight at 4°C with the secondary antibody diluted in PBS/1% BSA. Extravidin–alkaline phosphatase conjugate (1:30,000 in PBS/1% BSA) and BCIP/NBT solution (bromochloroindolyle phophate/nitroblue tetrazolium; both from Sigma) were used for spot development. The number of spots was quantified using a KS-ELISPOT-Reader (Carl Zeiss, Oberkochen, Germany).
Determination of serum IgG by ELISA
Microplates (96-well; Greiner Bio One) were coated with antigen (0.125 μg/ml mBSA), collagen type I (from rat tail tendon) and type II (10 μg/ml), and proteoglycans (10 μg/ml both from bovine cartilage) and left overnight, as described previously [14]. After washing, plates were incubated with serially diluted serum samples and the amount of bound IgG was determined using anti-mouse IgG-peroxidase conjugate (ICN, Eschwege, Germany) and ortho-phenylendiamine (Sigma) as substrate. Extinction was measured at 492 nm against 620 nm with an ELISA reader (Tecan, Crailsheim, Germany).
Cell transfer for in vivohoming assay
For in vivo homing assay, cells were sorted with a modified protocol and labelled with 111indium, as described elsewhere [10]. Briefly, CD4+ cells were enriched by negative selection. Enriched CD4+ T cells were stained with FITC-conjugated anti-CD4-F(ab) and anti-CD25-allophycocyanine and sorted into CD4+CD25+ or CD4+CD25- cells by FACS (BD). Cells were labelled with 111In (Indiumoxin; Amersham-Buchler) for 20 min at room temperature; 1 × 106 labeled cells were injected intravenously, and 24 hours later mice were killed and the distribution of radioactivity in various organs and the rest of the body was measured in a γ-counter (Wallac Counter, Turku, Finnland).
Alternatively, a proportion of these cells was labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) by incubation with 5 μmol/l CFSE (Molecular Probes, Leiden, The Netherlands) in RPMI 1640 for 5 min at room temperature. After washing, 1 × 106 cells were injected intravenously. Twenty-four hours later single cell suspensions were prepared from the draining and nondraining peripheral and mesenteric lymph nodes, the spleen and the peripheral blood, and stained with anti-CD4 and analyzed by FACS. Dead cells were excluded using propidiumiodide.
Statistical analysis
Data are expressed as mean ± standard error of mean, unless otherwise indicated. Experimental groups were tested for statistically significant differences with the Mann–Whitney U-test using SPSS 10.0 (SPSS Inc, Chicago, IL, USA).
Depletion of CD25-expressing cells exacerbates antigen-induced arthritis
Increased cellular and humoral immune responsiveness in CD25-depleted mice
For analysis of the cellular immune responses ex vivo, draining lymph node cells of arthritic animals were harvested 14 days after AIA induction, restimulated with mBSA, and analyzed for proliferative response and cytokine production. As expected from the increased DTH reaction, the proliferative response to mBSA was significantly increased in cells from CD25-depleted mice as compared with that in rat IgG-treated controls (Fig. 3b). Importantly, even without antigenic stimulation the lymph node cells from CD25-depleted mice proliferated fourfold as much as cells from mice treated with control IgG. These data imply that a substantial proportion of the T-cell compartment is still activated 14 days after intra-articular antigen challenge in the absence of Treg cells.
Compatible with these findings is that the production of cytokines in response to mBSA was greater in CD25-depleted mice. Importantly, both T-helper-1 (IFN-γ) and T-helper-2 (IL-4) responses were aggravated by depletion of Treg cells, indicating that both types of response are subject to suppression by Treg cells (Fig. 3c). Again, cytokine secretion from Treg-depleted animals was increased even without antigenic stimulus. In accordance with this, serum levels of IgG directed against mBSA as well as levels of the cartilage-specific autoantigens collagen type I, collagen type II and proteoglycans, were found to be increased in CD25-depleted mice (Fig. 3d).
Taken together, these data clearly demonstrate that CD4+CD25+ Treg cells regulate the severity of arthritis by limiting the cellular and humoral immune responses against the inducing antigen mBSA as well as some arthritis-related autoantigens.
Transfer of CD4+CD25+cells
It is known that Treg cells must be activated via their T-cell receptor to exert their suppressive function. Because we were unable to use antigen-specific (i.e. T-cell receptor transgenic) Treg cells, we opted to pre-activate the CD4+CD25+ cells by in vitro culture in the presence of anti-CD3, anti-CD28 and IL-2 in order to increase their suppressive potential. Transfer of 1 × 106 pre-activated cells significantly suppressed both knee joint swelling and histological arthritis score (Fig. 4c). This effective suppression of AIA development was a consistent finding in different experiments, even with the use of lower cell numbers (for instance 2 × 105 cells; data not shown).
Taken together, our data demonstrate that Treg cells can inhibit arthritis development when transferred at the time of arthritis induction. However, we were unable to demonstrate any therapeutic effect of Treg cell transfer (in numbers that are effective in prevention) when performed after disease onset.
Transferred CD4+CD25+ Tregcells do not suppress humoral or cellular immune responses
Homing properties of CD4+CD25+ Tregcells
Because the mechanism of suppression of Treg cells in vitro is cell contact dependent, localization of cells might be important for their regulatory activity. Therefore, we investigated the migration behaviour of CD4+CD25+ and CD4+CD25- cells in vivo. For these experiments CD4+ cells were enriched by negative selection and sorted by FACS into CD4+CD25+ and CD4+CD25- populations with preferential use of F(ab)-fragments or antibodies, which do not interfere with migration in vivo. Cells were labelled with 111In and injected intravenously into AIA mice 7 days after induction of arthritis. After 24 hours radioactivity was measured in different organs.
Our findings provide clear evidence that CD4+CD25+ Treg cells are critical for regulating the severity of AIA in mice. We showed this by manipulating the Treg cell numbers using two different approaches: depletion of CD25-expressing cells and transfer of purified CD4+CD25+ Treg cells. It is important to stress that we depleted CD25-expressing cells in the interval between immunization and AIA induction, because CD25-depletion before immunization profoundly increases the resulting humoral and cellular immune responses [3, 12]. These data are consistent with studies conducted in collagen-induced arthritis; however, in these experiments CD25-expressing cells were depleted before immunization with collagen type II, and the resulting more severe arthritis could be interpreted as the result of stronger immunization state [12]. With our experimental design, we were able to examine the effect of Treg cells in ongoing joint inflammation directly. Because CD25 is also expressed on activated conventional T cells, it could be assumed that injection of an anti-CD25 antibody would deplete not only Treg cells but also effector T cells, but the exacerbated AIA in CD25-depleted mice argues against such a depletion of effector T cells. Accordingly, in control experiments lymph node cells from CD25-depleted mice isolated at the time of induction of AIA were able to mount a similar anti-mBSA response in vitro as compared with control mice (data not shown). Furthermore, CD4+CD25+ cells isolated from immunized donors can suppress development of AIA (Fig. 4b). Taken together, these data imply that the CD25+ compartment in immunized mice largely consists of Treg cells.
AIA induction in CD25-depleted mice resulted in a much more severe arthritis in the acute and chronic stages of disease. We recently showed, with the use of a depleting anti-CD4 antibody, that this acute stage of AIA is already under the control of T cells [15]. Nevertheless, early AIA is dominated by cells of the innate immune system [19], and the exacerbation of arthritis in CD25-depleted mice could be due to a lack of suppression of these cells by Treg cells. In accordance with this view, CD4+CD25+ Treg cells are able to suppress innate immune cells in a model of bacteria-induced colitis [20].
In later stages exacerbated arthritis in CD25-depleted mice is accompanied by increased mBSA-specific proliferation and IgG production. This enhanced responsiveness emerged during arthritis development and is due to sustained T cell activation. Such prolonged T cell activation in the absence of CD4+CD25+ cells has also been described in other disease models [21] and is probably the cause of the increased AIA severity. Moreover, the PC61 antibody used in our study has a half-life of approximately 3 weeks in vivo (Sutmuller R, personal communication), which makes it possible that Treg cell function is not only impaired by depletion but also by blockade of IL-2 binding to CD25 by the PC61 antibody. IL-2 or IL-2 signalling via CD25 has been shown to be critical to the regulatory action of Treg cells [22, 23]. Also, activation-induced cell death of pathogenic T cells, which is regulated by IL-2, could be impaired by withdrawal of IL-2 signalling and therefore contribute to the observed high levels of cellular immune responses in our study [24].
The fact that depletion of CD4+CD25+ Treg cells enhances the immune response against the foreign antigen mBSA clearly demonstrates that their suppressive effect is not strictly limited to autoreactive T cells. Taking into consideration that Treg cells are also critically involved in the control of immune responses against pathogens [25, 26], their physiological function is not just to prevent autoimmunity but also to control the extent of inflammatory reactions in order to prevent tissue damage to the host. Further support for the influence of CD4+CD25+ Treg cells on arthritis development came from the transfer experiments. When transferred at the time of induction of AIA, CD4+CD25+ cells were able to ameliorate ongoing disease. Analysis of the recipients did not reveal a remarkable long-lasting suppression of systemic mBSA-specific immune reactions. Thus, prevention of AIA appears to be possible without inducing anergy or abrogating previously induced T-cell effector functions [27]. In contrast to this, transferred CD4+CD25- cells significantly enhance cell-mediated and humoral immune responses.
Furthermore, the homing data presented here demonstrate that CD4+CD25+ cells can migrate into the arthritic knee joint. Functional Treg cells have repeatedly been found within such effector sites and/or draining lymph nodes, for instance in tolerated allografts [28], in Langerhans islets and pancreatic lymph nodes in inflammation-induced diabetes [29], in chronically inflamed skin in a Leishmania infection model [30], and in the mucosa and mesenteric lymph nodes in inflammatory colitis in severe combined immunodeficient (SCID) mice [31]. Interestingly, two recent papers [32, 33] reported an accumulation of functional Treg cells in the inflamed joints of patients with RA, juvenile arthritis and other rheumatic diseases.
It is most likely that the transferred CD4+CD25+ Treg cells act in the draining lymph node as well as in the inflamed tissue. Within such a scenario, it could be possible that Treg cells inhibit the activation of effector T cells and their subsequent migration to the joints. Such a mechanism was recently speculated in modulation of virally induced immunopathology by T cells [26].
Huehn and colleagues [11] recently demonstrated that CD4+CD25+ Treg cells can be divided into subsets based on the expression of the integrin αEβ7. Moreover, this marker identifies CD25- Treg cells [34]. Both αEβ7-expressing subsets had better capacity to reach the inflamed joint and to prevent arthritis in the AIA model, as compared with αEβ7- Treg cells [10]. Thus, suppression at the site of inflammation is also an important part of the activity of Treg cells. How this effect is mediated is unclear but an involvement of IL-10 or transforming growth factor-β is possible [20, 35, 36].
If these hypotheses are correct, then they could explain why the transfer of Treg cells after arthritis induction is not effective. On the one hand, transfer of Treg cells 24 hours after intra-articular antigen challenge might be too late to inhibit activation of effector T cells and their migration to the joint. Indeed, T-cell activation is an early event in AIA because CD4+ T cell depletion ameliorates the acute stage of the model [15]. On the other hand, it could be possible that the suppressive function of regulatory T cells is switched off under the inflammatory conditions present in the inflamed tissue by factors such as IL-6 or glucocorticoid-induced tumor necrosis factor family-related gene (GITR) and GITR-ligand interactions, abrogating the suppressive effect of Treg cells [37]. With this in mind, it could be interesting to investigate whether the accumulated Treg cells in patients with arthritis function properly in vivo and whether these patients could really benefit from a therapeutic enhancement of Treg function, as suggested by some enthusiastic investigators in this field.
In this regard, data on the curative effects of Treg cells in experimental disease models are conflicting. To best of our knowledge, a curative effect of CD4+CD25+ Treg cells has only been demonstrated in the colitis model induced by transfer of CD45RBhigh T cells into SCID mice [31, 38]. In contrast, other authors were unable to demonstrate such an inhibitory effect of Treg cells on SCID colitis when they were transferred 1 week after administration of pathogenic CD45RBhigh T cells [39]. Because arthritis in the AIA model has a hyperacute onset, it could be assumed that the time window for an ameliorative effect of Treg cell transfer ends very shortly after intra-articular injection of antigen. However, further studies on the role of Treg cells in other arthritis models are clearly needed to clarify whether enhancement in Treg cell function might be beneficial in experimental arthritis and perhaps in human disease.
Our data show that Treg cells are critically involved in the control of immune responses that are responsible for the pathogenesis of chronic arthritis. Transfer of such cells can modulate the severity of ongoing inflammatory arthritis but they cannot suppress established disease. Thus, timing of Treg cell transfer for therapeutic purposes is of considerable importance.
antigen-induced arthritis
5,6-carboxyfluorescein diacetate succinimidyl ester
delayed-type hypersensitivity
enzyme-linked immunosorbent assay
fluorescence-activated cell sorting
fetal calf serum
- mBSA:
methylated bovine serum albumin
phosphate-buffered saline
rheumatoid arthritis
severe combined immunodeficient
- Treg:
regulatory T cell.
We thank T Kaiser and K Raba for FACS sorting; M Schinz and A Kaufmann for help with ELISPOT; H Börner, C Hüttich and R Stöckigt for their excellent technical assistance; and KW Pratt and D Szczawinska for critical comments on the manuscript. This work was supported by the Kompetenznetz Rheuma (Grant 01 GI 0344), Deutsche Forschungsgemeinschaft (Grant Br 1372/5-1) and the Interdisciplinary Center for Clinical Research (IZKF) Jena.
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- Green EA, Choi Y, Flavell RA: Pancreatic lymph node-derived CD4+CD25+ Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity. 2002, 16: 183-191. 10.1016/S1074-7613(02)00279-0.View ArticlePubMedGoogle Scholar
- Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL: CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature. 2002, 420: 502-507. 10.1038/nature01152.View ArticlePubMedGoogle Scholar
- Mottet C, Uhlig HH, Powrie F: Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol. 2003, 170: 3939-3943.View ArticlePubMedGoogle Scholar
- Cao D, Vollenhoven Rv R, Klareskog L, Trollmo C, Malmstrom V: CD25brightCD4+ regulatory T cells are enriched in inflamed joints of patients with chronic rheumatic disease. Arthritis Res Ther. 2004, 6: R335-R346. 10.1186/ar1084.PubMed CentralView ArticlePubMedGoogle Scholar
- de Kleer IM, Wedderburn LR, Taams LS, Patel A, Varsani H, Klein M, de Jager W, Pugayung G, Giannoni F, Rijkers G, et al: CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis. J Immunol. 2004, 172: 6435-6443.View ArticlePubMedGoogle Scholar
- Banz A, Peixoto A, Pontoux C, Cordier C, Rocha B, Papiernik M: A unique subpopulation of CD4+ regulatory T cells controls wasting disease, IL-10 secretion and T cell homeostasis. Eur J Immunol. 2003, 33: 2419-2428. 10.1002/eji.200324205.View ArticlePubMedGoogle Scholar
- Oida T, Zhang X, Goto M, Hachimura S, Totsuka M, Kaminogawa S, Weiner HL: CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism. J Immunol. 2003, 170: 2516-2522.View ArticlePubMedGoogle Scholar
- Green EA, Gorelik L, McGregor CM, Tran EH, Flavell RA: CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-beta–TGF-beta receptor interactions in type 1 diabetes. Proc Natl Acad Sci USA. 2003, 100: 10878-10883. 10.1073/pnas.1834400100.PubMed CentralView ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is cited.


text:
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Remembrance Day's @Gettysburg, date change for Nov. 2013
Discussion in 'Events, Living Histories, and Reenactments' started by M E Wolf, Jan 9, 2013.
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text:
Ah, Chris - sandy soil! An item I have little experience with.
'Minutissima' is so tiny that even if it were aggressive, I don't
think it would pose a problem. Yes, L. nummularia can move out in
the proper conditions. It doesn't do that much moving in the cracks
of the paving of my front patio because it gets hot and dry there, so
there's just a bit of it at the edges, but I put some in an adjacent
bed and it's gotten pretty happy - of course, it's in competition
with several other tough guys, so I just let them fight it out.
Most of my dry shade is due to tree and shrub roots - mostly trees.
In woodland conditions, tree roots suck up all available moisture
I agree - if you have columbines near the path, they'll end up in the
cracks as will everything else that makes seeds within at least a 5'
radius of the paths...plants love to seed into paving cracks:-)
Marge Talt, zone 7 Maryland
Editor: Gardening in Shade
Current Article: Corydalis
Complete Index of Articles by Category and Date
All Suite101.com garden topics :
> From: email@example.com
> Marge,
> I'll have to look for Lysmachia japonica 'Minutissima'. L.
> (Creeping Jenny) does well in the sun and part shade, but it's way
> aggressive. I have sandy soil, and the area where this garden is
located is
> under a blue spruce and red cedars. I'm guessing that my soil is
acidy as I
> have lots of moss occurring naturally on the property. Parts of
this shady
> area are dry due to tree roots. Other sections seem to have less
roots and
> things grow well there. I've top dressed with shredded mulch and
> manure when I've gotten around to it. As the mulch breaks down my
sandy soil
> improves somewhat. I might give moss a try or just leave a light
mulch cover
> between the stones. I'm sure that the columbines and other plants
will seed
> in between the rocks anyway!
> Chris
> Long Island, NY
> Zone 7
Support hort.net -- join the hort.net fund drive!


text:
- 2 x eSATA External Connectors
- Up to 6Gb/s
- $49.99 49.99
- $39.99 –
- Save: $10.00 (20%)
- Free Shipping (restrictions apply)
OK, the jury is back in 02/08/2013
This review is from: HighPoint RocketRAID 622 PCI-Express 2.0 x1 SATA III (6.0Gb/s) Controller Card
Faster than the default PCIe card that came with the unit. Write speeds have increased. Driver now appears to be stable, though it took a bit to get there.
At least on Linux, if you are willing to fight with the driver, it does get the job done. Note I am using for JBOD only.
Took a bit of trial and error to get the driver stable. Website table layout a bit confusing when getting the Open-source drivers.
Other Thoughts:
Note: I am only using this for JBOD, not using the RAID functionality. The following is abbreviated, just pointing out on how I got it to go.
Have TR5MB with PCIe card connected to CentOS 5.9 in-home server (Dell Precision 390) with 5-1TB disks installed. The card that came with the unit was plug and play but finally started to give up the go.
The 622 came with a driver disk, but only up to CentOS 5.5 (plus others). You can find the Open-source drivers on their website. Make sure you get the exact ones for this card, there are a few choices and the web site can be slightly confusing (this may have accidentally caused me some issues). In the end I made sure CentOS 5.9 had all the available patches, rebuilt the driver following the instructions supplied (make/make install worked without issue). I have been stable since then. This server runs 24/7. Ran a number of large file tests, no issues.
Of course, YMMV.
Did you find this review helpful? Yes / No Thanks for the valuable feedback you provided!


text:
Kõik hotellid asukohaga Baytown, Texas, Ameerika Ühendriigid
1 tuba, 2 täiskasvanut
6802 Garth Rd, Baytown, TX, 77521, Ameerika Ühendriigid, 800 004 4466 TASUTA lauatelefonist
Scottish Inn & Suites Baytown
Praktiline motell välibasseiniga, Baytown
- Tasuta kerge hommikusöök, tasuta wifi ja tasuta parkimine
Peamised teenused/mugavused
- 42 numbrituba
- Välibassein
- Ööpäevaringselt avatud vastuvõtulaud
- Kohv/tee saadaval ühiskasutatavates ruumides
- Õhukonditsioneer
- Igapäevane koristusteenus
- Iseteenindav pesumaja
- Köök
- Mikrolaineahi
- Külmik
- Privaatne vannituba
- Igapäevane majapidamis-/koristusteenus
- Pesumasin/kuivati
- San Jacinto ostukeskus (~ 0,2 km)
- Pirates Bay veepark (~ 2,9 km)
- Baytowni looduskeskus (~ 11,5 km)
- Port of Houston (~ 14,9 km)
- Sylvan Beach Park (~ 20,9 km)
Hotel had bed bugs and management not willing to work with business traveler. Will not…
Beds had hair in the sheets. Sheets appeared to be stained. AC was not on when we entered…
TripAdvisori reisijate hinne: 4
5 arvustuse põhjal
Scottish Inn & Suites Baytown
Teave hotelli kohta: 800 004 4466 TASUTA lauatelefonistalates 78€
- Standard tuba, 2 laia voodit
- Standard tuba, 1 ülilai voodi
Rohkem häid valikuid
Hotelli suurus
- Selles hotellis on 42 tuba
- Sisseregistreerimine alates 14.00
- Väljaregistreerimine 11.00
Sisseregistreerimisel vajalik
- Nõutav on krediitkaardi- või sularahadeposiit
- Nõutav on ametlik fotoga isikutunnistus
- Iseseisvalt sisse registreerivad külastajad peavad olema vähemalt 18 aasta vanused
- Lastevoodid ei ole saadaval
- Lemmikloomad ei ole lubatud
- Hotelli territooriumil saadaval tasuta wifi ja kaabliga internet
- Tubades on saadaval tasuta wifi ja kaabliga internet
- Tasuta iseteenindusega parkla
Lähedal asuvad 2 tärni hotellid Baytown
Toit ja joogid
- Tasuta kerge hommikusöök
- Kohv/tee saadaval ühiskasutatavates ruumides
Puhkus ja ajaviide
- Välibassein
- 24h vastuvõtulaud
- Pesumaja
Elamiseks vajalik
- Õhukonditsioneer
- Triikraud/triikimislaud (tellimisel)
- Pesumasin/kuivati
- Privaatne vannituba
- Ainult vann
- Föön
- 32-tollise ekraaniga LCD-telerid
- Kaabelkanalid
- DVD-mängijad
Ühendus maailmaga
- Laud
- Tasuta wifi
- Tasuta kaugekõned
Toit ja joogid
- Külmik
- Mikrolaineahi
- Köök
- Igapäevane majapidamis-/koristusteenus
Populaarsemad hotellid Baytown
Lähedal asuvad hotellid
Soovitatud hotellid
Viimased arvustused
Hea 3,2 /5 from 18 reviews
Scottish Inn & Suites Baytown
Not good
Scottish Inn & Suites Baytown
Clean but not relaxing
Overall the stay was just okay. The room seemed clean which may have been due to the very strong odor of cleansers. The lighting in my room was very stark. Bright overhead recessed lights that were really too bright for comfort. There were no lamps in the room at all so reading in bed required bright overhead lights and then you had to get out of bed, go to the wall and turn off the lights when finished. Not a huge issue but not the comfort expected in most hotels. My entire room was painted inside with a sea/beachscape mural on all walls and even the ceiling. This was a Jacuzzi room, (which I did not request - nor was I charged for) and that may be the reason it was painted so gaudily. The bed was comfortable and I had a good night sleep, I just would not say it was a relaxing environment while awake.
Scottish Inn & Suites Baytown
Dazzed and confused in Baytown
All was good... EXCEPT... someone was SMOKING in a nearby room we smelled smoke all night... and I am allergic to it... so that sucked.... headache and congestion for next few days Also... the proprietor wanted to charge my card again... I told him I had already paid thru the website... but he insisted... and his charge was considerable less than the website... I have not seen his charge show up yet on my card... but he processed it... so please reply as to why this happened and to the exact charges
Scottish Inn & Suites Baytown
where do I start
The hotel is nicely decorated and looks great. The woman behind the counter was polite ,but the window at the front desk is off-putting. There was dust/ smears on most of the surfaces, and hair in the tub so I went to the front desk. The woman offered to clean it for me, but she was looking after a 4 year old girl so I cleaned it myself. It's also strange my room had a kitchenette, but no plates, glasses, skillets, etc. When I went to the front desk the man spoke so little English and seemed incredibly angry/ bothered I was a the desk so it was not worth trying to explain. Finally, of the 2 days I stayed here for business the internet kicked me off the web more than I could access it. When I phoned the front desk it was the non English speaking angry acting man indicated he would reset it. I went to dinner and 75 minutes later still no web access. I couldn't even send email, totally unacceptable.
Scottish Inn & Suites Baytown
No internet for IPad.


text:
My reflection is gone!! Been looking for her all
day long!
Once she was there, She smiled too and her eyes were gleaming!
My reflection has died from too many nights I cry, You are gone my love,In_Love with each other once before.
My reflection is gone saddened by being ignored, Our love we just need to gain back and get it restored.
My reflection is gone and I want her back! I want her to smile and giggle so please hold me close and hear her too!
My reflection has left I bet you never even knew!
She is sad,She is hurt,She needs a new re-birth.
My reflection will be found I do believe I can find her but, Your help she will need and for her god's no mystery,So please be on the look out!
My reflection is lost so give me a shout let me know if you get a glimpse...maybe her heart will not be in pieces but,Whole again.
Maybe She will return home soon and she will feel
as happy as the stars and the moon.
© Copyright 2017 Lavendar. All rights reserved.
Status: Finished
Genre: Poetry
Poem / Religion and Spirituality
Poem / Poetry
Poem / Poetry
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
There was an error uploading your file.


text:
The return of the discredited methods of stop and search to British policing is most disturbing. Home Office figures show that the number of people being stopped and searched has risen twentyfold in six years. Last year some 100 people were being stopped each day. Why has a technique so long associated with heavy-handed policing - and widely blamed for exacerbating racial tension - been allowed to return? For the answer we must look to the Government's creeping authoritarianism and the opportunity afforded to illiberal ministers by the heightened terrorist threat.
Since the end of the old "sus" laws, the police must have a reasonable suspicion that someone has committed an offence before they can stop and search them. But the Terrorism Act, passed in 2000, shifted the ground back again. This grants police the right to stop anyone in an area designated as being under threat of terrorism, regardless of whether there is any reason to suspect that person of a crime. Senior police officers can authorise the use of such powers in specific areas for up to 28 days. More than 1,000 orders to establish such areas have been given the go-ahead in the past six years. Ministers, in fear of terrorist attacks, have even encouraged the police to exercise these new sweeping powers, particularly since the July 7 bombings in London.
The result is that numbers of stop and searches has risen sharply. And unsurprisingly, ethnic minorities are being disproportionately targeted again. One in five of those stopped last year was black or Asian.
If stop and search was succeeding in apprehending terrorists, a defence of the tactic might be mounted. But there is no evidence that this is the case. Only 455 people, out of more than 35,000 stopped and searched, were arrested last year through these powers. A small number of these arrests were for terrorism-related offences. But no terrorists themselves were stopped in this way. And there is certainly no reason to believe that major plots have been foiled by these methods. A large number of the arrests were for motoring offences. The reality is that stop and search has become a way for the police to harass innocent members of the public.
This law has been abused in other ways too. There are disturbing indications that it has been used to suppress legitimate political demonstrations. A stop and search zone was used to disrupt protests at an international arms fair in east London in 2003. And we should not forget the treatment of Walter Wolfgang, the pensioner who was arrested under this law after he heckled Jack Straw at last year's Labour conference.
No one would dispute that the police need some discretionary powers, but they cannot be as broad-brush as this law allows. Even Lord Carlile, the Government's independent adviser on terrorism legislation, believes that the use of stop and search could be halved without reducing public safety. Today the House of Lords will, rightly, try to tighten the legislation to limit the power of police to authorise blanket stop-and-search operations.
In recent years we have also seen the surreptitious creation of a DNA database and an explosion of anti-terrorist legislation, and now we are facing a national ID card scheme. Lord Falconer has let slip that the Government wants such cards to become compulsory. All this, combined with the powers of stop and search, means we could soon find ourselves in a position where we have to justify our existence to the state, rather than the other way round. This cannot be tolerated.
A tension between national security and civil liberties is to be expected. But the Government is taking this country down a road that will not only leave us less free, but less safe as well.Reuse content


text:
"You're going to get yours"
…and people wondered why anyone who lives in that town was terrified to use their real names when it comes to discussing this case!
"You're going to get yours. And if you don't get yours, somebody close to you will."
~Coach Reno Saccoccia
New York Times article: http://www.nytimes.com/2012/12/17/sports/high-school-football-rape-case-unfolds-online-and-divides-steubenville-ohio.html?pagewanted=all&_r=0


text:
NEW YORK, NY—Neon Trees' latest
album, Pop Psychology, debuted at
number one on Billboard's Top Rock
Albums chart warranting an extended
tour that will find 'The Trees' on the
road until late fall.
A good sound starts with
good input, and the band
tour s with a sizable mic
complement. Vocals are captured
with Audio-Technica AE6100
handheld mics for the leads and the
AE4100 model for the rest of the
band. Meanwhile, the guitar and bass
cabinets are miked with AT4040s and
a Sennheiser e 609, respectively. For
the drums, there's a Shure Beta91 on
the kick, and a Yamaha Subkick, too;
otherwise, the drums are surrounded
by Sennheiser e 604s on the toms,
Audio-Technica ATM450s on hat
and snare bottom, ATM650 on snare
top and AT4033s for overheads.
Rounding out the stage capturing are
a slew of Radial DI boxes.
For the U.S. leg of the tour, Duffy
has been using an Allen & Heath
iLive-144 modular console at the FOH
position with an iDR-10 and iDR-32
rack set up to fulfill opening act needs.
Also on-hand at FOH is a Focusrite
RedNet 4 preamp interface and a
Manley VoxBox for the lead vocals.
"I've been using Allen & Heath
for three years now," said Duffy. "It's
a stable and sonically beautiful surface…
then you have their tech support
and there really is no comparison."
"I'm at a gig and some people are
checking out my original compact iLive
T-112 console with comments, like
'What's that toy?' because of the small
footprint. I always say to them, 'listen
to my show, then we'll talk about it.' Afterwards,
they come back and want to
know everything about the 'toy!'"
At stageside, monitor engineer
Mike Bangs oversees another Allen
& Heath iLive console—this one a
T-112—with an iDR-48, which sends
mixes to a cross-stage monitoring and
front-fill solution. The unusual arrangement
places a Vue Audiotechnik
al-8 element and hs-25 subwoofer on
both sides of the stage facing inward.
A pair of subcompact al-4 elements
in front of each al-8/hs-25 combination
provide additional front fill.
American Music & Sound (A&H US Distributor)
TOP TEN: This week's most popular articles
DPA helps Moddi blend cassette technology into live gigs
Prolight + Sound 2017 presenting new 'Silent Stage'
Low Profile Ultra-slim Swingout Mount for Flat-panels, 65lbs Capacity by Premier Mounts. Model: AM65
Low Profile Ultra-slim Swingout Mount for Flat-panels, 95lbs Capacity by Premier Mounts. Model: AM95
12" 2-way Passive Loudspeaker by d&b audiotechnik Corporation. Model: 24S
Underground Swedish nightclub, Underbara, gets Yamaha overhaul
DiGiCo enters the Bear's Den
The Italian sound scene in 2017
53rd CAS Award Winners
API Announces Saul Walker Memorial Scholarship
Spotlight: Pro Tools | MTRX Interface


text:
Wego.com.sg recently found 4 hotels in Cranbourne, Australia. Book last minute hotel or find great hotel deals such as Comfort Inn Mahogany Park for S$198 per night. Save more on cheap hotels with rates from as low as S$198 per night.
Filters help you save time when choosing where to stay. Try clicking on a few now...
Cranbourne Hotels 3 Star Hotels Cranbourne Cheap Hotels Cranbourne Cheap Accommodation Cranbourne
Flights to Australia Flights from Australia Australia Airports Australia Airlines


text:
Supercar Experiences£49 instead of up to £119 for a junior or adult experience with 1 supercar, or £89 with 2 supercars at Drive Me, Stafford - save up to 59%
ADULT supercar experience inc. 4 laps in 1 car with Drive Me (was £119) OR redeem towards another Wowcher deal.You save 59% (£70)
JUNIOR supercar experience inc. 6 laps in 1 car with Drive Me (was £114) OR redeem towards another Wowcher deal.You save 57% (£65)
ADULT supercar experience inc. 12 laps in 2 cars with Drive Me (was £205) OR redeem towards another Wowcher deal.You save 57% (£116)
Save up to 59%
Promotion ends on
16 / 03 / 2014
- Enjoy a supercar experience for juniors or adults.
- Juniors get to drive 6 laps in one car, plus a 4x4 passenger ride.
- Adults get to drive 4 laps in one car.
- Or can upgrade & drive 2 cars, for a total of 12 laps!
- Cars include Nissan GTR, Ferrari F430, Audi R8 & more!
- Receive a certificate & driving analysis after your laps.
The Fine Print
- One per person.
- May buy multiples as gifts.
- Voucher valid until 30th Apr 2014.
- £20 surcharge for weekend experiences.
- Must be aged 12 years or over for junior experience.
- Must be aged 17 years or over with a full manual UK driving licence for adult experience.
- Must be between 4'10 and 6'6 with a max weight of 18.5 stone for adult experience.
- Adult and junior experiences do not run on the same day.
- Subject to availability.
Full Details
If you have a need for speed that the daily commute can't quite quench then we've got something which will get your blood pumping!
Today we're offering you a junior supercar experience including 6 laps in 1 car or an adult supercar experience including 4 laps in 1 car for just £49 or an adult supercar experience including 6 laps each in 2 cars for £89 - saving you up to 59% off Drive Me's prices.
Based near Stafford train station, Drive Me's purpose built track at Stafford Activity Centre is the ideal place to unleash your inner speed demon. If you're travelling by train you can even get a free lift to the site. When you arrive you'll receive a safety briefing and driving technique instruction. Then, when you're ready, you'll get in your chosen car, strap in and put the pedal to the metal. If you are over 12 years and fancy the junior experience, you'll get a quick driving lesson to go over the basics, before you take to the track to drive 6 laps in one car, followed by a 4x4 passenger ride. If you are an adult you can opt to drive either 4 laps in 1 car or 12 laps in 2 cars (6 laps in each car!). Both options also include a 4x4 passenger ride. Cars are subject to availability but include:
- Lamborghini Gallardo
- Ferrari F430
- Aston Martin V8 Vantage
- Audi R8
- Nissan GTR
- Subaru Impreza
- Mitsubishi Evolution
After tearing up the tarmac you'll receive a certificate and driving analysis, and there's also an option to purchase photos or prints. Brilliant!
The experiences run on selected midweek and weekend dates.
How to Redeem
- When you've bought we'll send you your unique wowcher code.
- Call Drive Me to book using the number we send you.
- Quote your full wowcher code.
Once purchased, you can redeem this wowcher towards another wowcher or claim a Wowcher Wallet Credit. Click here for more info.
Lavender Hotels Spa Day & Prosecco Afternoon Tea for 2 - 3 Locations!£29 instead of £71.90 for a spa day and Prosecco afternoon tea for two at one of three great Lavender Hotels in Preston, Burnley and Bury - save 46%
Level 1, 2 & 3 Sage 50 Computerised Accounts Course£16 instead of £299 for an online Sage level 1, 2 and 3 course or £399 for a classroom-based course in a choice of six UK locations with Love to Think - save up to 95%
Save 95%
Time left to purchase


text:
Portland Metro
SW Washington
Visit Lennar.com
Contact us
Portland Metro, Oregon
Southwest Washington
Lennar offers a wide variety of unique floorplan styles and price ranges to choose from as well as many quick move-in homes. We are proud to be building in only the most desirable locations within the Portland Metro and southwest Washington areas and providing buyers a wide selection of distinctive lifestyles.
*Offer available for a limited time. At closing you will receive a closing credit of not less than $4,000 for select homes in select Lennar communities, as determined by Lennar in its sole discretion. The closing credit will be reflected in the purchase and sale agreement you sign and can only be modified in writing by you and Lennar. Offer available only to qualified buyers financing through Universal American Mortgage Company and closing at designated closing agent. Not valid in conjunction with any other offer. Lennar reserves the right to withdraw or change the offer at any time and without prior notice. CCB#LENNAN1893QG. CCB#195307. Copyright © 2015 Lennar Corporation. All rights reserved. Lennar, the Lennar logo, the Everything's Included logo, and the Next Gen logo are registered service marks or service marks of Lennar Corporation and/or its subsidiaries. 4/16


text:
Home / Home & Garden / Home Decor / Tableware & Kitchen / Home Max- Savannah Serving Platter- Red
Home Max- Savannah Serving Platter- Red
Give your kitchen a southwestern update with the Savannah Red Serving Platter. Crafted with embossed tooled leather patterns, this serving platter makes a beautiful accent. Handcrafted ceramic 12 3/8"W x 12 3/8"H


text:
|Contact Us
Venture Mud has been providing drilling fluid products and technical services to the petroleum industry since 1996, with continuous customer satisfaction and success.
Venture Mud provides professional service for drilling and workover operations from fluid design to application at the drill site.
Our strength is derived through our people, who continually work hard at keeping costs low and drilling as efficient as possible.
If your next project is in Texas, New Mexico, Arkansas or Oklahoma, please give Venture Mud a call.
We look forward to the opportunity of working with you and your company.


text:
We'll I still think it would b fun to play with some of the people I really get along with on here. Mibs, shock, stylez, and a few others even if it never happens at this point it would b a lot of fun
Type: Posts; User: Bayareaboy
We'll I still think it would b fun to play with some of the people I really get along with on here. Mibs, shock, stylez, and a few others even if it never happens at this point it would b a lot of fun
It would be nice to get him just don't see it happening
Number 6 player n the league baby!!!
McDonalds mcdoubles with fries on them and a side of fries with acoke! Yum
Have to agree as long as our team wins I'm not worried about who thinks they are right n wrong.
I still think we should play that psd basketball pick up game
I think at this point klay comes off the bench it's worked great n preseason and he doesn't mind the role. He gives the bench a good offensive weapon as we'll
East is a different story lol
its like punching a brick wall...you get to let anger out, but you only cause yourself pain.[/QUOTE]
Nicely put
Palo Alto is such a nice area I'd love to live there but man it's expensive
Damn!!! I missed
The fight! Guess I'll just have to drop n more I kinda want in hahaha
I think even if he plays majority of the games this year Nadia huge for us his injuries already haunt him. So if we resign him itb way cheaper
I'm excited to see nedo more then anyone really. But at the same time I wanna see the improvement in bazemores game
Yea boi!!!
Call it an offseason man!! We are pretty solid all around. Great depth solid defense and of course good offense. I can't wait for the season to start
I'd like kamen to back up bogut and lee more the I would O'Neal
Agree klay should come off the bench
Lets start the (lets start Barnes over klay) thread and get this thing going!!!
I think it b awesome to get udoh back as our backup 4/5. Just don't see it happening
I agree stylez Barnes was everything he showed in the playoffs when we drafted him. He even showed flashes of it throughout the year but just seemed to hardly ever touch the ball or be a pet of the...
I'd also take a chance on Blair as a backup for our 4/5 positions
If like dalembert but he's prob cost us 2 much
I think he will also. Plus I feel he will help Barnes development with his offensive game also
I think it's a bit much but at the same time we got him cheaper then everyone else which is crazy. Also we use our trade exceptions. When webuy picks lacob uses them then
I'd like to get either hansborough or Byron mullens


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Harris Flotebote Sunliner 220 Boats for sale
Harris Flotebote Sunliner 220Grand Island, New York2016Request Price111
2016 Harris Sunliner 220 2016 Harris 220 Sunliner. Tri-toon performance w/ Mercury 150hp, hydraulic tilt steering, white diamond side panels w/ silver sport graphic, silver sport arch w/ bimini, dual rear facing loungers w/ club chair option, desert camel furniture, convenience pkg, depth finder, changing room, docking lights, rope seat base lighting, woven teak light flooring, ultralux helm seat, ski tow bar, Polk stereo and much more. Must see this boat in person! For a family of pontooning enthusiasts, the right luxury pontoon must be as versatile as it is elegant, as capable as it is stylish. After all, this is where you'll be catching up with friends, sunbathing in the golden summer sun, trolling for fish on a quiet morning and marveling at the riot of sunset color on the water. With ten available floorplans, the Sunliner 220 will surpass all expectations. The CS floorplan has a walk-through bow gate, aft swim-deck access, two lavish bow lounges and a soft L-shaped aft lounge with available sunpad. The DL provides dual rear-facing loungers, The FC appeals to anglers as a true "Fish and Cruise" model with its two stern fish seats, fully equipped workstation, aerated livewell and rod holders. In contrast, the FS model provides four fish seats — two in the bow and two in the stern. Naturally, when you're not fishing, your family and friends will relax in the gracious living space, enjoying the many thoughtful touches and opulent creature comforts that make Harris FloteBote cruising such an inspirational, rejuvenating experience.More…Seller Anchor MarineRequest Information* We weren't able to post your message, please check the highlighted fields and try again.
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post #31 of 53
11/22/08 at 7:08pm
No, my laptop by itself only has a hp out. That circuit has an amp for driving hps. If it was a line out, it would be a fixed voltage for an preamp/amp feed and not going through a hp amp. My docking station has analog rca out as well as digital outs. Feeding the amp through an hp out vs a line out will make a difference in sound quality.
So basically what your saying that you don't have any recommendations for the OP. This thread is about helping someone choose an amplifier. What is it that your recommending? Line out, Headphone out, neither is great source but at least with an amp he can listen to his music without distortion and clipping.
Wouldnt it be better to just say " I would recommend you get him a Dac as well" or "You should tell him to get a Dac to go with his amp" and then make a recommendation?
KECES DA-151, USB DAC never sounded so good on computer - eBay (item 150307893556 end time Dec-03-08 08:17:01 PST)
Well obviously he has other places to use the amplifier, he also has some sansui vintage big amplifier.....still i think he will probably find a way to use it better after i give it to him....:P
Now we're getting somewhere. So he can use this amp with more than his laptop.
Here is a nice little amp. It's been on here a while so offer him your top budget and see.
Do you know what in the hell your talking about? If you know what a volume control knob is, you will see one in the middle of the amp in the first picture.
Excellent condition single power PPX3. This particular unit does not have a volume control. The volume knob is there but it is not connected. I don't think you can just connect some wires to the main board. I used this amp with my 2 channel system with my preamp. You would need a way to control the volume.


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Thank you for visiting Speedo 7cm Sportsbrief Adult Swimwear on oakleysbikes.co.uk
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Match each Smurf to its appropriate sport! You will have 90 seconds to pair 9 different activities. Grab athletic props like basketballs, golf clubs, and weights. After matching the correct sport, the Smurf will perform an athletic animation and disappear!
The Smurfs is one of our selected Movie Games . Also very popular on this website right now are Dragonball Z Goku Dressup, Tiles Of The Simpsons, Comic Stars Fighting 3.5 and Dragon Ball Dress Up.
Report game as broken
Cancel report of broken game


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Federales is a Spanglish word used to denote certain organizations from Latin countries. The term gained widespread usage by English-speakers due to popularization in such films as The Wild Bunch, The Treasure of the Sierra Madre, and Blue Streak. The singular "Federale" is in fact incorrect, since the Spanish language uses "es" to pluralize nouns ending in a consonant; therefore, the correct singular form is "Federal" (with stress on the last syllable, unlike the English adjective), though this term is ambiguous in written English. The term is a cognate and counterpart to the slang "Feds" in the United States.
Law enforcement[edit]
A short term traditionally used for certain Mexican federal police agencies such as:
The Mexican Federal Police, and any of its predecessors
- Federal Preventive Police/Policía Federal Preventiva,
- Federal Highway Police/Policía Federal de Caminos, and
- Federal Fiscal Police/Policía Fiscal Federal.
The Federal Ministerial Police/Policía Federal Ministerial (PFM) and any of its predecessors:
- Federal Investigations Agency/Agencia Federal de Investigación and the
- Federal Judicial Police/Policía Judicial Federal).
Historically, "Federales" was also the common term used for the Mexican regular or Federal Army, especially during the 34-year rule of Porfirio Díaz until 1911. In part the expression served the purpose of distinguishing centrally controlled military units from provincial militias or rural mounted police (rurales). Following Díaz's overthrow by rebel forces led by Francisco Madero, the Federal Army remained in existence. The Federales were eventually disbanded in July and August 1914, after Madero's successor Victoriano Huerta was in turn defeated by an alliance of revolutionary forces.[1]
- ^ P. Jowett, pages 32–41 The Mexican Revolution 1910–1920, ISBN 1-84176-989-4


text: Home Gallery Video EA Sports Madden '09 - Leon EA Sports: Madden '09 - Leon Published on Aug 25, 2008 Editor's Pick Rate this Ad You must be registered to rate the ad. Please Login or Register Now About CreditsDateAug 25, 2008Client:EA SportsAgency:Wieden + Kennedy PortlandExecutive Creative Director:Jelly HelmExecutive Creative Director:Steve LukerCreative Director:Jed AlgerCreative Director:Aaron AllenCopywriter:Caleb JensenArt Director:Taylor TwistAgency Executive Producer:Ben GrylewiczAgency Producer:Monica RanesAgency Producer:Jeff SelisDirector:Errol MorrisExecutive Producer:Robbie FernandezExecutive Producer:Lizzie SchwartzProducer:Julie AhlbergPost Production:Joint EditorialEditor:Kyle ValentaEditor:Matthew HilberOnline:Vendetta PostOnline Editor:Jim BohnAudio Post Production:Lime StudiosSound Designer:Charlie Keating Tagsgames North America Latest Creativity News on Ad Age Community Films Signs Young, Winecoff Joins Epoch Murillo to Lead McDonald's Team at Alma, Mariano Joins Zulu Alpha Kilo Have You Entered Our 40 Under 40 Contest Yet? Related work EA Sports: Madden NFL 17 - Catchin' With Flair Ft. Rob Gronkowski Heat EA Sports: Madden NFL 17-Start Me ft. Von Miller Heat San Francisco EA Sports: The Madden 16 - Pilot Heat


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Tooled-Up.com78 Suez RoadBrimsdownEnfieldMiddlesexEN3 7PSTel : 0208 805 3535


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Butland joins Robins
Birmingham, Updated:
[.[views].], BY ClubCall
Birmingham starlet Jack Butland has completed an initial one-month loan switch to Cheltenham in order to further his development.
The goalkeeper, who is only 18 years of age, made a successful debut for England's Under-21 side in their 6-0 romp over Azerbaijan last Thursday.Butland is yet to make a first-team appearance for Blues but it is hoped he will get the chance to gain vital matchday experience at Whaddon Road, in a Cheltenham side managed by former City midfielder Mark Yates.The Bristol-born shot-stopper will challenge Scott Brown for Town's number one jersey and could potentially make his debut in Saturday's home clash against Macclesfield.


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Presenter Rajan Datar will be roaming across India for a special edition of the travel news show. He will be in Mumbai to report on its growing nightlife as well as looking at how technology is changing tourism, and visiting the city's first theme park Adlabs Imagica. Then it's on to Kochrab Ashram in Ahmedabad where you can live like Gandhi for a week. Moving to Goa Rajan meets volunteers of Clean Up India, before visiting the coast of Karnataka to ask if it will can be the new Goa. And in light of recent incidents Rajan will also explore how safe India really is female travellers.
Last on
New Year's Day 2015 22:30 GMT


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Labour's conference has passed the under 18 voter remit…where they want the votering age lowered to 16…this crazy stuff….the Labour party is now going to peddle their stuff at the school gate and allowing unionized teachers to teach socialism in the classroom.
Basically Labour can't connect with adults. This is a very worrying remit to pass.
I am very concerned that teachers will be given the rights to preach left wing politics under the guise of curriculum and then will be audited by the ERO to ensure they have delivered the left wing politics.
There is no way this measure should even be contemplated until proper impartial Civics classes are initiated in our schools, but watch the teacher unions oppose that too.
We now have the strange situation where Labour wants to raise the drinking age but lower the voting age…I think they have priorities ass-backwards.
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Hi Internationsee, I am looking for motivated, self- starting people to join me for businesses here. Feel free to take a look at my website below and let me know if you are interested to learn more. Take a leapppp bizzz :))
Carrie Protected content


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NYLO "Indigo Summer" Exclusive Preview
An exclusive look at the upcoming mixtape, Indigo Summer from fledgling music prodigy, Nylo.
BBC/ICECREAM 2013 Holiday Collection Preview
Mass Appeal stops by Pharrell's BBC and ICECREAM showroom for a preview of the holiday 2013 collections.
Andreis Mikael Costa "Cut With The Kitchen Knife" Opens Tonight
Tonight artist Andreis Mikael Costa will exhibit a series of collage works on paper in his first exhibition in Los Angeles titled Cut With The Kitchen Knife at BOA/SOA Gallery.


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Safe Online Payments
|Bus Operator
|Last Bus
Mettupalayam to Hosur Bus Service
|Number of buses :
|Starting Fare :
Mettupalayam to Hosur Bus Distance
|Time taken :
|4 hours 49 mins
|Total operators :
Long journey by bus from mettupalayam to hosur is made effortless by redBus, one of the eminent bus ticketing platform fulfilling most of the travel requirements. Numerous operators run their buses in this route creating ample amount of convenience for many people in India. Secured online ticket reservation process and easy booking techniques help every traveler to undergo smooth booking.
If you are also looking for return checkout, please ensure to checkHosur to Mettupalayam Bus Tickets.
A number of prominent operators ply buses from mettupalayam to hosur ensuring punctuality and safety for its passengers. Major operators offering disparate kinds of bus services are,
Most of the buses pass through various boarding terminals in the route, a few of the many popular points are:
Most of the buses stop at several drop points in the route, a few of the many popular ones are:
Various types of buses serve from Mettupalayam to Hosur ensuring safety and comfort for travelers, a few buses services are:
You can now book route tickets from mettupalayam to hosur at redBus.in. Apply time to time redBus offers on online bus ticket booking. Make some money and memories too!
Mettupalayam is a hill station which is situated on the base of the Nilgiri Hills on the bank of Bhavani River. It is very close to Ooty. It is one of the most developing places. The fabulous walking spot is the Kotagiri where one can witness deer and elephants. There are loads of temples like the Idugampalayam Aanjineyar, Dhasampalayam Vishnu temple, Nana Bathrakali Amman Kovil, Madeshwaran kovil, Subramaniya Swamy temple etc and many more. The Black Thunder Water Park is a great attraction because of its great rides. The place has a lot of natural beauties so there are many films that have been made here.
The city of Hosur is geographically located in Tamil Nadu but is more accessible from Bangalore and is in fact more associated with Bangalore because of its proximity to the IT hub of India. Hosur is now a highly developed district due to the presence of a very successful industrial belt comprising of some of the well known and reputed industries of India and the world.


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On News 88.9 KNPR
On Classical 89.7
1289 S. Torrey Pines Dr.Las Vegas, NV 89146
Main Number: 1-702-258-9895Toll Free: 1-888-258-9895
More contact info
an member station
Erika Yowell writes about art in Las Vegas. The public is invited to the unveiling of NightLight Friday at 4 p.m. in the parking garage off 4th street in Las Vegas.
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Does this restaurant offer highchairs for toddlers?
Is this restaurant good for dinner?
Is this restaurant good for local cuisine?
Is this primarily a coffee shop?
Is this an American restaurant?
Is this restaurant good for dining on a budget?
Is this restaurant good for lunch?
Are the prices at this restaurant mid-range / moderate?
Can a vegetarian person get a good meal at this restaurant?
Can a gluten free person get a good meal at this restaurant?
Thanks for helping!
We visit every time we come to my husbands hometown of Oakley. Best ice cream I have ever had in any state I've ever been to!
Superb and tasty ice cream, delectable chocolate!
Service was friendly and attentive, shop was well organized, decor fitting the establishment.
I will be back!
I grew up around farmers that made ice cream with real ingredients....this is as close as it gets....if you give this place a poor review maybe it's not their issue....I have been going here for 25 years ever since I moved to Cin. and this is the only ice cream parlor in Cincinnati period. Great people great spot fabulous ice...
This is one of the best ice creams I had in my life, and it is up there with Savannah's Leopold's, on a category by themselves. The ambience of the place and the wonderful old gentleman that runs it, Mr Aglamesis himself, add to the experience. (and for Johnny Cash fans like me: Rumour has it that the man himself...
The debate will go on forever. But in my opinion Aglemesis ice cream is the best. Graeters is outstanding also. And we are so lucky to have 2 of the top 5 nationally rated Ice cream makers right here in Cincinnati.
Walking into Aglemesis in Oakley takes you back to the days of the old fashioned fountain soda shops.
This place has the best hand dipped chocolates, and the best ice cream around. Everything I have ever gotten there has been delicious.
The ice cream is delicious in an old time ice cream parlor.the best ice cream. Small place that is paced in the summer. Even the usual flavors are anything but ordinary
I know many people here think the "other" place has better ice cream but I disagree. However, I am a Gold Star fan also so who knows for sure. In any case, I love the store, love the old fashioned parlor feel of Aglamesis, love the table service - all above and beyond. Their ice cream is amazing but they...
Aglamesis was founded in Cincinnati and is one if my favorite local ice cream stores. I have been to the Oakley and Montgomery stores many times and they offer a quaint ice cream experience where they serve their own French pot ice creams.
I grew up on Graeter's, but the mint chocolate chip ice cream I ordered at Aglamesis Brothers was faaaaar and away better than Graeter's (which has become so commercial and modern! Where's the atmosphere?) The parlor wasn't maybe wildly clean, but it has great personality. I think the mint ice cream was made with real peppermint oil, and there was...
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Spice Jet toll free number is 1-800-180 3333 or you can directly call them at 09871803333
Make use of the toll free number to get accurate information on a timely basis without spending a dime.
Spice Jet toll free number is 1-800-180 3333 or you can directly call them at 09871803333
Make use of the toll free number to get accurate information on a timely basis without spending a dime.
- Rajesh
We Compliment you for becoming a part of mega family of more than 12 Lakh Smart Traders who are getting benefitted with our services worldwide. We assure You that we will deliver more than what we promise. Do remember to leave questions and Feedback. Your Success is Guaranteed with us!
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Did I ever tell you about the time my MOTHER got mad at an armadillo and
tried to shoot it with my dad's handgun? It was in the middle of the
night. She held the gun too close to her face and when the gun kicked,
she got a black eye. The armadillo...is still at large. I just looked at
her and said, "mother, some people should not play with guns...tsk."
Surprised the neighbors didn't call the police on her.
Jesse R. Bell
"Xena for President"
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Helping brands acclimate to the rapidly changing, consumer-driven social web through storytelling, social media management and digital production.See All Services
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Turning valuable consumer data, conversations and patterns into actionable insights to improve brand value, loyalty and sales.Discover Latest Insights
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A Traveler, Explorer, Millenial 20-Something and Digital Nomad, I'm on a mission: Finding the extraordinary in the everyday ordinary.Go Seek Adventure
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From web page development to creative design to SEO integration to content marketing to video promotion, no day is EVER the same for a digital producer. But the rewarding part is, at the end of the day, all those efforts can maximize a business or brand's impact at every digital touchpoint.
Social Media Management
It's true: managing social isn't all kittens and unicorns. It takes hard work, great strategic planning and hawk eyes. But if done well, happy consumers can lead to vital insights on the consumer's path-to-purchase journey that never existed… until today.
Great copy should be short, sweet and satiably enduring. It should stir the soul, awaken the mind, move the body. "Make it simple. Make it memorable. Make it inviting to look at. Make it fun to read."
–– Leo Burnett
Amazing Brands & Clients I've Worked With…
"Either write something worth reading, or do something worth writing." –– Ben Franklin
Stand Atop Broken Hill, Torrey Pines.
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#746: Birthday On A Denver Rooftop.
#745: Pass A Bottle to Mr. Jones.
#744: Eat A SlapFish.
#743: Taco Me Tuesday, San Diego.
#742: Take Every Foodie Liberty Here.
#741: Join An SD Writer's Group.
#740: Vote In A California Election.
"Lauren is a brilliant strategist and "ideator" for social media. Her copywriting skills can transform a seemingly dull piece of data into a great story. She is an endless fountain of ideas for big brand brainstorming. Lauren is the perfect blend of meticulous organization and an innovative talent in the social media sphere and always aimed to turn out great work. I highly recommend Lauren to any company looking to hire a knock-out writer and social media extraordinaire."
Jessyca Hix
Recruiting Agent/Client Services Manager, Vitamin T
"Working with Lauren is a delight because she is focused and polite, and ready to roll up her sleeves and get campaigns launched. She values efficiencies in workflows and has her eye on the bigger picture. Lauren was my client and she was great at communicating her needs to the Percolate team and collaborate with us to help her J&J team run faster toward their goals."
Kayla Flaherty, Percolate, Inc.
Account Executive, Mid Market
"Lauren was invaluable in guiding our strategy and execution with all things Facebook and social. She proactively pitched in time and again to help out with things that may or may not have even been in her scope. We will greatly miss her behind-the-scenes leadership and expertise."


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Did you blink and miss the newest New York housewife? You're not alone!
Yes, "Real Housewives of New York" fans, the newest housewife Jennifer Gilbert made her debut last night, Thursday, April 29. She was the event planner that Jill hired for her holiday party.
Poor thing. When Sonja Morgan debuted in the last episode, the Red Sea parted, angels sang and Bravo inserted a segment introducing her. Jennifer? She didn't even get one of those title cards where she gets to pose while her family stands in the far background.
So, who's Jennifer Gilbert? She recently spoke to IVillage.com and our pals at LATimes.com. Here's what we gleaned:
- She has been married for seven years and she has three kids: A five-year-old daughter name Blaise and two-year-old twin sons Saxton and Grey.
- She owns Save the Date, an event planning company, and she's co-owner of Portamee, a baby-carrier producer.
- Now 41, Ernst & Young named her entrepreneur of the year when she was 29 years old.
- She had no relationship to any of the other housewives before Jill hired her to plan her holiday party.
- She's over the Bethenny – Jill feud and claims she doesn't belong to either team.
We fully expect the show to roll out the red carpet for her soon.
For now, what are your initial thoughts on the new housewife?
Follow Zap2it and TheRealJethro on Twitter and Zap2it on Facebook for the latest TV, movie and celebrity news.
'Real Housewives of NJ': Andy Cohen hosts live premiere party online
'Real Housewives of NJ': Danielle Staub has a second sex tape?
'Real Housewives NJ' on 'Regis and Kelly': You can't fake this, Regis!
Jay Leno to 'Real Housewives of NJ': Where's the 'prostitution-whore'?
Video Credit: IVillage.com


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Jenni Murray and Jane Garvey are joined by leading chefs and food writers who share their secrets for perfect home-cooked dishes. From BBC Radio 4's Woman's Hour.
September 7th, 2016
Episode 186 of 192 episodes
Chetna Makan's twist on a traditional recipe
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R.K. Builders Hawaii
Signs and Carvings
Need a Wooden Sign for your business? Add interest to your doors or cabinets with carved images!
Merrie Monarch Invitational Hawaiian Arts Fair
Ray Kobayashi, Hawaii (Big Island) General Contractor / Realtor
Craftsmanship & Experience
We have built over 500 residential homes throughout East Hawaii including a 20-unit townhouse (Kawili Regency) and an 18,000 sf warehouse in the Shipman Industrial Park in Keaau. Beginning with our first home in Hawaiian Paradise Park in 1972, we have probably built a home in almost every subdivision in Puna and Hilo.
As both a building contractor and a Realtor, Ray Kobayashi combines his experience and knowledge to construct homes that weld his customers' desires with what they can afford to build. He clearly remembers how hard it was to obtain a loan for an existing pre-built home, which made him decide to build his own. Working an average of 12 hours a day, seven days a week for nearly 40 years, Ray approaches his construction projects with the same vigor and enthusiasm of his very first project. More about our construction services »
Specializing in Hawaiian Hardwoods
We purchased an ohia forest and built a wood mill on the property, which allows us to focus on using local hardwoods in home construction. R.K. Builders, Inc. can take a newly cut koa, ohia, mango or eucalyptus tree, cut it into useable sizes, dry it in kilns, then trim the wood into pieces that can create original crafted walls, flooring, ceilings, moldings and cabinets. Click here for our native wood gallery. Before you grade your lot we would be happy to salvage any useable trees to integrate into your new home!
R.K. Builder's recent projects, Lelepau Street and Kaiwiki Road, both in Hilo, are magnificent examples of Ray's craftsmanship and vision.
If you are looking to build residential or commercial, interested in Hawaii island real estate or custom Hawaiian hardwood furniture or finishes please contact us!
Saeko & Masa Hayashi


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Pokémon Red and Blue Versions
|Pokémon Red and Blue Versions
|Game Freak
|Game Boy
|Release Date(s)
| JP: February 27, 1996
NA: September 30, 1998
EU: May 10, 1999
AUS: November 1, 1998
|Generation I
|Compatible with
|Generation I, Generation II
|Preceded by:
|Succeeded by:
|Pokémon Yellow Version
Pokémon Red and Blue Versions are part of a series of RPG games called "The Pokémon Series", and is part of the first generation of Pokémon games. As a young Pokémon Trainer, your goal is become the greatest Pokémon Master ever, by capturing and training up to 151 Pokémon. Not all Pokémon are available in one version, and you'll need to trade with friends using a Game Link cable to catch 'em all!
Although used on a Gameboy Color, the main pallet is red, black, and gray.
Contrary to popular belief, Pokémon Red and Blue weren't the first games released in the series, Japan actually received two games before this, which weren't shipped internationally, those titles were Pocket Monsters Aka and Pocket Monsters Midori (Pokémon Green and Pokémon Red).
A Pokémon Green ROM was released, however it was translated into English exactly from Japanese, and therefore is quite hard to understand, with captions such as "but unable to determine smoothly!", and "CATAP's firm tO assail!"
The main point of this game is to collect all 8 badges and become a Pokémon champion. thought this isn't attained easily as you need to train your Pokémon, and set them with the right tactics to be able to defeat them all and be the strongest.
Through out the game, you will fight your rival (default: Gary Oak). He is always trying to become a better Pokémon trainer than you, and finally becomes the Pokémon Champion. He is the last storyline boss, as well as a mid-boss many times as well.
- Pokémon Red, is compatible with Pokémon Blue, Green, Yellow, Crystal, Gold, and Silver.
- 139 catch-able Pokémon in the game (the rest can be obtained by trading with friends who have Blue/Yellow version)
- Game Link Cable to battle and trade Pokémon with friends!
- Insert game into a GameBoy Color and Pokémon Red will appear in Red Filters.
- Insert game into a Super GameBoy and Pokémon Red will feature a special red Pokéball border.
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