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Scratching triggers skin flares in atopic dermatitis (AD). We demonstrate that scratching of human skin, and tape stripping of mouse skin, causes neutrophil influx. This influx in mice was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1−/− mice, and required expression of BLT1 on both T cells and non-T cells. Co-transfer of WT neutrophils, but not neutrophils deficient in BLT1 or the LTB4 synthesizing enzyme LTA4H, restored the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.
Eicosanoids are biologically active lipid mediators that are rapidly generated at sites of injury or inflammation. Leukotrienes (LTs) and prostaglandins are two classes of eicosanoids generated by the metabolism of arachidonic acid through the 5-lipoxygenase (5-LO) pathway and the cyclooxygenase pathway, respectively (Funk, 2001). 5-LO expression and LT generation are generally restricted to myeloid leukocytes, particularly neutrophils, eosinophils, monocytes, macrophages, and mast cells (Werz, 2002). LTs consist of LTB4 and of cysteinyl leukotrienes (cys-LTs), which include LTC4, LTD4, and LTE4. The relative amounts of LTB4 and cys-LTs depend on the relative expression of the 5-LO distal enzymes LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), respectively. LTB4 is generated from innate immune cells such as neutrophils, macrophages, and mast cells in response to a variety of stimuli (Peters-Golden and Henderson, 2007). It primarily acts on myeloid leukocytes causing activation of integrins, adhesion to endothelium walls, and chemotaxis (Goodarzi et al., 2003; Huang et al., 1998; Patcha et al., 2004).
Two receptors for LTB4 have been identified: BLT1 and BLT2. BLT1 is a high-affinity receptor for LTB4 predominantly expressed on neutrophils, macrophages, eosinophils, and activated T cells, with little expression, if any, on resting T cells. BLT2 exhibits low-affinity binding for LTB4 and is ubiquitously expressed (Friedrich et al., 2003). All LTB4 effects on both neutrophils and T cells are lost in Ltb4r1−/− mice (Tager et al., 2003; Tager et al., 2000). LTB4 has been implicated in the pathogenesis of allergic diseases (Ohnishi et al., 2008). Nocturnal concentrations of LTB4 and cys-LTs are elevated in the bronchoalveolar lavage (BAL) fluid of patients with nocturnal asthma (Wenzel et al., 1995). Zileuton, a specific inhibitor of 5-LO, has been shown to decrease LTB4 concentrations in BAL fluid, raising the possibility that its beneficial effect in asthma may involve decreased production of both cys-LTs and LTB4 (Wenzel et al., 1995). Studies on Ltb4r1−/− mice have suggested an important role of the LTB4-BLT1 pathway in the recruitment of effector CD4+ and CD8+ T cells to antigen-challenged airways in a classical mouse model of asthma (Miyahara et al., 2005; Tager et al., 2003; Terawaki et al., 2005).
Atopic dermatitis (AD) is an inflammatory skin disease characterized by itching and scratching, dermal infiltration with Th2 cells and a systemic Th2 cell-mediated response with elevated serum IgE and eosinophilia. Neutrophils from AD patients have elevated LTA4H activity (Okano-Mitani et al., 1996), and release more LTB4 in response to several stimuli (Hilger et al., 1991). LTB4 concentrations are elevated in lesional skin of patients with AD (Fogh et al., 1989; Koro et al., 1999; Ruzicka et al., 1986; Thorsen et al., 1990). However, little is known about the role of LTB4 in AD. To this end, we have developed a mouse model of allergic skin inflammation with many similarities to AD. In this model, repeated epicutaneous (EC) sensitization of tape-stripped skin with ovalbumin (OVA) results in a Th2 cell dominated systemic immune response characterized by dermal infiltration of CD4+ T cells and eosinophils and increased local expression of Th2 cell-derived cytokines (Spergel et al., 1998; Spergel et al., 1999). We have used this model to examine the potential role of LTB4 in allergic skin inflammation. Our findings indicate that LTB4-mediated attraction of neutrophils to skin subjected to tape stripping, a surrogate for scratching, is essential for the development of allergic inflammation at cutaneous sites of antigen introduction.
Mechanical injury to the skin by scratching triggers flares in patients with AD (Fleischer and Boguniewicz, 2010). We examined whether scratching triggers a cellular infiltrate in the skin. There were virtually no detectable neutrophils in the dermis of unscratched skin. In contrast, neutrophils, which were easily differentiated from other inflammatory cells by their characteristic multilobed nucleus, accumulated in the dermis of scratched skin (Fig. 1A,B). There was also an increase of one cell layer in epidermal thickness in scratched skin sites (Fig. 1A). LTB4 is a potent chemoattractant for neutrophils, which express the LTB4 receptor BLT1 (Peters-Golden and Henderson, 2007). Levels of LTB4 and BLT1 mRNA were significantly elevated in scratched skin compared to control unscratched skin (Fig. 1C,D).
We examined whether tape stripping, a surrogate for scratching, causes neutrophil accumulation in mouse skin. Shaved dorsal skin of C57BL/6 mice was tape-stripped six times with Tegaderm and examined 24 hrs later. Histologic examination revealed that there were virtually no detectable neutrophils in the dermis of shaved non tape-stripped skin. In contrast, neutrophils were abundant in the dermis of tape-stripped skin (Fig. 1E,F). There was an increase in epidermal and dermal thickness in tape-stripped skin sites compared to non tape-stripped skin (27.24±1.4 mm vs. 8.04±0.4 mm, and 252.9±11.4 mm vs. 154.9±11.9 mm, n=4, p<0.001 each). The amount of LTB4 and BLT1 mRNA were significantly elevated in tape-stripped skin compared to non tape-stripped skin (Fig. 1G,H). An influx of neutrophils was also observed in ear skin 24 hrs after tape stripping six times with Tegaderm, as determined by flow cytometry analysis of cell suspensions from trypsin digested skin for the presence of CD11b+Gr-1+ cells (Fig. 1I).
Neutrophil influx, LTB4 accumulation, and expression of BLT1 mRNA in tape-stripped skin were comparable in mast cell-deficient WBB6F1/J-KitW/KitW-v (KitW/KitW-v) mice and WT WBB6F1 controls (Fig. 2A-C). Neutrophil depletion in C57BL/6 mice by Gr-1 mAb resulted in the loss of >99% of the CD11b+Gr-1+ cells from the blood (Fig. S1A), and strongly impaired the accumulation of neutrophils, LTB4 and BLT1 mRNA in tape-stripped ear skin, compared to treatment with isotype control antibody (Fig. 2D-F). The role of neutrophils in the accumulation of LTB4 and BLT1 mRNA in tape-stripped skin was confirmed in mice depleted of neutrophils by treatment with cyclophosphamide (CTX) (Fig. 2G-I), which resulted in >98% depletion of circulating blood neutrophils (Fig. S1B). These results suggest that the accumulation of LTB4 and BLT1 mRNA in tape-stripped mouse skin is largely dependent on neutrophils.
Kinetic analysis revealed that neutrophil accumulation in tape-stripped ear skin of WT mice, peaked at 24 hrs and waned by 72 hrs (Fig. 3A), as recently reported (Gregorio et al., 2010; Guiducci et al., 2010). There was markedly less accumulation of neutrophils in tape-stripped ear skin of Ltb4r1−/− mice compared to WT controls at all time points (Fig. 3A). The residual accumulation of neutrophils in Ltb4r1−/− mice may be due to robust induction by tape stripping of mRNA for neutrophil chemoattracting chemokines, such as CXCL2, interleukin-1β (IL-1β and IL-6; both the rapid induction (within 6 hrs) of these cytokines and neutrophil influx into tape-stripped skin, were blunted in mice lacking myeloid differentiation primary response gene 88 (Myd88) (Fig. 3B,C). These results indicate that neutrophil influx in tape-stripped mouse skin is largely dependent on BLT1.
To examine whether BLT1 expression by neutrophils is important for their accumulation in tape-stripped skin, we compared the ability of adoptively transferred neutrophils purified from bone marrow of CD45.2+ WT and Ltb4r1−/− mice to accumulate in tape-stripped ears of CD45.1+ WT recipients depleted of neutrophils by CTX. Because of the short half-life of neutrophils, the recipients' ears were tape-stripped 20 hrs before neutrophil transfer to allow for expression of inflammatory cytokines in injured skin. Adoptively transferred WT neutrophils, but not Ltb4r1−/− neutrophils, accumulated in recipient ears 4 hrs, and, to a lesser extent, 24 hrs, post transfer (Fig. 3D). The rise in blood neutrophil count and the percentages of CD45.2+ donor neutrophils in the spleens 4 hrs post transfer were comparable in recipients of WT and Ltb4r1−/− neutrophils (data not shown), indicating that the general circulation of Ltb4r1−/−neutrophils was not impaired.
Neutrophil accumulation in ear skin 24 hrs after tape stripping was severely diminished in Lta4h−/− mice compared to WT controls (Fig. 3E), and in WT mice treated with the LTA4H inhibitor bestatin (Fig. 3F). Since most of the LTB4 that accumulated in tape-stripped skin was derived from neutrophils, we examined whether neutrophil-derived LTB4 was important for the optimal accumulation of neutrophils in tape-stripped skin. There was minimal influx of adoptively transferred CD45.2+ Lta4h−/− neutrophils, which cannot synthesize LTB4, into tape-stripped ears of CTX-treated CD45.1+ WT recipients, compared to CD45.2+ WT neutrophils (Fig. 3G). The percentages of CD45.2+ donor neutrophils in the spleens were comparable in recipients of Lta4h−/− and WT neutrophils (data not shown).
Neutrophil-derived LTB4 may act in an autocrine and/or paracrine manner to promote neutrophil recruitment to tape-stripped skin. To address this issue, we performed co-transfer experiments in which Lta4h−/−, Ltb4r1−/− or WT neutrophils loaded with CMTMR dye (red) were mixed with equal numbers of WT neutrophils loaded with CMFDA dye (green) and adoptively transferred into CTX-treated WT recipients. Four hours later, ear cells were analyzed and the homing index of Lta4h−/− and Ltb4r1−/− neutrophils was calculated relative to that of WT neutrophils, set at 1. Co-administration of WT neutrophils partially, but not completely, restored the influx of Lta4h−/− neutrophils to tape-stripped skin (Fig. 3H), indicating both paracrine and autocrine roles of neutrophil-derived LTB4 in the recruitment of neutrophils to mechanically injured skin. Ltb4r1−/− neutrophils co-transferred with WT neutrophils accumulated significantly less than Lta4h−/− neutrophils co-transferred with WT neutrophils (Fig. 3H), supporting a paracrine component to the action of neutrophil-derived LTB4.
The role of LTB4 in neutrophil recruitment to the skin was not limited to mechanical injury induced by tape stripping. Treatment of mice with bestatin resulted in a significant reduction in neutrophil recruitment to sites of cutaneous infection with S. aureus (Fig. S2A) and in increased bacterial counts and development of larger lesions at these sites (Fig. S2B,C). Bestatin treatment also significantly inhibited the recruitment of neutrophils in response to sterile wounding of the skin (Fig. S2D), and resulted in significantly larger skin lesions on day 1 post-wounding (Fig. S2E).
BLT1 expression on T cells has been shown to be important in a mouse model of antigen-driven allergic airway inflammation (Tager et al., 2003). Consistent with this finding, Ltb4r1−/− mice failed to develop allergic skin inflammation following EC sensitization with OVA, as evidenced by their failure to increase dermal infiltration by CD4+ T cells and eosinophils, and upregulate IL-4 and IL-13 mRNA expression in OVA-sensitized skin (Fig. S3A,B). Impaired skin inflammation in Ltb4r1−/− mice was not due to failure to mount a systemic immune response to EC sensitization as their OVA specific antibody concentrations and cytokine secretion by OVA-stimulated splenocytes were comparable to WT controls (Fig. S3C,D).
To investigate the role of BLT1 expression on T and non-T cells in allergic skin inflammation, CD4+ donor T cells from EC-sensitized WT and Ltb4r1−/− mice were examined for their capacity to transfer allergic skin inflammation to WT and Ltb4r1−/− recipients. OVA stimulated CD4+ splenocytes from EC-sensitized mice were adoptively transferred by intravenous (i.v.) injection to naïve recipients, which were challenged with OVA on shaved and tape-stripped back skin on days 0 and 3 after transfer, and examined on day 7. As previously reported in BALB/c mice (He et al., 2008), CD4+ T cells from C57BL/6 WT donors EC-sensitized with OVA transferred allergic skin inflammation to WT recipients, as evidenced by significant increases in epidermal and dermal thickness, number of CD4+ T cells, and IL-4 and IL-13 mRNA expression at sites of OVA challenge compared to sites of saline challenge (Fig. 4 and data not shown). CD4+ T cells from Ltb4r1−/− donors EC-sensitized with OVA failed to transfer allergic skin inflammation to WT recipients. Unexpectedly, CD4+ T cells from WT donors EC-sensitized with OVA failed to transfer allergic skin inflammation to Ltb4r1−/−recipients (Fig. 4A-D), indicating a critical role for BLT1 expression on non-T cells in our model. As expected, CD4+ T cells from Ltb4r1−/− donors EC-sensitized with OVA failed to transfer allergic skin inflammation to Ltb4r1−/− recipients. Recipients of CD4+ T cells from OVA-sensitized donors failed to develop allergic skin inflammation in response to skin challenge with saline (data not shown). Transfer of CD4+ T cells from saline-sensitized mice failed to elicit detectable skin inflammation in OVA-challenged skin of recipients (data not shown).
To determine whether BLT1 expression on neutrophils is important for the development of allergic skin inflammation, we compared WT and Ltb4r1−/− neutrophils for their capacity to rescue the transfer allergic skin inflammation by OVA-activated CD4+ effector T cells from EC-sensitized WT mice to Ltb4r1−/− recipients. Neutrophils were administered i.v. (107 per mouse) at days 0 and 3 of the one week-long epicutaneous challenge with OVA (Fig. 5A). Administration of WT neutrophils rescued the ability of WT CD4+ T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. This was evidenced by significant increases in epidermal and dermal thickness, numbers of CD4+ T cells in the dermis and expression of IL-4 and IL-13 mRNA, compared to skin of mice that received only WT CD4+ T cells (Fig. 5A-D). In contrast, administration of Ltb4r1−/− neutrophils failed to rescue the ability of WT CD4+ T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. These results demonstrate that BLT1 expression by neutrophils is critical for the development of allergic skin inflammation.
Failure of Ltb4r1−/− neutrophils to rescue the ability of WT CD4+ T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients prompted us to directly investigate the role of BLT1 expression by neutrophils in the accumulation of antigen specific effector CD4+ T cells into cutaneous sites of antigen challenge. We examined the ability of CD4+ OT-II T cells, activated with OVA peptide and antigen presenting cells (APCs) for 4 days in vitro, to accumulate into the tape-stripped ear skin of recipients following challenge with OVA+cholera toxin (CT) or CT alone, as control. Vα2+Vβ5+ WT OT-II effector T cells accumulated significantly more in antigen-challenged skin than in control challenged ear skin of WT recipients (Fig. 5E). In contrast, WT OT-II effector T cells failed to accumulate in antigen-challenged skin of Ltb4r1−/− recipients (Fig. 5E), indicating that BLT1 expression by non-T cells is important for the homing of CD4+ effector T cells to the skin. Ltb4r1−/− OT-II effector T cells failed to accumulate in antigen-challenged skin of WT recipients (Fig. S4A), despite normal proliferation to OVA (data not shown) and normal expression of the skin homing receptor CCR4 and memory marker CD44 (Fig. S4B,C), confirming the role of BLT1 expression on T cells in homing to the skin.
Co-administration of WT neutrophils i.v. significantly increased the accumulation of adoptively transferred WT OT-II effector T cells into antigen-challenged skin of WT recipients (Fig. 5F). More importantly, it completely rescued the ability of WT OT-II effector T cells to accumulate in antigen-challenged skin of Ltb4r1−/− recipients (Fig. 5F). These results demonstrate that BLT1 expression by neutrophils is critical for the accumulation of CD4+ effector T cells into cutaneous sites of antigen challenge and for allergic skin inflammation.
We next investigated whether LTB4 production by skin-infiltrating neutrophils important for the development of allergic skin inflammation. To this purpose, we examined whether Lta4h−/− neutrophils rescued the ability of OVA-activated CD4+ effector T cells from EC-sensitized WT mice to transfer allergic skin inflammation to Ltb4r1−/− recipients. Because Lta4h−/− neutrophils failed to migrate to tape-stripped skin, neutrophils from Lta4h−/− mice and WT controls were injected intradermally (i.d.) on days 0 and 3 at the site of EC challenge. Intradermal administration of WT neutrophils rescued the ability of WT CD4+ T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients, evidenced by significant increases in epidermal and dermal thickness, CD4+ T cell infiltration in the dermis, and expression of IL-4 and IL-13 mRNA, compared to antigen-challenged skin of mice that received only WT CD4+ T cells (Fig. 6A-D). In contrast, i.d. injection Lta4h−/− neutrophils in Ltb4r1−/− recipients failed to rescue the ability of WT CD4+ T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. In addition, i.d. administration of Ltb4r1−/− neutrophils, which have an intact ability to produce LTB4, caused an increase in the accumulation of OT-II cells in EC challenged ear skin of Ltb4r1−/− recipients, and in ear scratching, comparable to those caused by i.d. administration of WT neutrophils (Fig. 6E,F).
LTB4 induces itch-associated scratching response in mice (Kim et al., 2007). Tape stripping induced significantly more scratching in WT mice than in Ltb4r1−/− and Lta4h−/− mice, suggesting that LTB4 is important in the scratching response to mechanical injury (Fig. S5A). EC-sensitized with OVA exhibited increased scratching (Fig. S5B), and LTB4 concentrations were elevated in OVA sensitized mouse skin sites (Fig. S5C), as in AD skin lesions. These results suggest that generation of LTB4 by infiltrating neutrophils is critical for their ability to promote T cell mediated allergic skin inflammation.
Cutaneous exposure to antigens provokes skin flares in AD patients (Nosbaum et al., 2010; Sicherer and Leung, 2007). Therefore, we next investigated whether administration of bestatin to EC-sensitized mice prevented the development of allergic skin inflammation following cutaneous antigen challenge. C57BL/6 mice were EC-sensitized with OVA for 7 weeks then immediately challenged by application of OVA to a previously unsensitized shaved and tape-stripped skin site on the back. Mice were treated by intraperitoneal (i.p.) injection with bestatin (0.4 mg/day) or vehicle control, daily for 7 days beginning 2 days before the seven-day challenge (Fig. 7A). Mice treated with vehicle alone developed allergic skin inflammation in response to cutaneous OVA challenge, as evidenced by significantly increased epidermal and dermal thickness, dermal infiltration with CD4+ T cells, and expression of Th2 cell-derived cytokines, compared to challenge with saline (Fig. 7B-E). In contrast, mice treated with bestatin failed to develop allergic skin inflammation in response to cutaneous OVA challenge. These results suggest that inhibition of LTB4 production prevents allergic skin inflammation driven by cutaneous antigen exposure in previously sensitized mice.
In this study, we have identified a critical role for neutrophils in allergic skin inflammation in a mouse model with features of AD. We demonstrated that LTB4 production by neutrophils was essential for their accumulation in mechanically injured skin and for the recruitment of effector CD4+ T cells to cutaneous sites of antigen exposure and development of allergic skin inflammation.
Mechanical skin injury by scratching in humans, and its surrogate, tape stripping in mice, caused robust infiltration of neutrophils and accumulation of LTB4 and mRNA for its receptor, BLT1, in the dermis. The slight increase in epidermal thickness in injured human and mouse skin is possibly due to the release of cytokines that promote keratinocyte proliferation, which include IL-6 (Werner et al., 2007). Accumulation of neutrophils and BLT1 mRNA was substantially greater in tape-stripped mouse skin than in scratched human skin, possibly because mouse skin is much thinner than human skin and might be more susceptible to mechanical skin injury. The relatively comparable elevation of LTB4 concentrations in tape-stripped mouse skin and scratched human skin suggests a more rapid washout of LTB4 from injured mouse skin. The baseline concentration of LTB4 may reflect a background signal intrinsic to the assay used and/or be derived from resident cells in the skin and may have not been biologically active, since neutrophils were not detected in unmanipulated skin. Studies in Lta4h−/− and Ltb4r1−/− mice demonstrated that LTB4 and BLT1 are both important for neutrophil accumulation into tape-stripped mouse skin. The role of LTB4 in neutrophil recruitment to the skin is supported by the observation that epicutaneous application of LTB4 in normal healthy volunteers results in local neutrophil infiltration (de Jong et al., 1992). LTB4 induces itch-associated scratching response in mice (Kim et al., 2007). Tape stripping induced significantly more scratching in WT mice than in Ltb4r1−/− and Lta4h−/− mice, suggesting that LTB4 is important in the scratching response to mechanical injury.
Neutrophils, but not mast cells, were the major source of the increased LTB4 in mouse skin 24 hrs following mechanical injury. LTB4 failed to accumulate in mice depleted of neutrophils using Gr-1 mAb or CTX, but accumulated normally in the mechanically injured skin of mast cell-deficient KitW/KitW-v mice. Although mast cells could release LTB4 early after mechanical injury, mast cell-derived LTB4 was clearly not essential for neutrophil influx into mechanically injured skin. This is consistent with our previous observation that allergic skin inflammation elicited by EC sensitization of tape-stripped skin with OVA is intact in mast cell-deficient KitW/KitW-v mice (Alenius et al., 2002).
Adoptive transfer experiments demonstrated that neutrophil influx in tape-stripped mouse skin was largely dependent on BLT1 and on both paracrine and autocrine actions of neutrophil-derived LTB4. LTB4 played a role in neutrophil recruitment to the skin in response to bacterial infection and sterile wounding. In addition, neutrophil-derived LTB4 is required for the recruitment of neutrophils to the joints and for the development of inflammatory arthritis (Chen et al., 2006; Chou et al., 2010; Kim et al., 2006). These observations suggest that LTB4 generated by neutrophils is important for their recruitment to the skin in response to a variety of inflammatory stimuli, as well as for their recruitment to other tissues.
Additional factors, which include inflammatory chemokines and cytokines, produced by resident skin cells may contribute to the initial neutrophil influx in the tape-stripped skin, as suggested by the residual neutrophil infiltrate observed of Ltb4r1−/− mice. Furthermore, production of IL-1b and IL-6 by skin infiltrating WT neutrophils may explain the finding that Ltb4r1−/− neutrophils could be detected in tape-stripped skin when co-transferred with WT neutrophils, but not when transferred alone. Both the rapid induction (within 6 hrs) of CXCL2, IL-1b and IL-6 mRNA and neutrophil influx into tape-stripped skin, were blunted in Myd88−/− mice. This observation suggests that signaling via TLR receptors, possibly by endogenous alarmins (Guiducci et al., 2010) and/or signaling via IL-1 receptor (IL-1R) family members released from damaged keratinocytes, plays an important role in the initial neutrophil influx into injured skin.
A central finding of the present study is that CD4+ T effector cells from OVA-sensitized WT mice fail to transfer allergic inflammation to Ltb4r1−/− recipients. Taken together with the observation that Ltb4r1−/− CD4+ T cells fail to transfer allergic inflammation, this result indicates that the expression of BLT1 on both non-T cells and T cells is important for allergic skin inflammation. Co-transfer of WT neutrophils, but not Ltb4r1−/− neutrophils, rescued the ability of CD4+ effector T cells to accumulate in antigen-challenged skin of Ltb4r1−/− recipients and cause allergic skin inflammation. Intradermal administration of WT, and Ltb4r1−/−, but not Lta4h−/−, neutrophils rescued the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. This finding demonstrates that neutrophil-derived LTB4 is critical for the ability of CD4+ effector T cells to cause allergic skin inflammation. These observations demonstrate that LTB4 production by neutrophils is critical both for the BLT1 dependent accumulation of neutrophils and the BLT1 dependent recruitment of antigen specific effector CD4+ T cells to cutaneous sits of antigen introduction.
The LTB4 synthesis inhibitor bestatin blocked neutrophil influx into tape-stripped skin and inhibited the development of allergic inflammation at sites of cutaneous antigen challenge in mice. This result suggests that blockade of LTB4-BLT1 interaction may be a useful strategy in preventing acute flares that are precipitated in quiescent skin in patients with AD by scratching and introduction of previously encountered antigen.
LTB4 concentrations were elevated in OVA sensitized mouse skin sites, as in AD skin lesions, and mice EC-sensitized with OVA exhibited increased scratching. However, unlike AD skin lesions, which contain few, if any, neutrophils (Leiferman, 1994), EC-sensitized mouse skin sites exhibit neutrophil infiltration (Spergel et al., 1998). Neutrophils, like eosinophils, may not persist intact in chronic AD lesions. It thus remains possible that scratching established skin lesions induces a transient acute neutrophil influx, which triggers LTB4-dependent recruitment of BLT1+ effector T cells, resulting in the exacerbation of allergic skin inflammation.
Scratching is a hallmark of AD and causes further disruption of an already abnormal skin barrier, allowing cutaneous introduction of antigen. Our finding that the LTB4-BLT1 axis is implicated in two sequential steps in allergic inflammation at sites of cutaneous antigen exposure, namely recruitment of neutrophils and neutrophil-dependent recruitment of effector T cells, make the LTB4-BLT1 axis a potential therapeutic target in AD.
Two healthy non-allergic adult subjects were asked, after obtaining informed consent, to scratch the inner side of their forearm, for a total of 12 times over a period of 24 hrs (every hour 8 times on day 0, and 4 times on day 1) using each time twenty strokes of their index fingernail. A 4 mm punch biopsy was obtained an hour after the last scratching from the scratched site and from the corresponding unscratched site of the contralateral forearm. Histologic examination was performed on H&E stained sections by an experienced dermatopathologist who was blinded to the source of the material.
Ltb4r1−/− and Ltb4r1−/− OT-II mice on C57BL/6 background, were previously described (Tager et al., 2003). Lta4h−/− mice, a kind gift of Dr. Koller (Byrum et al., 1999) were bred on C57BL/6 background for 10 generations. WT C57BL/6 mice were obtained from Charles River Laboratory. J-KitW/KitW-v (WBB6F1) mice and congenic WBB6F1 WT mice, OT-II, and CD45.1+ mice on C57BL/6 background were obtained from the Jackson Laboratory. LysEGFP reporter mice on a C57BL/6 genetic background were previously described (Faust et al., 2000).
The animals were kept in a specific pathogen-free environment. All procedures performed on the animals were in accordance with the Animal Care and Use Committee of the Boston Children's Hospital or UCLA Chancellor's Animal Research Committee.
To cause mechanical injury, the shaved back skin or the ear skin were tape-stripped 6 times with Tegaderm (Westnet Inc, Canton, MA). For neutrophil depletion, WT mice were treated with CTX or PBS control as described previously (Zuluaga et al., 2006). In selected experiments, WT mice were treated by i.p. injection with 100 μg Gr-1 mAb (RB6-8C5, ebioscience) or isotype control at day -3 and -1 of tape stripping. Neutrophil depletion was confirmed by flow cytometry at day 0 before tape stripping. Mice were treated by i.p. injection with 0.4 mg/day bestatin (Cayman Chemical) or vehicle control, daily for 2 days before tape stripping.
EC sensitization of six- to eight-week-old female mice was performed as described previously (Spergel et al., 1998). Briefly, the skin of anesthetized mice was shaved and tape-stripped 6 times by transparent IV dressing (Tegaderm®; Owens & Minor Inc., Franklin, Massachusetts, USA). One hundred micrograms of chicken egg ovalbumin (OVA Grade V; Sigma-Aldrich, St. Louis, Missouri, USA) in 100 μL of normal saline, or placebo (100 μL of normal saline), were placed on a patch of sterile gauze (1 × 1 cm), which was secured to the skin with a transparent bio-occlusive dressing. Each mouse had a total of three one-week exposures to the patch separated from each other by two-week intervals. In the acute skin challenge model, mice were EC-sensitized with OVA for 7 weeks then challenged on days 0 and 3 after sensitization (days 50 and 53 from the start) by means of application of OVA or saline to shaved and tape-stripped, previously unsensitized skin and were studied on day 7 of challenge (day 57 from the start). Mice were treated by i.p. injection with 0.4 mg/day bestatin or vehicle control, daily for 7 days beginning 2 days before the seven-day challenge as shown in Fig. 7A.
Ear skin was used for flow cytometry analysis due to a technical difficulty of preparing cell suspension from the dorsal skin. Cells from ears were isolated as described previously (He et al., 2008). Briefly, ears were separated in two halves and floated on trypsin-EDTA (Cellgro) for 30 min at 37°C to allow dissociation of the epidermis. Both epidermal and dermal sheets were incubated for a further 1-2 hr at 37°C. The resulting cell suspensions were stained with the appropriate antibodies and analyzed by flow cytometry.
For surface staining, the following antibodies were used: fluorochrome-labeled anti-CD11b, Gr-1, CD4, CD44, Vα2, Vβ5, CD45.1 and CD45.2 (all from eBioscience). Cells were analyzed on FACSCanto and the data was analyzed using Flowjo software.
LTB4 concentrations in human and mouse skin were determined using solid phase extraction followed by EIA kit measurement according to the manufacturer's instructions (Cayman Chemical).
Mouse neutrophils were prepared from the femur and tibia of WT and Ltb4r1−/− mice using discontinuous Percoll density centrifugation as previously described (Jia et al., 2007). Preparations were >90% pure as determined by flow cytometry analysis with double staining using Gr-1 and CD11b mAbs.
CD45.1+ WT recipient mice were treated with CTX as described above and ear skin were tape-stripped 20 hrs before neutrophil transfer. 1 × 107 bone marrow neutrophils were isolated from CD45.2+ WT, Ltb4r1−/−, and Lta4h−/− mice and i.v. injected into CTX-treated WT mice. The percentage of adoptively transferred neutrophils recruited to the ear skin was analyzed 4 hrs and 24 hrs after transfer using a FACSCanto (BD Biosciences).
In co-transfer experiment, 5 × 106 neutrophils from WT and Ltb4r1−/− mice were labeled with 1 μM CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) and 15 μM CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (Invitrogen) at 37°C for 10 minutes. Ltb4r1−/− or WT neutrophils loaded with CMTMR dye (red) were mixed with 5 × 106 WT neutrophils loaded with CMFDA dye (green) at an input ratio of ~1:1 and adoptively transferred by i.v. into CTX-treated WT recipients whose ears were tape-stripped 20 hrs earlier. Four hours later, the ratio of green:red fluorescent cells in the ears was determined, adjusted to the input ratio and the relative skin homing index was calculated.
Splenocytes from EC-sensitized WT or Ltb4r1−/− mice were cultured with OVA for 5 days, then CD4+ T cells were purified by AutoMACS and injected i.v. into naïve recipients (5 × 106 CD4+ T cells per mouse) which were challenged the same day by application of 100 mg OVA to shaved and tape-stripped dorsal skin. Skin challenge was repeated on day 3 and the skin site was examined on day 7. In selected experiments, 5 × 106 splenic CD4+ T cells from WT mice EC-sensitized with OVA were injected i.v. into naïve Ltb4r1−/− recipients on day 0 and WT, Ltb4r1−/−, or Lta4h−/−neutrophils were administered i.v. (1 × 107) or i.d. (1 × 106) on day 0 and day 3 of one-week EC challenge.
CD4+ T cells were prepared from the spleen of OT-II mice on WT or Ltb4r1−/− background and activated with irradiated syngeneic splenocytes (3000 rads) (1:10 ratio) as antigen-presenting cells (APCs) in the presence of OVA (100 mg/ml) and IL-2 (10 ng/ml) for 5 days. 5 × 106 activated transgenic CD4+ T cells from WT or Ltb4r1−/− background were injected i.v. into naïve recipients followed by application of OVA+cholera toxin (CT) or CT alone to tape-stripped ear skin as previously described (He et al., 2008). In selected experiments, recipients received WT or Ltb4r1−/− neutrophils i.v. (1 × 107) or i.d. (1 × 106) on day 0 and again on day 3 of one-week EC challenge. At the end of one-week EC challenge, single cell suspension was prepared from ear skin as described above, stained with the appropriate antibodies, and analyzed by flow cytometry.
Total RNA was extracted from homogenized skin tissue or from cultured cells with RNAqueous extraction kit (Ambion Inc) following the manufacturer's instructions. cDNA was generated with iScript cDNA synthesis kit (Bio-rad Laboratory). Quantitative real-time PCR was done using Taqman Gene Expression Assay, universal PCR master mix and the ABI Prism 7300 sequence detection system with commercial primers and probes, all from Applied Biosystems. Fold induction of target gene expression was calculated using the comparative method for relative quantitation by normalization to the internal control β2-microglobulin, as described previously (He et al., 2007). For histological analysis, skin specimens were fixed in 10% buffered formalin and embedded in paraffin. Multiple 4 mm sections of skin were stained with hematoxylin and eosin (H&E). Neutrophils were counted in ten randomly chosen areas 25 × 25 μm in size. CD4 staining of skin sections was performed as previously described (Spergel et al., 1998). Epidermal and dermal thickening, eosinophils, and CD4+ T cells were counted blinded in 10-15 high-power fields (HPFs) at a magnification of 400×.
Mice were bled, and serum concentrations of OVA-specific IgE, IgG1, and IgG2a Abs were measured using modified OVA-specific ELISAs as previously described (He et al., 2008).
Single-cell suspensions from spleen of EC-sensitized mice were cultured in complete RPMI 1640 (Invitrogen) supplemented by 10% FCS, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin at 3 × 106/ml in 24-well following the stimulation with OVA (100 μg/ml) for 4 days. Supernatants were collected for cytokine measurements by ELISA.
Two-tailed Student's t-test or one-way ANOVA was used to determine statistical differences between groups. A p value smaller than 0.05 was considered statistically significant.
This work was supported by the Atopic Dermatitis Research Network NIH/NIAID contract HHSN272201000020C (RSG), USPHS grants AR-047417 (RSG), AI 065858-03 (DML) HL-085100, AI-076471, HL-092020, GM-076084 and a Research Scholar Grant from American Cancer Society (HRL) and AI-050892 and AI-040618 (ADL), R01 AI078910 (LSM), T32 AR058921 (JSC), R24 CA92865 (UCLA Small Animal Imaging Resource Program), and the Children's Hospital Boston Faculty Career Development Fellowship (MKO). David M Lee is currently an employee of Novartis Pharma AG and owns stock/options >$10,000, and has ownership and consulting income >$10,000 from Synovex Corp. Mei Chen is currently an employee of Pfizer Inc. We thank Drs. Michel J. Massaad and Hans C. Oettgen for reading the manuscript. We also thank Ms. Jacqueline Beaupre for her technical assistance.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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text:
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text: Potent Daggamalt Potion Duration - 60 seconds Effect - Heals 930 Effect over time - Heals 155 every 1 second Retrieved from "http://aoc.wikia.com/wiki/Potent_Daggamalt?oldid=37461" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Missing Icon Potion Unconverted Items Consumable Add category Cancel Save


text:
SonicVision buttresses the audio mixes of KRUA 88.1's DJs with eye-popping digital animations created by the American Museum of Natural History and MTV2 as well as visualizations from the Fractal Foundation's Fractal Pack. No two performances feature the same music and visuals, making SonicVision a unique sensory buffet at every show. On February 25, DJ Aqua Black spins dreamy lo-fi with a taste of sounds from artists such as Tame Impala, Avey Tare of Animal Collective, and Beach House. Or check out MC Kennett on March 18, who may blast electro-acoustic tracks from Cut Copy, Animal Collective, or Handsome Furs. On April 18, DJ Wildebeest blasts his own electronic house beats alongside music from Justice and The Postal Service, and on April 22, The Captain feeds ears with indie and electronic soul, spinning tunes by Bloc Party, Legion of Doom, The Faint, and more.
The UAA Planetarium offers free parking on Fridays, and recommends leaving at least 10 minutes to park. UAA begins releasing seats 10 minutes prior to showtime, so make sure to arrive early.
Tickets to all UAA Planetarium shows are free for Planetarium members, and $5 for UAA students and children up to 18 years old. Children under the age of 13 are not permitted.


text:
'Tees victorious to start 2012 season
Jungmann wins pro debut in 11-4 rout of Daytona
By Frank Longobardo / Brevard County Manatees | April 5, 2012 7:02 PM ET
The Brevard County Manatees opened the 2012 campaign with an 11-4 win over their rival the Daytona Cubs on Thursday night, in front of 4,637 fans at Space Coast Stadium.
2011 first round pick Taylor Jungmann earned a win in his professional debut as he went five innings and allowed one run on four hits, while walking four and striking out three. Catcher Shawn Zarraga led the way at the plate as he went 4-for-5 with a double and four RBI.
Jungmann received run support early and often as the 'Tees scored seven runs of their 11 runs in the first two innings and chased Daytona starter Matt Loosen after just a third of an inning.
Brevard County benefitted from four walks in the first, en route to a three-run inning, including a back-to-back bases-loaded walks by Mike Walker and Shea Vucinich. Following the back-to-back walks, Zarraga drove in Scott Krieger with a RBI single and Loosen's night was finished.
The Manatees put another crooked number on the scoreboard in the second as they added four more runs on four hits and two more walks.
Reggie Keen also drove in four runs as he went 2-for-5 on opening night. Vucinich may have had the stat line of the night as he was 0-for-3 with two runs scored and two RBI on bases-loaded walks.
Shortstop Nick Shaw was 2-for-4 with two runs scored in the leadoff spot in his Brevard County debut.
Greg Holle, Eric Marzec, Alan Williams and Casey Medlen all pitched an inning each, in relief of Jungmann, with Holle and Medlen throwing scoreless frames.
The Manatees will continue their three-game series with the Cubs on Friday night at 7:05 p.m. in Daytona Beach at Jackie Robinson Ballpark.
2010 second rounder Jimmy Nelson will take the mound in his Manatees' debut. Brevard County will return home on Saturday to finish the three-game series.
This story was not subject to the approval of the National Association of Professional Baseball Leagues or its clubs.View More


text:
Fox at the Oscars: Homosexuality among the "darker themes this year"
While introducing a segment about the Academy Awards, Fox News' Gregg Jarrett said that the films nominated in the Best Picture category carry "darker themes," such as "homosexuality; homicide bombings; political assassinations."
While introducing a segment on the 78th Annual Academy Awards [1] on the March 5 edition of Fox News' Fox News Live, anchor Gregg Jarrett [2] noted that the films nominated in the Best Picture category dealt with themes such as "homosexuality; homicide bombings; political assassinations," and that Hollywood was "going with a lot of darker themes this year." As Jarrett spoke, onscreen text read: "Hollywood walks on the darker side."
From the March 5 edition of Fox News' Fox News Live:
JARRETT: They're the best movies of the year, allegedly. Most of us, though, haven't seen them. Films dealing with homosexuality; homicide bombings; political assassinations -- the list goes on and on. Hollywood, going with a lot darker themes this year, and they're courting some controversy.
- Posted In
- Diversity & Discrimination [4], LGBTQ [5]
- Network/Outlet
- Fox News Channel [6]
- Show/Publication
- FOX News Live [7]


text:
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text:
The deadline for signing up as a partner with the regional economic initiative "Campaign for One Acadiana" is Saturday.
The push to create a multiparish economic development organization began last year, including the fundraising goal of securing $3 million a year over the next five years.
Jason El Koubi, president and chief executive of the Greater Lafayette Chamber of Commerce that now runs the nine-parish One Acadiana, said contributions have been coming in but the group has not quite reached what's needed yet.
"We are quickly closing in on our $3 million annual budget goal," El Koubi said. "In order to cross the finish line, we need business leaders across the region on board with us as co-investors."
The list of contributors will be named after Saturday, the Jan. 31 deadline set by the chamber.
Contributors become part of a tiered system, with those contributing the most having the most influence.
Backers of One Acadiana, the current name for an organization that has yet to be formally named, say the organization will help the region speak with a stronger voice to pull in business and federal funds.
Anyone wishing to join can still contact the organization's Investor Relations Director Missy Bienvenu Andrade at missy@oneacadiana .org or call (337) 408-3652.


text:
Naugatuck Valley Financial (NVSL) in Connecticut has named a longtime financial manager to become its interim chief financial officer.
Thomas Van Lenten will serve as interim CFO at the holding company and Naugatuck Valley Savings and Loan. The Office of the Comptroller of the Currency did not object to Van Lenten's appointment, the $559 million-asset company said Wednesday.
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text:
It's a nice and clean Motel and the prices are ok. The staff was ok too. Pool was out of order. Bed was comfortable. Only bad thing was the vodka bottles we've found under the mattress the next morning when I was searching for my...More
Getting you more information on this room
Getting you more information on this room
"make sure a selection smoking vs no-smoking when choosing a room. Also, request a room away from the elevator..."
"Travel one mile to the south and you have a large selection of rooms that offer much more for the same price."
"Park their early because once the construction crews park in the lot, there is very little parking left."


text:
Os Principais blocos econômicos Mercosul Brasil Uruguai Argentina Paraguai Venezuela Chile Bolívia União Europeia França Alemanha Reino
Unido Itália Grécia Bélgica Dinamarca Portugal LUXEMBURGO Holanda Suécia Finlândia Áustria Espanha Irlanda Canadá EUA México Nafta Peru Equador PACTO ANDINO Colômbia China Japão Coréia do Sul Taiwan Vietnã Filipinas Brunei Indonésia Tailândia Malásia Autrália Nova Zelândia 4001 Informática APEC
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Thanks for sticking up for me guys, but I shouldn't of lashed out on the thread, I'm just extremely tired and should of done it privately so there wasn't such confusion, anyways..
Twitch.tv layout I've been working on for pinkusagi (I had to do it since I love Sailor Moon). I think I'm getting the hang of these twitch pages now.
I want to start a Signature request. I know that I'm a 0post user... I'm more a reader ;-)
Visible text: Fylona (Charactername), if it's not to much than: EU-Eredar Horde (server) and <Immortalitas> (guild)
Armory link/Facial screenshot: http :/ /eu.battle . net/wow/de/character/eredar/ fylona/ simple
Gear set: Like my armory Transmog (T12 Heroic Rogue)
Helm visible: Yes please
Your pose: If any1 could implemt my 2 Fistweapons (Claw+Tallon of the Phoenix) it would be nice.
General colour scheme: dark violett/black like my tier set
Any extras: I would prefer a MMO-C version and a 500x115/125 version for our guild forum
and respect to all the amazing graphic heroes here
That Twitch layout is absolutely stunning!
Request from yesterday that I claimed:
(Please download the full size image from my DA page using the download button there)
I don't feel like I'm all that great at wallpapers, however I'm going to open my signature queue and might possibly agree to take on a few wallpapers as well. I might look into learning how to make Twitch layouts soon.
I'm looking for one Twitch request to do this weekend. All these shiny ones made recently have left me feeling inspired.
Examples of my work can be found by clicking the deviantArt link in my signature space.
Use the template found in the first post.
PM me.
Usual blah blah blah.
I say Twitch, but I can stretch to a wallpaper. But no signatures (hated my recent ones so working on reinventing my signature style, again). Kthx.
Gotten too many PM's. No more please.
Last edited by ELYPOP; 2012-11-08 at 01:32 PM.
Elyaan! I have a request for you. I need a logo for my twitch stream to block my chat. I want it to be of my guild <TF> of Hyjal. Here's our website: http://www.thefabled.net/
General color theme is MoP (green/yellow/a bit of purple in there). General Theme is for MoP pandas!!
As for Text: I'd like to have "The Fabled of Hyjal" be there in ninja-esque font like the one on our website.
I'm really not that creative with background images or whatnot, just make it look MoPpy! Thanks in advance.
Posted a few weeks ago, no1 have picked it up yet. If it lacks information send me message and i will try to to my best. If you don't feel like doing the whole thing a background is enough because i can manage to make a somewhat decent offline banned and header myself.
I'm looking to start streaming but i'm terrible at photoshop, and was hoping that someone here could help me
What you want for you page:
I want the whole set
Visible text:
On the background i want my name "Haydn", my guild "Custom" and my server "Twisting Nether". On the offline banner, it should say something like "offline" "follow to see when i go online!", As for the header it should just be a nice background with the text "Haydn"
It's Haydn on Twisting Nether
General colour scheme:
I have no idea, i'm not a artistic person. I think it would be better if you decided, would probably be better
Specifics/Extras: I like the t12 hc gear that i have transmoged to (Volcanic Vestments), if you want to have my character on the background use that set + bulward of azzinoth.
Would be really awesome if someone had the time to do this "
Visible text: Character name, guild and server. (Pi, Dysfunction, Kel'Thuzad - US)
Armory link/Facial screenshot: http://us.battle.net/wow/en/characte...uzad/Pi/simple
Gear set: Monk Challenge Mode Set
Helm visible: Yes
Your pose: I really like a lot of the close up toon profile signatures that have been posted here. So, whatever pose that would be best for that.
General colour scheme: Cyan (General mistweaver colors)
Any extras: I would love if a Pi symbol could be incorporated or placed strategically somewhere within the signature.
Cinder Kitty with words: Got Cinder?
Could someone make this for me? Could be pretty simple I would assume.


text: Day of Destiny Printings/Rarity: Cost: CMC: 4 Card Type: Legendary Enchantment Oracle Text: Legendary creatures you control get +2/+2. Flavor Text: "Rise like the sun, stand like the mountain, charge like the lion, die as a hero."—General Takeno Retrieved from "http://mtg.wikia.com/wiki/Day_of_Destiny?oldid=66043" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Betrayers of Kamigawa Rare Cards CMC: 4 White Legendary Enchantment Legendary Enchantment


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Beauty in the World MP3
Abercrombie & Fitch May 2010 - Macy Gray - Beauty In The World (Cutmore Remix Edit) MP3
A&F Playlists - All the Abercrombie & Fitch tracks ever. http://www.anfplaylists.com.
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House dance remix with piano playback done by Cutmore. Comes from official "Louboutins" CD 2010.
Macy Gray - Beauty In The World Cutmore Radio Edit MP3
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Francisco Toscano - I want to feel free (Cutmore Club Mix) MP3
EP for SALE on JULY 16th, 2012! Available in iTunes here!: UK: http://bit.ly/MSSM6x, USA: http://bit.ly/NvsNDZ, México: http://bit.ly/Ls2zRH, France: ...
Crush Gone Wrong (Cutmore Club Mix) MP3
Provided to YouTube by DANCE ALL DAY Musicvertriebs GmbH Crush Gone Wrong (Cutmore Club Mix) · Kady Z Progressive House Anthems 2015 ℗ 2015 ...
CANDYMAN 187 ft. Snoop Dogg-CUTMORE Killa Club Mix.m4v (Danny Morris video remix) MP3
Candyman 187 ft. Snoop Dogg's Billboard Club Hit, "High off the Fame" (currently D49*-39*- Billboard Club!) taking off with NEW MIXES by CUTMORE AND ...
Mig & Rizzo Feat. Heidi - Love Slave (Cutmore Full On Club Mix) MP3
K'naan feat. Nelly Furtado - Is Anybody Out There? (Cutmore Club Mix) (Audio) (HQ) MP3
(HQ) Official Remix.
Beauty in the World (Cutmore Extended Remix) MP3
Provided to YouTube by Universal Music Group North America Beauty in the World (Cutmore Extended Remix) · Macy Gray Beauty In The World ℗ 2010 ...
Ke$ha - C'mon (Cosmic Dawn Club Mix) MP3
C'mon (Cosmic Dawn Remix) Ke$ha ℗ 2012 Kemosabe Records.
Mega Mix Justin Bieber - Portal Teen MP3
Track List : 1. Boyfriend (Joe Gauthreaux Peter Barona Remix)/ Main Version 2. As Long As You Love Me 3. U Smile (Brosill Remix) 4. Next 2 You (Jyvhouse ...
Weekly Spotlight - DJ Cutmore MP3
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K'naan Ft Nelly Furtado - Is Anybody Out There (Cutmore Club Remix) MP3
"Is Anybody Out There" is Co-written and performed by Somali-born rapper K'naan, featuring Canadian singer-songwriter Nelly Furtado, from his first extended ...
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For the people in America as it is not yet available to buy on the U.S.A iTunes. Please buy the EP on iTunes and get them to no.1!! Love you boys!


text:
'At home it was a choice of either housework or sexual abuse' Heartbreaking statements from victims of vile abuser
The victim impact statements from the brothers who were horribly abused by their mother's partner makes for harrowing reading.
Earlier today it was confirmed that the Wicklow man who abused them would be sentenced next year for assaulting the two boys hundreds of times.
The abuse included forcing the boys to commit incest with each other as well as allowing a friend to abuse the boys.
In powerful victim impact statements the two victims of a "paedophile rapist" described the "harrowing" effects of the abuse.
The elder victim, now aged in his 30s, said: "From the age of nine when the abuse started I felt confused and frightened at what was happening. He made it seem like it was normal but something was telling me it wasn't normal."
"When I realised it wasn't I felt embarrassment and shame. I had to keep it a secret. I would dread finishing school and having to go home."
"I felt I didn't have any friends because it took my self esteem and knocked the confidence out of me. Now I'm anxious all the time, have panic attacks. I'm socially awkward, find it very hard to trust or talk to new people.
"I feel like he robbed me of my childhood." He said he was afraid to bring friends back to the family home. From the age of 13 he began working part time jobs both in the mornings and into the night to avoid being at home.
"I was scared getting home that I was going to be abused. Most of the time he'd be in bed but sometimes he'd wait up to abuse me".
He said he got into trouble at school because "there was a lot of anger in me". He said he wrote a suicide note when he was 15 and tied a sheet around the banister upstairs to hang himself.
"I was so depressed. I didn't go through with it cos there was a little voice said: "No, don't let him win".
At the age of 21 he moved out and then started to "go off the rails". He said: "I couldn't cope. I wasn't ready to come forward and ask for help at that stage. I started to hit the drink very hard. I was drinking everyday after work."
"To this day I have no confidence in myself, it's affected my relationships. I find it hard to be intimate with someone 'cos I feel like he's standing over my shoulder, 'cos of the things he made me do. I suffer from physical problems of being intimate. I suffer from nightmares."
He said he is paranoid and overprotective when it comes to his own children.
His younger brother said he and his brother did not get the protection and teaching that parents are meant to give their children.
"Instead we got daily abuse from our abuser which we were asked to keep to ourselves for 22 years."
"It took our childhood and most of our adult life due to trying to hide it, the damage. It took so much away from me, me being a child and the closeness and bond I should have with my brother."
"We both knew what was happening to us but we were ashamed to talk about it and unable to be in each other's company."
"I have no friends due to no life skills as I don't have confidence. At home it was a choice of either housework or sexual abuse."
He said sleeping at night is hard because he is scared of the dark.
"As 80% of the abuse happened in my bedroom I have to sleep during the day where I can and where there is natural light. My daily battle is getting up to face another day ahead of me so the vicious cycle beings."
"Why not kill myself? I tried before to end it all. I didn't succeed. This situation is consuming every bit of my life where a simple task such as brushing my teeth is the hardest thing for me to do."
"I am living with mental health issues, anxiety, panic attacks, wondering will I ever have a normal life. Living what can only be described as hell on earth, not working enough to pay rent."
"I cannot wear clothes for more than one day. I have to have my hair trimmed every week in order to feel clean. When having a shower I need to have two towels as I feel the need to hide my body.
"Because I feel embarrassed of my body I have felt the necessity to get my body tattooed – to change my appearance."
By Declan Brennan


text:
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text: There is no accent on the bottle label, but I often see the word written with an accent over the "e" which makes me believe the emphasis is on the first syllable. A friend in the wine business tells me the emphasis is, instead, on the "trus." Which one of us is correct? Many thanks!


text:
Greeley-Evans school officials want extra money, just without the strings
February 10, 2014
Ranelle Lang would never turn down extra money for Greeley-Evans School District 6.
But since added money for the schools next year looks like it will come with strings attached, she and several dozen other Colorado superintendents have told the state legislature enough is enough.
The state needs to give back the money it's cut over the past few years before it starts adding more mandates, the school leaders say.
Lang and about 60 other superintendents submitted their own proposal for how to use the money, which includes $275 million in funds this year to restore some of what's been cut over the past five years. They ask that the remaining balance be restored over the next few years.
"A significant portion of the $275 million should be used to reduce the 'negative factor,' " the proposal reads. "… A portion of the $275 million allocation should be directed to students with significant risks of not making adequate educational progress (for example, English language development, poverty, special education or literacy)."
Lang said she doesn't support any bill currently on the table and won't support any future bills that don't address the negative factor, which was the mechanism used to reduce funding allocated to public schools that circumvented voter-approved legislation to raise education spending by 1 percent yearly.
"Every single district made choices about how they were going to meet the budget cuts, and when they try and restore them we will all make different decisions," Lang said. "But the notion that continues to be prevalent in Denver is 'I have a pet idea, therefore, I will apply it to all districts regardless of what local community A wants or B needs.' That is the part that really reached a frustration with superintendents."
Lang likens her budget, which has lost $23.5 million over the past few years, to being on a forced diet.
"Everything we have in our district is living on 500 calories a day," said the Greeley-Evans School District 6 superintendent. "It would be really nice to give every program at least 1,000 calories a day."
When Lang's hopes of doing just that were dashed with the downfall of Amendment 66 last fall, she immediately began to dread what next year's funding might look like and ponder what was left to cut.
But with more than $1 billion of excess money sitting in the state education fund, there were initial hopes that legislators would use some of it to bump up the governor's projected $200 million public school funding for 2014-15. However, the bills being introduced all have strings attached.
"They are dashing about trying to earmark the money," Lang said. "They are saying, 'OK, we'll give you more money, but you have to use it for additional transparency or to develop the average daily membership (a record -keeping program) or for whatever their pet project is at the time."
Sen. Scott Renfroe, R-Eaton, who is also the chairman of the West Ridge Academy Charter Board of Directors and a member of the state education committee, agrees with Lang and the others.
But he said he doesn't know if he can get enough support from other elected officials, with most of the bills being proposed taking bits and pieces of last year's failed school finance reform bill known as Senate Bill 213 and proposing them as individual bills or lumping several together.
"There are a lot of opportunities this year," Renfroe said. "There is quite a bit of money. But I understand the superintendents are not happy. There are a lot of strings tied to the money. Legislators complain of strings to federal government dollars but that's what we're looking at doing with this. I'd like to find a way to move that money back to the district and push the decision to the local level."
Rep. Dave Young, D-Greeley, said school finance is much more complicated and he's not ready to decide one way or another, but suspects it will be a tough sell to just give the money back.
"It is still unclear how much of the money is in ongoing (revenue)," he said. "So much of education is people. If a large percentage of that money is in one-time funds we can't just give it back to hire someone one year to be fired the next."
Young, a retired District 6 math teacher, said he appreciates the superintendents' position and he will do as much as he can but said there are a lot of things they can do with one-time money such as more technology.
"Everybody wants us to help," he said. 'But we want to be sure we're being thoughtful of where were putting the money."
District 6 Board of Education President Roger DeWitt echoed Lang's and Renfroe's remarks.
"The money that would help us do the extra things with our English language learners and (children) living in poverty, that's the money that disappeared," DeWitt said. "There are little bits of hemorrhaging going on … There are a variety of things we're not doing. There are programs and supports in some buildings and not in others. I'm excited about the idea of making a wish list of things we're not doing."
He added, "But I'm not holding my breath we'll get it, and I know there will be extra things they will ask us to do for the good of the order."
Lang said they need to restore funding they've cut before they begin to make more mandates on districts.
"We just want cuts restored to rebuild whatever it was in our district we reduced to make the budget," Lang said. "There are 178 different districts with 178 different things that happened. Our communities know best what they want."
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Damien Rice - Creep (Rare Acoustic) MP3
live from KCRW.
Creep - Damien Rice MP3
Creep by Damien Rice.
Damien Rice - Creep MP3
Creep by Damien Rice. - - - [LYRICS] When you're here before, Couldn't look you in the eye. You're just like an angel, Your skin makes me cry. You float like a ...
Creep (cover of Damien Rice's cover of Radiohead) MP3
In which I cover a cover.
creep -radiohead (damien rice) cover MP3
rough cover, procrastination from study, enjoyyyyy.
Damien Rice - Creep (Radiohead Cover) MP3
This is a beautiful song called "Creep" covered by Damien Rice, originally written and recorded by Radiohead. Lyrics: When you were here before Couldn't look ...
Damien Rice - Creep (Cover) MP3
Basically, the lyrics apply amazingly and I love this song... soo I thought I would cover it, also I really wanted to make a YouTube video as I haven't made one in ...
Damien Rice - Creep (Lyrics) MP3
Ein wunderschöner und vielsagender Song. :)
Damien Rice & Glen Hansard - Blower's Daughter + Creep (Busking on Grafton St Christmas Eve) MP3
Damien Rice and Glen Hansard busking on Grafton St, Dublin for Simon Community on christmas eve. 24/12/11.
Radiohead - CREEP Cover MP3
A cover of the song Creep, covered by Damien Rice, covered by Monkeyguitarist. The song "Creep" won't catch a cold this winter.
Damien Rice - The Blower's Daughter/Creep (Cover) MP3
I felt ambitious. Headphones are recommended and mistakes are plentiful.
[HD] Damien Rice — The Blower's Daughter & Creep (Live At Roma, 30-07-2012; HQ-Sound) MP3
All Credits: http://www.warnerchappell.com Video: http://www.youtube.com/watch?v=Ta2zsOWTdPc&list=UUlTQwvy8t2l5HU6GfXlq6zA&index=173.
Damien Rice - Creep cover MP3
Disclaimer: You can find the photo here http://laurazalenga.deviantart.com/art/Into-a-Dark-99213349 It's obvious I don't own the song. When you were here ...
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Damien Rice - Creep ( cover Radiohead ) lyrics MP3
I`am a creep.
Damien Rice - The Blower's Daughter & Creep (Sommerfest 2010) MP3
Sommerfesten, Giske, Norway - July 31, 2010 Part 6/7: Damien Rice - The Blower's Daughter & Creep.
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https://www.facebook.com/djthesmith songs and news! Radiohead's cover by Damien Rice. Enjoy it!
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Damien Rice - Creep vs The Blower's Daughter live @ Rock Werchter Festival (01-07-07)
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text: a female is alot bigger then a male Retrieved from "http://answers.wikia.com/wiki/How_do_you_tell_the_sex_of_a_spider?oldid=4472170" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Animals Answered questions Spiders Add category Cancel Save


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Brian S O'Neill" <brone...@earthlink.net>
|Re: [lang] FastStringBuffer?
|Thu, 07 Aug 2003 14:02:18 GMT
The big advantage that the StringBuffer has is that it's defined in the java.lang package. As a result, it has access to package private members of the String class. Calling toString on StringBuffer does not allocate a copy of the characters, but rather passes the reference to it's char[]. With any FastStringBuffer class, a copy will be created. This will likely hurt performance much more than removing synchronization. ----- Original Message ----- From: "Chris Feldhacker" <firstname.lastname@example.com> To: "Jakarta Commons Developers List" <email@example.com> Sent: Wednesday, August 06, 2003 08:30 P Subject: [lang] FastStringBuffer? > Maybe I'm missing something, or maybe this has been discussed before, but... > > Is there a replacement for StringBuffer someplace, perhaps a > "FastStringBuffer" class? > > I only ask because StringBuffers are used everywhere, even internally by the > JVM. However, all of the methods on the StringBuffer class are > synchronized. In my experience (and in looking through most of the lang > code), 99% of the time StringBuffer is only used as a method variable to > compose a message String; therefore, it is not possible for multiple threads > to access the same StringBuffer. So, why not eliminate the synchronized > method calls and increase performance? > > > > --------------------------------------------------------------------- > To unsubscribe, e-mail: firstname.lastname@example.com > For additional commands, e-mail: firstname.lastname@example.com >
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Details released Monday at a Scotland Yard news conference indicated that four men who attempted to bomb three subway trains and one double-decker bus ran through city streets but eluded capture after their lunchtime attacks failed. Police have identified two of the suspects. (Video: Police ID men)
Thursday's attempted attacks came two weeks after bombings on three subway trains and a double-decker bus killed 56 people, including the suicide bombers, and injured more than 700.
London remained tense Monday as the manhunt continued. On Friday, police — exercising their shoot-to-kill authority — killed a 27-year-old Brazilian whom they mistook for a bomber. (Related stories: Brazil demands explanation | Protests mount)
Prime Minister Tony Blair apologized Monday but defended the policy. "We are all desperately sorry for the death of an innocent person," he said. "But we also have to understand that the police are doing their job in very, very difficult circumstances."
Police again appealed for the public's help in the search for the bombing suspects. "I hope that by setting out some of what we have been able to learn over the past few days, the public may be able to contribute even more to the progress of the investigation," Peter Clarke, head of Scotland Yard's anti-terrorism branch, said at Monday's news conference. Scotland Yard said police have received more than 500 telephone tips on its anti-terror hotline and 80 e-mail messages since Friday.
Clarke said all four men carried a knapsack or a sports duffel bag holding a homemade bomb packed in a plastic food container. A fifth bomb discarded in a park near one subway station was in an identical container. The attack, he said, was coordinated. Three of the men entered the Stockwell subway station together at 12:25 p.m. Thursday. (Related story: Bombs yield clues)
He asked shopkeepers who may have sold five or more of the clear plastic containers with white lids to call police if they can remember who purchased them. The plastic containers, imported from India, are sold in about 100 stores in Britain.
Police have arrested five people, including two Monday, in connection with the bombings.
Clarke identified the bus bomber as Muktar Said Ibrahim, also known as Muktar Mohammed Said. Said, 27, went into the Stockwell Underground station and was seen heading toward the train platform. At 12:53 p.m. Thursday, he boarded the No. 26 bus carrying a gray-and-black knapsack and sat toward the back of the bus. Said placed the bag next to him and got off the bus 12 minutes later after he attempted to detonate the bomb, Clarke said.
He named Yasin Hassan Omar, 24, as the man who tried to set off a bomb on a train between Oxford Circus and the Warren Street station. Clarke said Omar entered the Stockwell station at 12:25 with a purple knapsack. At 12:40 p.m., he was spotted without the knapsack in the Warren Street station. He vaulted the ticket barrier and ran toward the exit, Clarke said.
The nationalities of Said and Omar were not disclosed.
Armed officers raided a London apartment that Said was believed to have visited recently. Forensic officers in white overalls searched the apartment in Curtis House, a concrete high-rise in the city's northern suburbs.
Resident Sammy Jones, 33, said she thought she recognized a man who visited the building as Said. She said he stayed with an African named George who lived in a ninth-floor apartment. Jones said two men of Somali appearance also visited the apartment.
"They used to come in at all hours of the night," she said.
She said a few weeks earlier she had seen George and the man she thought was Said filling an elevator with small brown cardboard boxes. When she asked what was in them, they replied "wallpaper stripper."
Metropolitan Police also said Monday they had arrested two people on suspicion of terrorism in the area but not at the raided address. Three other suspects are already being questioned at a high-security London police station "on suspicion of the commission, instigation or preparation of acts of terrorism" in connection with the July 21 attacks.
Police did not identify the man who attempted to set off a bomb on a train traveling toward the Oval station. The man, who wore a shirt emblazoned with "New York," left the station at 12:35 p.m. chased by passengers, police said. He ran toward Brixton and left the T-shirt at an intersection. He was last seen at 12:45 p.m.
A fourth man wearing a dark blue baseball cap entered a different station at 12:20 p.m. He tried to detonate his bomb on a train traveling toward Shepherd's Bush. Police say he escaped the train by climbing through a back window and running down the tracks. He ran past the British Broadcasting Corp. building and was last seen under the A40 highway.
Forensic examination of the four partially detonated bombs found "clear similarities" with a fifth bomb found under a bush by a passerby in a park near the Shepherd's Bush station in west London, Clarke said.
Police Commissioner Ian Blair said police were mounting "an absolutely brilliant operation, and it is, of course, racing against time."
The police commissioner has indicated he believed al-Qaeda-linked terrorists were involved in both the July 7 and July 21 attacks. Asked if the attacks were connected, he said: "We have no proof that they are linked, but clearly there is a pattern here."
Police were reportedly investigating whether some of the July 21 suspects may have visited the same Welsh whitewater rafting center as two of the July 7 suicide bombers, Mohammed Sidique Khan and Shahzad Tanweer. (Related story: Suspects linked to rafting)
The men went rafting there June 4, according to the National Whitewater Center. Police have refused to comment on media reports that a rafting center brochure was found in the explosives-laden knapsack found on a bus July 21.
Center director Paul O'Sullivan told The Associated Press the two identified July 21 suspects were not registered on the same rafting trip as the July 7 suspects.
Contributing: Wire reports


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dandysplay... Photographer
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Credit NotesAdinda Nada Shofa Nasution
Mayhem #2482420
Follow me on twitter @nadaSN: http://www.twitter.com/nadaSN
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Front Teeth + Teeth Whitening Q&A
How can I clean the yellow brown spots from my teeth? (photo)
They have been since my childhood. Will cleaning help? I'm scared to try cleaning coz ppl say that it will cause gums to loose their grip. Pls help READ MORE
Why are my teeth translucent/transparent? Is there a way to fix? (Photo)
Ive been noticing that my top two front teeth are translucent from the edges a little to the bottom middle too i can notice it whenever i get close in... READ MORE
What Would Cause Only 1 Tooth to Stain Much Faster Than the Others? (photo)
I have 1 front tooth that is suddenly staining quickly yet the rest of my teeth appear fine. What would cause 1 tooth to stain so quickly while the... READ MORE
How may I able to get rid of brown spots on the back of my front teeth? (photos)
I have brown dirt which doesn't come off at the back of my front teeth at the back, I have recently been drinking lots of green tea, could that been... READ MORE
Front Tooth Yellow Again After Internal Bleaching Should I Get It Done Again or a Crown Etc? (photo)
I've had root canal and internal bleaching in the past but the tooth after a year has turned yellow again. Should I keep getting internal bleaching or... READ MORE
How are my front two bottom teeth so yellow & is there a way to whiten them? (Photo)
My 2 front bottom teeth are extremely yellow while the rest of my teeth are white. I know I had a eating disorder when I was young and dumb so I don't... READ MORE
I chipped the front surfaces, top layers, of my 2 front teeth and the chips are now discolored. What can I do at home to whiten?
I chipped the front surfaces, or top layers, (Is this my enamel?) of both my front teeth. Both chips are each about the size of a sewing pin head and... READ MORE
Which treatment for yellow stains and uneven teeth should I go for? (photos)
As you can see my front upper teeth are protruding out and lower jaw is smaller then the upper, i am 22 years old. what kind of treatment should i... READ MORE
Can a Tooth/crown Be Excluded when Making a Mold?
I have crowns on my front teeth (crowns are 15 yrs old). One of the crowns has a small nick off the front face (invisible to anyone but myself and my... READ MORE
What should I do about my discolored front teeth? (photos)
When I was a kid I fell on concrete and chipped one of my front teeth, and it seems damaged both. One tooth has a yellow hue while the other a dark... READ MORE
My teeth has a yellow stain and can't remove it. Any suggestions? (photos)
2007 was when I first noticed the stain on my 2 front teeth. I am currently 18 years old and this Potential could ruin my life and a chance of getting... READ MORE
My upper front tooth is getting yellow. What are the measures to stop it? (Photo)
I know that my tooth is broken (not like chipped ) from its root. I checked it by heating a spoon and placing it on the tooth and I felt nothing, but... READ MORE
Is it safe to use a whitening tray AFTER getting teeth bonded?
I had my front 2 teeth bonded about 3 weeks ago. I have a whitening tray with whitening gel that I use occasionally. It comes with a small LED light... READ MORE
My composite having black spot on front tooth 1 week after procedure. Any suggestions? (photos)
Hi. I got a composite I my front two teeth almost 3months back for cosmetic reasons.however after 5days from procedure I had a streak of blood on one... READ MORE
What is the best whitening system to lighten a dark front tooth (non-vital) on a teen? (photos)
Front tooth was injured two weeks ago and is already getting darker. What is the best way/system to whiten her teeth so the dark front doesn't stand... READ MORE
What can i do for a Non-vital front tooth that is not painful or infected?
I went for my normal cleaning, and I'm getting married in Sept, so they give you a free whitening kit for home use as a "gift" They looked at my teeth... READ MORE
What is the best course of treatment for a dark front tooth that won't respond to bleaching? (photo)
My two bottom center front teeth were damaged in an accident. they both have had root canals but over the years one tooth has turned very dark and... READ MORE
My four teeth are porcelain and they are a shade lighter than the rest. What can I do?
Pain during/after internal bleaching (for 2 days)
I am getting some sore pain in my front teeth which has been treated with internal(walking) bleaching after root canal therapy. My dentist has assured... READ MORE
How can I whiten my teeth at home? Does baking soda work to whiten teeth? (photos)
Right now I can't afford any expensive teeth whitening treatments, maybe when I am older. How can I safely whiten them at home for less than about... READ MORE