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Conceived and designed the experiments: PRE UW MAF TJ SFM. Performed the experiments: PRE CD. Analyzed the data: PRE CD SFM. Contributed reagents/materials/analysis tools: FDL CMS MAF. Wrote the paper: PRE SFM.
Allergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated skin disease induced by protein-reactive organic and inorganic chemicals. A key feature of contact allergens is their ability to trigger an innate immune response that leads to skin inflammation. Previous evidence from the mouse contact hypersensitivity (CHS) model suggests a role for endogenous activators of innate immune signaling. Here, we analyzed the role of contact sensitizer induced ROS production and concomitant changes in hyaluronic acid metabolism on CHS responses.
We analyzed in vitro and in vivo ROS production using fluorescent ROS detection reagents. HA fragmentation was determined by gel electrophoresis. The influence of blocking ROS production and HA degradation by antioxidants, hyaluronidase-inhibitor or p38 MAPK inhibitor was analyzed in the murine CHS model. Here, we demonstrate that organic contact sensitizers induce production of reactive oxygen species (ROS) and a concomitant breakdown of the extracellular matrix (ECM) component hyaluronic acid (HA) to pro-inflammatory low molecular weight fragments in the skin. Importantly, inhibition of either ROS-mediated or enzymatic HA breakdown prevents sensitization as well as elicitation of CHS.
These data identify an indirect mechanism of contact sensitizer induced innate inflammatory signaling involving the breakdown of the ECM and generation of endogenous danger signals. Our findings suggest a beneficial role for anti-oxidants and hyaluronidase inhibitors in prevention and treatment of ACD.
Allergic contact dermatitis (ACD) is a T cell-mediated delayed type hypersensitivity reaction, which is induced by protein-reactive organic chemicals or metal ions. In mice, the contact hypersensitivity (CHS) model mimics the processes occurring in human ACD. The first skin contact with sensitizing allergens results in activation and migration of allergen-bearing skin DCs to the skin-draining lymph nodes where they complete maturation and present the antigen to allergen specific naive T cells. Subsequently, in a second phase, re-exposure to the same sensitizer results in the recruitment of effector T cells to the inflamed skin and their cytotoxic action on skin cells [1], [2]. The adaptive immune response in ACD is elicited mainly by activation and expansion of cytotoxic CD8+ Tc1 or CD4+ Th1 cells and Tc17/Th17 cells in a multi-step process [3], [4], [5]. In murine CHS the main effector cells are cytotoxic Tc1 cells.
A crucial step for efficient priming of naïve T cells is the induction of a maturation process in DCs. However, in contrast to pathogen induced TLR triggering and subsequent activation of the MyD88 and TRIF dependent signaling pathways by invading pathogens [6], DC activation caused by contact sensitizers is incomplete. The in vitro exposure of DCs to 2,4,6-trinitrochlorobenzene (TNCB) leads to the up-regulation of co-stimulatory molecules, but fails to induce a cytokine response [7]. In this case, for full activation of DCs a secondary signal is necessary that is derived from the tissue microenvironment [8], [9], [10], [11], [12]. This signal might be provided by endogenous ligands activating pattern recognition receptors (PRRs). Indeed, our observation that double deficient mice lacking expression of functional IL12Rβ2/TLR4, IL12Rββ2/TLR2 or TLR2/TLR4 are resistant to CHS while expression of the above combination of receptors on murine DCs only is sufficient for the induction of CHS, strongly suggested a role for endogenous TLR2 and TLR4 ligands [7]. Several endogenous molecules, so-called damage associated molecular patterns (DAMPs), have been suggested to elicit immune-stimulatory effects - analogous to microbial pathogen associated molecular patterns (PAMPs) - by triggering TLR or NOD like receptor (NLR) signaling [13], [14]. Among these endogenous molecules are heat shock proteins, uric acid, ATP and ECM components such as biglycan and low MW fragments of hyaluronic acid (HA) [15], [16], [17], [18].
HA is a negatively charged glucosaminoglycan ubiquitously distributed in the ECM [19] and is primarily produced by dermal fibroblasts and epidermal keratinocytes and to a lesser extent by other cell types like smooth muscle cells [20]. Regarding its immune-modulatory effects, the size of HA plays an important role. High MW HA (<1×106 kDa) is anti-angiogenic, anti-inflammatory and immunosuppressive [21], [22], [23]. In contrast, breakdown products occurring in the range from 1.2 to 500 kDa that are generated during inflammation or tissue damage induce pro-inflammatory innate immune responses [24] presumably via TLR2 and/or TLR4 in immune cells like macrophages or DCs [10], [25] and play a pro-inflammatory role in lung inflammation [8], [26]. HA fragments can be generated enzymatically by a group of hyaluronidases, and non-enzymatically by ROS, especially at sites of inflammation, tissue injury and tumorigenesis [27].
The fact that sensitization to the contact sensitizer TNCB was significantly reduced in germ-free mice pre-treated with an inhibitor of HA function (Pep-1) underlined the role of HA in the induction of skin inflammation by contact sensitizers [7].
In the present study, we further investigated the role and metabolism of HA as a putative endogenous activator of innate immune signaling necessary to trigger full activation of DCs in vivo. We suggest a new mechanism for the generation of a pro-inflammatory milieu by organic contact sensitizers. In contrast to the direct human TLR4 activation by the metal ion nickel [28], organic sensitizers such as TNCB induce ECM degradation thereby providing endogenous activators of the innate immune response. Understanding the underlying molecular mechanisms of this xenoinflammation which is a crucial prerequisite for the development of CHS responses [13], [14] is essential for the development of reliable in vitro test systems for the identification of chemicals with skin sensitizing potential [29]. In conclusion, interference with contact sensitizer induced modulation of HA metabolism might help to prevent the innate inflammatory response that is instrumental both for the sensitization and elicitation phase and should, therefore, result in new therapies for ACD.
C57BL/10 mice lacking TLR2 and TLR4 are resistant to TNCB-induced CHS responses (Figure 1A) and, moreover, CHS responses to TNCB (Figure 1B) and oxazolone (data not shown) are successfully induced in germ-free TLR2- and TLR4-competent C57BL/10 ScSn wildtype mice. These data confirm our previous findings [7] and suggest strongly that endogenous activators of innate signaling such as low MW HA fragments are required in the sensitization and elicitation phase of CHS. Based on our previous demonstration of a role of HA in CHS we focused on HA and its metabolism [7].
Pro-inflammatory low MW fragments of HA can be produced by oxidative degradation of high MW HA by ROS [30], [31]. Over-expression of extracellular SOD inhibits this degradation and prevents experimental lung inflammation [32] and CHS in mice [33]. Therefore, we analyzed the ability of the strong contact sensitizer TNCB to induce ROS production in the murine keratinocyte cell line Pam212 and in the fibroblast cell line L929 in vitro (Figure 2A). We observed ROS production that was efficiently blocked by the plant derived antioxidant RF-40s [34] as well as by the antioxidant N-acetylcysteine (NAC) (Figure 2A). Furthermore, dose-dependent ROS production was observed in Pam212 cells also after treatment with the extreme sensitizer oxazolone and the moderate sensitizer mercaptobenzothiazole (MBT) [35], [36], but not with the non-sensitizing pro-hapten para-phenylene diamine (PPD) (Figure 2B). H202 treatment served as positive control (Figure 2B).
In addition, Figure 3A shows that ROS production is induced in vivo by treatment of mouse ears with TNCB and that this production can be blocked again by pre-treatment with RF-40s or NAC.
To determine whether contact sensitizers induce mitochondrial ROS production, we analyzed the TNCB- or LPS- (positive control) stimulated ROS response in Pam212 and L929 cells after addition of the mitochondria specific ROS detection reagent MitoSOX™. Both stimuli induced a mitochondria-specific ROS response in both cell types (Figure 3B). Moreover, treatment of primary human keratinocytes or fibroblasts with the strong sensitizer DNCB (Figure 3C), as well as treatment of bone marrow derived DCs (BMDCs) with TNCB (Figure 4A), shows that contact allergens induce ROS also in primary cells. To assess the contribution of NADPH oxidase-induced and mitochondrial ROS to total ROS production, Pam212 and L929 cells (Figure 4B,C) were pre-treated with the mitochondria specific antioxidant ammonium pyrrolidine dithiocarbamate (APDC) [37] before stimulation with TNCB. This treatment failed to reduce the overall ROS response. Similar results were obtained when Pam212 or L929 (Figure 4B,C) cells were treated with the NADPH-oxidase specific inhibitor diphenyleneiodonium (DPI).
Recently, gp91phox (Nox2) knock out mice, that lack the catalytic subunit of the superoxide-generating NADPH oxidase Nox2, have been reported to be less susceptible to dextran sulfate sodium induced colitis [38]. This effect seemed to depend on a reduced oxidative burst in the intestine. When we sensitized and challenged gp91phox knock out mice with TNCB, ear swelling was normal compared to responses in wild type mice (Figure 4D), indicating that abrogating only Nox2 dependent superoxide production is not sufficient to suppress the CHS response.
Collectively, our findings demonstrate that contact sensitizers induce ROS production in vitro and in vivo that can be blocked by addition of antioxidants like RF-40s or NAC.
To investigate whether contact sensitizers are able to induce HA degradation in vivo we visualized the HA content in murine abdominal skin by immunohistochemistry after contact sensitizer treatment. Figure 5A shows paraffin sections obtained 2, 4, 24 or 72 h after treatment with the strong contact sensitizer TNCB. In comparison to the solvent treated controls a significant decrease in epidermal HA content was observed 24 h after contact sensitizer application. 72 h after TNCB application, re-occurrence of HA and thickening of the epidermis was observed. Untreated skin showed the same HA content as solvent treated skin (data not shown). Disappearance of epidermal HA was also observed 24 h after application of the strong sensitizer DNFB and the extreme sensitizer oxazolone (Figure 5D). Similar effects were observed when analyzing human skin treated ex vivo with TNCB (Figure 5B). These data suggest that contact sensitizers induce the degradation of HA in the epidermal layer of the skin to low molecular weight HA fragments that may be important mediators of the contact sensitizer induced innate inflammatory response [7], [10], [25], [39], [40], [41].
To study the degradation of high MW HA to low MW fragments induced by contact sensitizers in the skin in vivo, we performed agarose gel electrophoresis of HA from punch biopsy samples taken from mice at different time points (untreated, 4 h and 24 h) after contact sensitizer application. Figure 5C shows the degradation of high MW HA 4 h after application of TNCB to lower MW fragments compared to the untreated control. The predominant occurrence of low MW fragments is still detectable 24 h after treatment. This confirms the ability of contact sensitizers to induce HA degradation in vivo.
In order to directly assess the oxidative degradation of high MW HA and the effects of antioxidants by SDS-electrophoresis, we made use of a ROS producing Cu(II)SO4/H2O2 system as recently described by Gao et al. [32]. Figure 6A shows the in chemico degradation of high MW HA by ROS (lanes 5–7). RF40s treatment resulted in a dose dependent inhibition of degradation (lanes 8–10). In contrast, treatment of HA with combined CuSO4 and H2O2 (lane 4) or additionally with solvent (lane 5–6) shows the ROS mediated degradation of HA and the only weak anti-oxidative effect of solvent even at high concentrations. Similar inhibition of ROS mediated HA degradation was observed for the standard antioxidant NAC (Figure 6B). Importantly, blocking ROS production with NAC in vivo also inhibited HA degradation 24 h after TNCB application on ear skin as observed by HA staining in the epidermis (6C). Degradation of HA was confined to the side of the ear that was treated with TNCB (upper side in the panels). These results show the ability of ROS to directly degrade high MW HA to low MW fragments as well as the ability of both RF-40s and NAC to prevent this oxidative HA degradation.
Due to the pro-inflammatory role of ROS and their ability to induce HA degradation, we next analyzed the effects of topical pre-treatment with antioxidants on the development of CHS to TNCB. As shown in Figure 7A, the topical pre-treatment of the murine ear skin with NAC, Trolox or α-Tocopherol after ethanol wiping, or with RF-40s 30 min before sensitization resulted in an inhibition of the CHS response as measured by increase in ear thickness after challenge with the sensitizer. To verify that the effects of RF40s are due to its major antioxidant component, the flavonoid luteolin [34], [42], we treated mice with purified luteolin (HPLC >98%) or the respective solvent (ethanol). A similar reduction of the CHS response as seen after treatment with RF-40s was observed (Figure 7B). To further analyze the effect of RF-40s on the CHS response, we treated mice with RF-40 extract dissolved in ethanol (RF-40e) instead of the nanoparticular solvent used in RF-40s. Again, a similar reduction of CHS response compared to RF-40s or pure luteolin were observed (Figure 7B). Thus, we provide in vivo evidence that antioxidants are able to inhibit CHS responses to TNCB. In addition, we clearly demonstrate that the use of RF-40s is as effective as pure luteolin.
To further investigate the role of ROS in the CHS response RF-40s was applied either up to 180 min before or after sensitization or up to 180 min before or after challenge (Figure 7C). Application of RF-40s within 30 min before or after sensitization inhibited CHS responses. In addition, the application of antioxidants up to 180 min before or after challenge of sensitized mice also reduced CHS responses, indicating the potential use of antioxidants for the causative treatment of CHS responses. Notably, the inhibition of CHS was also observed in the case of the extreme sensitizer oxazolone when sensitized mice were pre-treated with RF-40s 15 min before challenge (Figure 7D). These data show that ROS crucially contribute to contact sensitizer induced skin inflammation and that blocking ROS production effectively inhibits CHS responses both in the sensitization as well as in the elicitation phase.
We next investigated the ability of contact sensitizers to enhance hyaluronidase activity in the skin using hyaluronidase zymography. Figure 8A shows that extracts of skin biopsies obtained 24 h after TNCB treatment exhibited enhanced hyaluronidase activity, whereas those from controls exhibited lower activity. The increase in hyaluronidase activity after contact sensitizer treatment is ROS dependent as NAC efficiently blocks it. These results identify a role of ROS in the regulation of hyaluronidase activity and are in line with a recent study showing ROS and p38 MAPK dependent regulation of hyaluronidase 2 (Hyal 2) activity in the airway epithelium [43]. They further link contact sensitizer treatment with enhanced hyaluronidase activity levels observed in skin.
To address the potential importance of the enhanced hyaluronidase activity for the CHS response we analyzed the in vivo effect of the hyaluronidase inhibitor aristolochic acid (AA). For this purpose AA was injected subcutaneously at non-toxic concentrations into the ear pinna of C57BL/6 mice 15 min before sensitization. Figure 8B shows that AA treatment prevented the CHS response to TNCB. To rule out that the effect of AA is due to toxicity or interference with skin penetration and to address the specificity of the AA effects we co-injected excess amount of active hyaluronidase with AA. This co-injection restored the CHS response to the level of control without AA (Figure 8B). Interestingly, injection of heat-inactivated hyaluronidase failed to do so (Figure 8C). These results show that functional hyaluronidase activity is a necessary prerequisite in the sensitization phase of CHS responses.
To further analyze the link between the contact sensitizer mediated generation of ROS and the increased hyaluronidase activity for CHS, we pre-treated ears of mice with the topically applied antioxidants NAC or RF-40s and co-injected active or heat inactivated exogenous hyaluronidase before sensitization with TNCB. Figure 8C shows the decreased CHS response in antioxidant treated compared to solvent treated mice. However, when mice were pre-treated with active hyaluronidase the inhibitory effect of the antioxidants was overcome and CHS responses were similar to untreated mice. For this effect, the enzymatic activity was crucial as heat inactivated hyaluronidase had no rescue effect for CHS in antioxidant treated mice (Figure 8C).
In order to study the potential role of p38 MAPK in the ROS mediated regulation of hyaluronidase activity mice were treated with the p38 MAPK inhibitor SB203580 before sensitization (Figure 8C). This treatment prevented the development of CHS to TNCB. Interestingly, also in this case active but not heat inactivated hyaluronidase reverted this effect indicating the involvement of p38 MAPK activation in the activation of hyaluronidases in vivo and, thereby, the induction of CHS responses (Figure 8C).
Preparation of ear sheets from C57BL/6 mice by mechanical separation of skin layers resulted in a trauma-induced production of the NF-κB dependent pro-inflammatory cytokine IL-6. This IL-6 production was abrogated by addition of AA to the ear sheet cultures indicating that hyaluronidase activity was required. In vitro treatment of the ear sheets with water-soluble 2,4,6-trinitrobenzene sulfonic acid (TNBS) resulted in reduced IL-6 production. Additional AA treatment further reduced IL-6 levels (Figure 8D). Similar effects of AA on IL-6 production were observed with ear sheets from NMRI mice treated in vitro with TNBS (Figure 8E). Interestingly, addition of active hyaluronidase reverted the inhibitory effects of AA. However, hyaluronidase addition to TNBS treated ear sheets did not increase IL-6 production further (Figure 8E).
In summary, these data indicate that contact sensitizer induced ROS production is crucially involved in the p38 MAPK-dependent up-regulation of hyaluronidase activity in vivo. Our findings demonstrate a critical role for contact sensitizer induced oxidative and enzymatic HA degradation in the induction of skin inflammation in CHS. Of note, the injection of high doses of hyaluronidase interferes with CHS (Figure 8F). This may be the result of excessive HA degradation which may prevent the timely generation of pro-inflammatory HA fragments required in the sensitization process.
Skin inflammation mediated by the innate immune system is a crucial step in the sensitization to contact allergens [13], [14], [27], [44]. Recent work has shown that contact sensitizers trigger innate immune mechanisms involved in anti-infectious responses [7], [18], [45], [46], [47]. These mechanisms are acting in a non-redundant collaborative manner [13], [48]. We have shown that CHS induced by organic contact sensitizers such as TNCB and oxazolone is absent in TLR2/TLR4 double deficient mice [7]. Since CHS responses develop normally in germ-free mice, we hypothesized that organic sensitizers utilize endogenous skin-derived TLR ligands to generate a pro-inflammatory tissue microenvironment [7]. This hypothesis is supported by the fact that TNCB and other organic contact sensitizers up-regulate maturation marker expression by DCs but fail to induce NF-κB dependent cytokine production by DCs in vitro [7]. Candidates for endogenous TLR2 and TLR4 activators are low MW HA fragments [8], [9], [10], [25], [39], [40]. High MW HA prevents TLR2 and TLR4 triggering but inflammation results in the generation of pro-inflammatory low MW HA fragments [8], [10]. Combined engagement of TLR4 and the HA receptor CD44 is needed for a full inflammatory response to HA fragments in vitro [40].
A putative role for HA in CHS has been described by us recently [7]. Blocking HA function with a peptide inhibitor prior to sensitization significantly reduced the CHS response to TNCB in germ-free mice. Therefore, we assumed that also in CHS high MW HA must be degraded to low MW fragments that provide necessary endogenous activators of innate inflammatory signaling in the skin.
We now show the degradation of HA in the epidermis of mice following application of contact sensitizers. This may trigger the release of soluble low MW HA and promote inflammation as described [39], [40]. Although to our knowledge no direct interaction of HA with TLR2 or TLR4 has been demonstrated so far, our data provides strong in vivo evidence that the degradation of HA to low molecular weight fragments is essential for the induction of a pro-inflammatory tissue micromilieu. In combination with the data that TLR2/4 expression is crucial for the induction of CHS our data therefore suggests that the inflammation caused by HA degradation involves TLR signaling. So far, we have unfortunately been unable to show an in vitro activation of DC using commercial low molecular weight HA fragments ranging from 2 to 12mers obtained from different suppliers, none of these fragments showed DC activating capacities (data not shown). This may be due to the fact that the exact HA fragment size required for TLR2/4 activation has not been clearly defined using synthetic material. Moreover, the activating structure in vivo may be different from the synthetic fragments, for example it may contain HA binding proteins as found in the ECM. Previous publications showing in vitro activation of DC and a role for HA in lung injury have all used their own HA preparations which make a direct comparison of the data difficult and leave open the possibility that the HA fragments from biologic material might contain either other TLR2/4 ligands or ligands for other TLRs. Biochemical data on the direct interaction of fragmented HA with TLR2/4 are urgently needed.
HA fragments can be generated either by the activity of HA degrading enzymes, the hyaluronidases, or by oxidative de-polymerization induced by ROS [31], [49], [50], [51], [52]. As HA degradation also occurs in human skin when contact sensitizers are applied ex vivo, our data indicate for a species spanning mechanism.
Inhibition of ROS induced HA degradation was linked to a reduction of inflammation in both bleomycin- and asbestos-induced models of pulmonary fibrosis [30], [32] as well as in a reduction of inflammatory gene expression in alveolar macrophages and epithelial cells (35). Remarkably, contact sensitizers induce ROS production both in human keratinocyte cell lines [53] as well as in DCs [54].
We demonstrate here that contact sensitizers induce ROS dependent degradation of high molecular weight HA in the skin. Our data did not show clear inhibitory effects of APDC treatment or gp91phox deficiency. This suggests a role for ROS from different cellular sources most likely including NADPH-oxidase dependent and mitochondrial ROS production. It remains to be determined whether these different ROS sources are redundant, additive or synergistic. In addition, we detect upregulation of hyaluronidase activity by contact sensitizers in the skin that promotes HA degradation. Functional inhibition of hyaluronidases by the hyaluronidase inhibitor AA abrogates not only trauma induced IL-6 production in vitro, but, more importantly, prevents sensitization for CHS. This inhibition is reverted when exogenous active hyaluronidase is co-administered with AA. Interestingly, inhibition of p38 MAPK activation also prevents CHS responses. This effect is at least in part dependent on the activity of hyaluronidases as co-administration of exogenous hyaluronidase reverts the inhibitory effect of the p38 MAPK inhibitor. These results are in line with a recent study showing that activation of p38 MAPK in lung inflammation results in enhanced hyaluronidase activity which in turn leads to the generation of low MW HA fragments and exaggerated inflammation [43].
So far, the functional role of HA degradation in ACD is not fully understood. In the present study, we underscore the pro-inflammatory role of HA breakdown in CHS and show that contact sensitizers modulate HA metabolism. Our data support the concept that contact sensitizer induced DAMPs serve as endogenous danger signals that are perceived by innate immune receptors [13], [48]. Thus, contact sensitizers induce HA breakdown which may result in TLR2-, TLR4- and CD44-dependent DC activation. In CHS, this HA mediated signal is delivered in the tissue microenvironment of the skin and is required for full DC activation in addition to the TLR independent induction of co-stimulatory molecules such as CD86 [7], [11]. Our data are in line with recent reports showing that congenital over-expression of hyaluronic acid synthetase 2 (HAS2) in Shar Pei dogs results in reoccurring breakdown of HA into low MW fragments, leading to the inflammatory hereditary periodic fever syndrome [55].
HA breakdown in CHS seems to be initially ROS mediated given the rapid induction of ROS by contact sensitizers. The central importance for ROS in chemical induced skin inflammation has been highlighted by the prevention of CHS involving a block of Langerhans cell migration upon keratinocyte directed over-expression of extracellular superoxide dismutase [33]. This may be due to the prevention of the oxidative and enzymatic HA degradation in the absence of sufficient amounts of ROS since ROS also regulate p38 MAPK dependent up-regulation of hyaluronidase as recently described for lung inflammation [43].
Thus, contact sensitizers induce endogenous danger signaling by triggering ROS and hyaluronidase mediated HA degradation. This process is crucial for CHS since sensitization is completely prevented by pre-treatment of the skin with antioxidants or the hyaluronidase inhibitor AA. In both cases, the inhibitory effects can be overcome by active hyaluronidase. However, at least regarding IL-6 production in ear sheets, the trauma induced IL-6 production occurring due to the mechanical separation of the ear sheets seems to result in a maximal cytokine production that can neither be significantly enhanced by addition of TNBS nor by hyaluronidase. Most interestingly, antioxidant application also prevents elicitation of CHS in sensitized animals. The role of HA induced signaling in the challenge phase of CHS remains to be determined. In vitro signaling studies with cells from contact allergen sensitized skin are, however, hampered by background problems due to the cell isolation procedure. Moreover, the deletion of the tissue context may significantly change cell behavior. Therefore, in vivo approaches using signaling inhibitors as initiated by us for p38 MAPK may provide more relevant results that could be translated into the development of novel treatment strategies.
Our findings add to an emerging, more general scheme highlighting an important functional role for ECM components as endogenous regulators of inflammation [9], [56]. Enhanced (or exaggerated) ECM degradation that disturbs the homeostasis of ECM turnover signals danger to the innate immune system. HA and biglycan are implicated in the activation of TLR2 and TLR4 signaling as well as in inflammasome activation [10], [17], [25], [57]. As shown here, blocking HA breakdown can prevent sensitization for CHS. This finding encourages the search for inhibitors of ECM degradation or for antagonists of the pro-inflammatory function of ECM components, which can be used in the prevention and therapy of inflammatory skin diseases such as ACD.
All of the experimental procedures were in accordance with institutional, state and federal guidelines on animal welfare. The animal experiments were approved by the Regierungspräsidium Freiburg and supervised by the Animal Protection Representatives of the University Freiburg Medical Center or the MPI.
C57BL/6, C57BL/10 (ScSn) and NMRI mice were purchased from Charles River Laboratories or provided by the breeding facility of the Max-Planck-Institute (MPI) for Immunobiology and Epigenetics in Freiburg, Germany. TLR2/4 deficient mice [58] were also provided by the MPI. C57BL/6 gp91phox mice were purchased from the Jackson Laboratories (Bar Harbor, ME) [59]. Mice were used at the age of 6–10 weeks. All of the experimental procedures were in accordance with institutional, state and federal guidelines on animal welfare. The animal experiments were approved by the Regierungspräsidium Freiburg and supervised by the Animal Protection Representatives of the University Freiburg Medical Center or the MPI.
The contact sensitizers 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (DNFB), and 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone (oxa)) were from Sigma-Aldrich, 2,4,6-trinitrochlorobenzene, (TNCB) was from VeZerf Laborsynthesen GmbH. Their classification as extreme, strong, moderate, weak is based on the Local Lymph Node assay [35], [36]. Pronase, aristolochic acid (AA), N-acetyl cysteine (NAC), ammonium pyrrolidine dithiocarbamate (APDC), diphenyleneiodonium (DPI) and the biotinylated HA binding protein (bHABP) were from Sigma-Aldrich. Hyaluronic acid (Streptococcus sp., low endotoxin) was obtained from Merck KGaA. Hyaluronic acid molecular weight markers (Select-HA™ HiLadder and LoLadder) were obtained from Amsbio. Antibody diluent with background reducing components, AEC chromogene and Dako Ultramount were from Dako Cytomation. Purified luteolin (HPLC >98%) (Carl Roth GmbH + Co. KG) was dissolved in ethanol. The Reseda luteola extract RF-40 (Batch 0506.06) was provided by Nahrungs-Ingenieurtechnik-GmbH (NIG) and solubilized by Aquanova AG (RF-40s) as described previously [42]. Here, "solvent" designates the nanoparticular polysorbate micelles used for the solubilization of the RF-40 extract. For a detailed description of the solubilization process and a HPLC characterization of the RF40 extract see Casetti et al. [60]. Alternatively, RF40 was solubilized in ethanol (RF40e).
Determination of intracellular oxidant production was based on the oxidation of CM-H2DCFDA (Sigma) by intracellular ROS, resulting in the formation of the fluorescent compound 2′,7′-dichlorofluorescein (DCF). A protocol for cellular staining was adapted from Liu et al. [61]. In brief, Pam212 or L929 cells were seeded at 5×105 cells/200 µl Dulbecco's modified Eagle medium (DMEM) in black µClear 96-well plates (Greiner Bio-One). After cultivation O.N., cells were loaded with 5 µM CM-H2DCFDA. ROS production was induced by addition of chemicals 30 min later and determined by measuring the fluorescence of the deacetylated, oxidized DCF at OD485/528 every 10 min for 1 h using a Tekan ELISA Reader. For flow cytometric detection of ROS production in BMDC, cells were seeded at 2×105 cells per well into 96 well plates. Cells were pre-treated by addition of 2.5 mM NAC or equivalent volumes of PBS (solvent control) for 15 min. After addition of contact sensitizer, cells were incubated for 1.5 h at 37°C, 5% CO2 before addition of 5 µM CM-H2DCFDA and further incubation for 30 min. After washing with PBS, DCF fluorescence was analyzed using a FACSCantoTMII (BD).
Ears of mice were topically treated with antioxidants and respective solvents either 15 min (RF-40s) or 1 h and in addition 15 min (NAC) before topical application of TNCB. Before NAC or PBS application, ears were wiped excessively with ethanol to allow penetration of NAC. 15 min after TNCB application, ears were taken after euthanasia and incubated ex vivo for 30 min with 5 mM DHE (Sigma) in DMSO. 8 mm punches were applied to glass slides and ROS production was monitored using fluorescence microscopy as oxidation of DHE results in the generation of the fluorescent 2-hydroxyethidium.
Pam212 or L929 cells were seeded at 4×105 cells per well in 200 µl DMEM in 8-well Lab-Tek II glass chamber slides (Nalgene Nunc). After cultivation overnight, cells were stimulated for 1 h with chemicals and subsequently loaded with 200 µl MitoSOX™ (Invitrogen) (5 mM Stock in ethanol dissolved in HBSS to working solution of 5 µM) for 10 min. After three washing steps with HBSS, cells were fixed in ice cold acetone for 2 s, nuclei were counterstained with DAPI and slides were covered with Fluoromount (Dako Cytomation) before detection of mitochondrial ROS formation as red staining by fluorescence microscopy.
Skin samples were fixed in HA-fixative as described by Lin et al [62]. Paraffin sections (3 µm) were fixed on slides overnight at 60°C and deparaffinized by Rotihistol (Carl Roth) treatment. After antigen demasking in citrate buffer in a steamer at 100°C for 30 min, permeabilisation was carried out with PBS/0.5% Tween 20 for 15 min at RT. After washing in TBS, endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 for 10 min at 4°C. Following three washing steps with TBS for 5 min each, unspecific receptor binding was blocked by incubation for 1 h with TBS/1% BSA at RT. The sections were then incubated overnight at 4°C with bHABP (25 µg/ml) diluted in antibody diluent with background reducing agents (Dako).
Detection of the bHABP was carried out using the DCS Chromoline Diagnostic System (DCS Innovative Diagnostik-Systeme). In brief, slides were incubated for 20 min with streptavidin conjugated horse-radish peroxidase (120) (HRP-Label, DCS), washed three times with TBS for 5 min and incubated at RT with 3-amino-9-ethylcarbazole (AEC) substrate solution until distinct red coloring was observed. After washing with dH2O for 2 min, sections were counterstained with haematoxylin and mounted with Dako Ultramount.
ROS were generated by the Cu(II)/H2O2 system as published [32]. In brief, both superoxide anion and hydroxyl radical generation was induced by incubation of 0.1 M NaH2PO4 (Merck) pH 7.4 containing 50 µM CuSO4 (Sigma-Aldrich) with 100 µM H2O2 (Carl Roth). Different concentrations of RF-40s or solvent were added to 20 µg high molecular weight hyaluronic acid (Calbiochem) and ROS inducing agents for 1 h at room temperature. Samples were analyzed by electrophoresis through a 7% polyacrylamide gel and visualized by overnight staining with 0.005% Stains-All dye (Sigma-Aldrich) in 50% ethanol. Bromophenol blue served as tracking marker for sample movement.
CHS was induced as described [7]. In brief, mice were sensitized by application of 150 µl Oxazolone or 100 µl of TNCB (both 3% w/v unless otherwise indicated) or respective volumes of solvent control on the shaved abdomen of 3–5 mice/group. Ear thickness was measured on day 5 and mice were challenged by application of 20 µl contact sensitizer (1%) or respective solvent control to the ears. 24 h later, the increase in ear thickness was measured.
Ear skin of mice was pre-treated by topical application of 20 µl NAC (5 mM) or RF40s (5.24 µM) or respective solvents 30 min before sensitization with 20 µl 3% TNCB. Alternatively, antioxidant treatment was done in sensitized mice on day 5 at different time points as indicated. Hyaluronidase inhibitor aristolochic acid (AA, 20 µl, 50 µM) or p38 MAPK inhibitor (SB203580, 20 µl, 0.02 µM) was injected into the ear pinna 15 min before sensitization with 20 µl TNCB (3%) on the back side of the ears. Mock sensitization with acetone served as control. Five days later, mice were challenged by application of 20 µl TNCB (1%) on the back side of contralateral ears. Increase in ear thickness was measured 24 h later with a thickness gauge (Mitutoyo).
Non-toxic AA concentrations were determined by LDH assay ex vivo on ear sheet culture samples and live/dead fluorescence microscopy (data not shown). Some treatment groups were co-injected with hyaluronidase (660 U/ml, active or heat inactivated (5 min, 99°C)) to assess the reversion of the inhibitory effects of antioxidants, the p38 MAPK inhibitor or AA on the CHS response. This treatment also revealed the non-toxic effects of AA in vivo.
Ears of mice were treated with chemicals for 24 h. Incubation of 8 mm punch biopsies was done in lysis buffer (50 mM Tris pH8.0, 1 mM EDTA, 150 mM NaCl2, 0.5% Na-desoxycholate, 1% Nonidet P40). Zymographic detection of hyaluronidase activity was carried out as described [63]. In brief, samples were electrophoresed through 10% SDS-polyacrylamide gels containing 0.17 mg/ml of high MW HA. After electrophoresis, gels were washed in 50 mM HEPES buffer pH 7.4 containing 3% Triton X-100 and subsequently incubated in 0.15 M NaCl2/0.1 M Na-formate, pH 3.5 at 37°C for 18 h. Gels were then stained with 0.5% Alcian blue in 3% acetic acid where hyaluronidase activity was visualized as clear bands on the blue background. For the assessment of relative activity, intensities of the bands were recorded using a Epson Biostep View Pix 700 with SilverFast-SE software (LaserSoft Imaging AG,) and analyzed using Quantity One software (Bio-Rad).
Ears of mice were separated in dorsal and ventral halves using forceps. Culture of the ear sheets was carried out in 6 well plates with 1.5 ml RPMI +10% FCS/well in an incubator at 37°C and 5% CO2. IL-6 production was analyzed 24 h after treatment with TNBS, AA and hyaluronidase or PBS as control as indicated using a commercial IL-6 ELISA kit (OptEia-Kit, Becton Dickinson) according to manufacturer instructions and a Tekan ELISA Reader.
Detection of the molecular size of HA samples derived from abdominal skin either untreated or treated with TNCB (3%) for 4 h or 24 h was carried out according to the protocol by Lee et al. [64]. In brief, 10 mm punch biopsies of skin samples were digested for 16 h at 55°C in Pronase (5 U/10 mg sample) in a total volume of 200 µl according to a protocol by Wang et al. [65] before inactivation of Pronase by heating to 100°C for 10 min. Afterwards, samples were mixed with loading buffer (2 M sucrose +0.02% bromophenolblue) and electrophoresed through a 0.5% agarose gel in TAE buffer for 8 h at a constant current of 2.5 V/cm (50 V). HA was visualized by staining in 0.005% Stains all (Fluka) overnight in the dark and destaining under normal light in water until distinct bands were visible.
Data were analysed using the unpaired Student t -test (two-tailed), and statistical significance was established at P≤0.05.Data are expressed as mean ± SD if not indicated otherwise.
We are grateful to Verena Weber for excellent technical assistance and to Dr. Eva Oswald for careful reading of the manuscript and excellent discussion.
Competing Interests: The Competence Center skintegral holds a patent on the production of luteolin rich extracts from Reseda luteola and their use. (Dr. Schempp, Dr. Wähling, Dr. Lange (2008) Method for the production of flavonoid-containing compositions and use thereof. PCT WO 2004/075816 A2. European patent EP2004/001598, 2004-09-10.) Otherwise no conflict of interest exists. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Funding: This work was supported in part by a grant of the European Commission as part of the project "Novel Testing Strategies for In Vitro Assessment of Allergens (Sens-it-iv)", LSHB-CT-2005-018681 (Dr. Martin) and by a grant from the German Federal Ministry of Economics and Technology, grant no. KF2556501SK0 (Dr. Schempp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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This property has been removed by the agent. It may be sold or temporarily removed from the market
Land for saleLeckie Estate, Gargunnock, Stirling, Stirlingshire, FK8
Under Offer 1,546 ac. | £3,380,000
Property Description
Commercial information
- 1,546 acres (625.6 hectares)
Key features
- Leckie House (5 beds)
- 1,547 acres in total
- Established Pheasant Shoot
- Grouse Moor
- Commercial and amenity woodland
- Stock farm
- Farmhouse & 3 cottages
- Indoor pool & all weather tennis court
- Two lochs
- EPC Rating = F
Full description
Tenure: Freehold
A Beautiful Estate in the heart of Scotland
Leckie is a beautiful estate which is well managed and immaculately maintained and continues to be a much cherished family home. The estate occupies an enviable position in the heart of Scotland and offers a little of everything you could wish for in an estate. Its appeal in part is its simplicity and the ability to run the estate without considerable time and physical effort or financial input.
Leckie is situated in the heart of Scotland. The estate enjoys a splendidly sheltered and private setting, with the Gargunnock Hills to the south and wonderful views northward to Ben Ledi and Ben Vorlich.
The pivot of the motorway network is only 7 miles to the east near Stirling, with the M80 giving quick access to Glasgow and the M9 leading directly to Edinburgh. Glasgow Airport is 36 miles via the Erskine Bridge and Edinburgh Airport is 39 miles via the M9. Both airports offer regular services to London and other UK cities, as well as services to an increasing number of international destinations.
Loch Lomond and The Trossachs National Park are within easy reach to the west. Gleneagles Hotel and its associated Country Club (22 miles) provides an extensive range of sports and leisure facilities including three golf courses - the King's, the Queen's and the PGA Centenary Course (designed by Jack Nicklaus). The golf courses of Aberfoyle, Callander, Sheriffmuir and Stirling are also nearby. In addition to the sport on Leckie, grouse and pheasant shooting can be taken in the area with salmon fishing on the River Teith.
Gargunnock lies one mile to the east and is an attractive village which has a primary school, pub/restaurant and a local shop. Stirling (7 miles) is the major city of central Scotland, with its historic castle and excellent shops and services, including a choice of supermarkets, a large shopping centre, a cinema and swimming pool. There is a Park & Ride terminus on the outskirts of Stirling linking to regular bus and train services to Edinburgh and Glasgow in addition to a direct service to London King's Cross (including sleeper service).
Independent schools in the area include Beaconhurst at Bridge of Allan and Ardvreck in Crieff. Glenalmond, Strathallan and Kilgraston are also within easy reach. Bridge of Allan is also home to Stirling University which has a sports centre and the MacRobert Arts Centre with a theatre and cinema.
Leckie Estate extends in total to 1,547 acres and comprises at its core, Leckie House, which is surrounded by a productive mixed farm, stands of amenity and commercial woodland and a grouse moor at the top of the Gargunnock Hills. At present both the farmland and grouse moor are let, thus avoiding the necessity for direct involvement or management. The family enjoys a day's shooting on the moor as part of the present arrangement, in addition to being fully paid up members of the shooting syndicate who run and manage the low ground pheasant shoot.
Leckie has a rich and varied history. This dates back to ancient times, with the vestiges of an iron age broch , above the wooded ravine of the Leckie Burn .
Leckie was originally a much larger estate and belonged to The Crown , before being transferred in part to Malcolm de Leky in the 1350s, during the reign of Kind David II, son of Robert the Bruce. The Leckies remained in possession until 1668 when the estate was sold to David Moir of Craigarnhall , in whose family it remained until 1906. Bonnie Prince Charlie visited the estate in the romantic, but ultimately doomed Jacobite Rebellion of 1745.
Picturesque stone bridges remain, dating from the seventeenth and eighteenth centuries and there are the ruins of old mills. The former Leckie House (now called Watson House) was built in 1836 and is now in separate ownership , having been converted into apartments.
Leckie was purchased in 1906 by George Younger of the eminent family, who had been brewers in Alloa since the early 1700s. George was a leading Conservative MP, becoming a Baronet in 1911 and a Peer in 1923 ,with the title Viscount Younger of Leckie.
The 'new' Leckie House was built in 1962 and from 1992, formed the much loved family home of George, 4th Viscount Younger, who had been Secretary of State for Scotland and also Minister of Defence under Mrs Thatcher. His widow, Diana , Viscountess Younger of Leckie, died in November 2015.
Barony Title
The title "Barony of Leckie" is believed to belong to the owners of Leckie Estate. Further details are available from the selling agents.
Constructed of harled brick under a slate roof, Leckie House occupies a commanding elevated position with stunning views to the west and north towards The Trossachs. The house is approached via a tarmac drive which leads off the private estate road and sweeps through the lightly wooded gardens to a parking area adjacent to the house. The formal gardens are enclosed by a high beech hedge and are enjoyed from the south facing paved terrace which is accessed directly from both the drawing room and indoor swimming pool complex. An enclosed all weather tennis court lies to the east of the garden. The layout of the house and the gardens make Leckie House a comfortable family home which is also ideally suited to accommodating and entertaining guests.
The accommodation is on three floors as shown on the accompanying floor plans. The principal reception rooms lie off the mai n entrance hall which also gives direct access to the Clive Christian fitted kitchen which has a four door gas fired Aga. Beyond lie the utility/laundry and drying rooms and a tiled hallway to the indoor pool with changing rooms and showering facilities. The heated pool (8m x 3.7m) is run on an ozone system. The integrated double garage has wooden sliding doors and also contains a WC with wash hand basin.
On the first floor the master bedroom suite includes an en suite shower room with dressing room/single bedroom adjacent. There are two further bedrooms with wash basins, a bathroom and study (with lift). Coombed ceilings feature on the second floor which includes a playroom/sitting room with fitted bookshelves, two further bedrooms and bathroom.
To the rear of Leckie House, enclosed by a high beech hedge, is a gravelled area in which a rotary clothes dryer, two Calor gas tanks, a wooden garden store and log store are sited.
Whitehill Cottage
Situated on the rear drive to Leckie House, Whitehill Cottage is a single storey detached bungalow built of timber clad brick under a tile roof which is currently subject to a service occupancy. The accommodation comprises: sitting room, kitchen, four bedrooms and bathroom. There is an enclosed garden to the front with drying green to the rear and a wooden garage for a single car.
Knock-o-Ronald Farm
Knock-o-Ronald Farm lies at the heart of Leckie Estate and encompasses all of the agricultural land (with the exception of Lot 2). In total the farm extends to 986.7 acres with the farmhouse, adjacent cottage and farm buildings centrally situated at the end of the private tarred drive which runs through the estate.
The farm is currently let on a Limited Duration Tenancy (LDT) until May 2018 and is run as a productive commercial stock unit with 760 breeding ewes and a suckler cow herd of 45. There is a good relationship with the tenant who has farmed at Knock-o-Ronald for the past 25 years. For those not wishing to farm the land themselves this is a ready-made, extremely workable arrangement for any new owner, with a tenant who is highly cooperative and supportive of the estate. Further details are available from the selling agents.
Knock-o-Ronald Cottage lies outwith the LDT and is currently let on a Short Assured Tenancy.
Constructed of stone under a slate roof, the farmhouse lies to the west of the farm steading with a range of traditional buildings adjoining, which offer potential for redevelopment/expansion. The accommodation is over two floors as shown on the accompanying plans and includes sitting room, fitted kitchen, external utility room, study, office, pantry, four bedrooms and bathroom. To the rear lies an enclosed garden and washing green.
Knock-o-Ronald Cottage
Situated to the west of the farmhouse, Knock-O-Ronald Cottage is a single storey timber building on a brick base under a tile roof. The accommodation comprises a kitchen, sitting room with wood burning stove, two bedrooms and a bathroom. The cottage enjoys a southerly aspect with views to the north.
Farm Buildings
The buildings at Knock-O-Ronald Farm are situated conveniently close to the farmhouse and provide good housing for both cattle and sheep. To the north lie the sheep handling facilities and an area of hard standing for storage.
Feed and Bale Store (13.14m x 8.74m) - steel portal frame, partial concrete floor, block walls with corrugated cladding and roof.
Cattle Court (32.19m x 22.28m) - steel portal frame, block/concrete panel walls, corrugated roof. Raised central feed passage. Lean-to with steel frame with feed barrier under corrugated roof. Earth floor.
Tupp Shed (9.48m x 5.18m) - Pole lean-to with block walls and profile sheet cladding and roof.
Dutch Barn (18.44m x 9.13m) - Steel frame with corrugated cladding. Earth floor. Adjoining Sheep Shed/Pens (9.17m x 6.44m) constructed of a steel farm with stone/block wall under corrugated roofing. To the rear is a pole lean-to (16.66m x 4.81m) with sheet roof. Earth floor.
Bull Pen - situated in a former stone bothy under a slate roof with modern kennel runs adjacent.
The land at Knock-o-Ronald is broadly divided into two parts as defined by the natural topography of the dramatic cliffs which are a feature of the estate: to the north lies gently undulating productive grassland; with permanent pasture and areas of rough grazing extending up the higher ground to the top of the Gargunnock Hills. The land is classified as class 4 and 5 by the James Hutton Institute and is predominantly utilised for the production of fodder crops and grazing for cattle and sheep. The majority of the fields have stock proof fencing and water is supplied to stock by fresh open water burns or troughs.
The woodlands on the estate are certified under the UK Woodland Assurance Scheme (UKWAS) and extend to 250.19 acres (101.25 ha) with a further 37.95 acres (15.36 ha) of open woodland.
The overall amenity of Leckie Estate is enhanced by its well maintained hedgerows and fences. There are delightful woodland walks adjacent to the Leckie & Easter Blackspout Burns which run through the estate, passing a ruined broch and the former mills at Old Leckie. The Old Sawmill which contains obsolete hydro-electric turbine & switchgear could have potential for conversion, subject to obtaining the necessary planning consents.
In 2015, 23.08 acres (9.34 ha) of commercial woodland were felled under the Forestry Grant Scheme. The remaining 123.11 acres (49.82 ha) of commercial conifer plantations and 104 acres (42.08 ha) of amenity woodland are all managed under the estate's Long Term Forest Plan (LTFP). Further details can be obtained from the selling agents.
Sport / Pheasant Shoot
The shooting over the low ground estate is let to a syndicate who run a very successful and enjoyable pheasant shoot. Working in conjunction with the farming tenant, approximately 1500 pheasant poults are put down annually in pens throughout the wooded areas across the estate. The natural contours and mix of vegetation and cover provide for exciting drives which are enjoyed every fortnight over the shooting season. The current arrangement, which includes an annual payment and two family days, is due to expire on 5 April 2017.
Loch Logan to the north of Loch Logan Wood to the west of the estate, and the Leckie Reservoir in Whitehill Wood on the same latitude provide exciting duck flighting and trout fishing in the reservoir. Roe stalking on the estate has been let separately on an annual basis.
A modern steel portal framed with box profile cladding Forestry Shed (8.5m x 8m) is located adjacent to the hill road in a woodland clearing and is ideal for the storage of estate and game rearing equipment.
The policy fields extend to approximately 43 acres (17.55 ha) and are let on a Short Limited Duration Tenancy (SLDT) until 14 May 2020. The land is divided into two enclosures and comprises 26.03 acres arable/ploughable pasture and 17.35 acres permanent pasture. Lying immediately to the north is an area of amenity woodland lying either side of the former drive to Watson House. There is considered to be development potential for a house site immediately adjacent to the former East Lodge.
The grouse moor adjoins Knock-o-Ronald above the cliffs at the top of the Gargunnock Hills and extends to 494 acres (200 ha) of heather hill and rough grazing. The hill can be accessed by foot from Knock-o-Ronald and offers the most spectacular panoramic views encompassing much of Stirlingshire from Ben Lomond in the west to the new Forth River Crossing in the east.
The hill grazings form part of the Knock-o-Ronald LDT. Since 2011, the sport has been leased to a local syndicate who have managed and keepered in conjunction with the adjacent moors of Burnfoot, Touch and Hillhead to provide 12 days driven grouse shooting across the four moors. Prior to this arrangement, the 5 year average bag over the Leckie Moor was 48 brace. The current lease will expire at the end of the grouse season (10th December 2016).
To the south lie the Earlsbu rn reservoirs and the Earlsburn Wind Farm which has recently been extended by the erection of a further eight turbines (24 in total) on land immediately adjacent to Leckie Grouse Moor. There may be potential on land belonging to the estate for the further development of this site utilising the infrastructure now in place.
Situated just off the private tarred estate road leading to Knock-o-Ronald Farm is a detached single storey traditional cottage of harled stone under a pitched slate roof which occupies an attractive south facing spot surrounded by mature woodland leading down to the Leckie Burn. Privacy is enhanced by a high beech hedge which bounds the front garden to the west with a gravel parking area to the rear.
The accommodation comprises sitting room, dining room, kitchen, three bedrooms and a bathroom as shown on the floor plan.
Strictly by appointment with Savills Edinburgh office (0131 247 3720).
Fixtures & Fittings
All light fittings, fitted carpets and curtains in Leckie House are included in the sale. Certain items of furniture, white goods and estate equipment may be made available by separate negotiation. The contents of Whitehill Cottage, Knock o Ronald Farmhouse and Cottage, and Sawmill Cottage are specifically excluded from the sale.
Local Authority - Stirling Council - Viewforth, 14-20 Pitt Terrace, Stirling, FK8 2ET. 01786 404040
Environmental Designations
There are two scheduled monuments: the Leckie Broch in the woodland 400m east of Knock-O-Ronald Farm; and the cairn at the summit of Carleatheran (both Lot 1). The estate lies within The Central Scotland Green Network Area.
Services, Council Tax, EPCs and occupancies
All properties have mains water and electricity and private drainage.
Sporting Rights
The shooting rights are included in the sale.
There is a housekeeper whose employment may be transferred to the purchaser of Lot 1 under the TUPE Regulations 2006.
Servitude Rights, Burdens, Wayleaves And Statutory Public And Other Access Rights
The property is sold subject to and with the benefit of all existing servitude and wayleave rights, including rights of access and rights of way, whether public or private. The property is also sold subject to the rights of public access under the Land Reform (Scotland) Act 2003. The purchaser(s) will be held to have satisfied themselves as to the nature of all such servitude rights and others following their solicitors' examination of the title deeds. If the property is sold in lots appropriate rights of access, servitude and wayleave will be granted and reserved as appropriate.
Forestry Grant Schemes and Timber
All standing and fallen timber is included in the sale. The purchaser(s) will be obliged to continue with the existing Forestry Commission Native Woodland Expansion and Restoration Scheme which runs until 2027 and will be responsible for the repayment of any grants previously received as a result of any failure to comply with the conditions of the scheme.
Rural Development Contract
The Estate is currently subject to a Rural Development Contract for the control of grey squirrels which is due to expire on 31st December 2017. The incoming purchaser will be bound to fulfil the obligations of the existing agreement.
Basic Payment Scheme
There are no Basic Payment Scheme entitlements available.
Less Favoured Area Status
The estate lies wholly within a Less Favoured Area. It is classified as severely disadvantaged.
Mineral Rights
The minerals are included within the sale insofar as they are not reserved by statute or common law to third parties.
Reservation of Benefit
A clawback will be reserved in favour of the sellers, equating to 25% of any uplift in value/income resulting from any wind farm development given planning approval within a period of 10 years from the date of entry.
Purchaser Obligations
In the event of Lots 1 & 3 being sold separately, the purchasers of each lot will be required to enter into a binding agreement in respect of serving the required noti ces to end the LDT over Knock-o-Ronald Farm.
The purchaser of Grouse Moor (Lot 3) will also be obliged if required to erect a stock proof boundary fence along the boundary of Lot 3 as highlighted on the sale plan.
The incoming purchaser of Leckie Estate will assume all and any landlord obligations in respect of any waygoing claims relating to the expiry of the SLDT and LDT agreements.
Entry & Possession
Entry by arrangement, subject to existing sporting lets and tenancy agreements: Knock-O-Ronald Farm (Lots 1 & 3) is subject to a LDT which will expire on 27 May 2018; the policy fields (Lot 2) are let on a SDLT which will expire on 14 May 2020.
Offers in Scottish Legal Form are to be submitted to the selling agents, Savills, Wemyss House, 8 Wemyss Place, Edinburgh, EH3 6DH. A closing date for offers may be fixed, and prospective purchasers are advised to register their interest with the selling aents following inspection.
Square Footage: 6,500 sq ft
Acreage: 1546 Acres
From Glasgow take the M80/A80 east, following signs to Stirling, merging onto the M9. Leave the M9 at Junction 10. Take the first exit at the roundabout onto the A84 and continue west towards Callander / Crianlarich / Safari Park. After Blair Drummond (3 miles) turn left onto the B8075 signposted to Gargunnock. Turn right at the T junction onto the A811 and continue for 1 mile, taking the first road on the left signposted to Beild Farm. Follow this minor public road which sweeps left below Beild Farm. The entrance gates to Leckie Estate lie on the right immediately adjacent to Beild Farm.
From Edinburgh take the M9 north past Stirling. Leave the M9 at junction 10 and continue as above.
The postcode is FK8 3BN - please beware, this does not lead directly to Leckie House.
Edinburgh Airport (www.edinburghairport.com) or Glasgow Airport (www.glasgowairport.com).
Railway Stations
The nearest station is Stirling. For general timetable enquiries telephone 08457 484950 or visit www.scotrail.co.uk
More information from this agent
To view this media, please visit the on-line version of this page at www.rightmove.co.uk/property-for-sale/property-54594592.html
Energy Performance Certificate (EPC) graphs
- View Knock-O-Ronald Cotta for this property See full size version online
- View Knock-O-Ronald Farm for this property See full size version online
- View Leckie House for this property See full size version online
- View Sawmill Cottage for this property See full size version online
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Listing History
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Market Info
Disclaimer - Property reference EDS160028. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Savills, Edinburgh Country. Please contact the selling agent or developer directly to obtain any information which may be available under the terms of The Energy Performance of Buildings (Certificates and Inspections) (England and Wales) Regulations 2007 or the Home Report if in relation to a residential property in Scotland.
Map data ©OpenStreetMap contributors.

subset: commoncrawl


text:
Bike Ride
9 ft
- Begins in:
- South Bend, IN, United States
- Created By:
- jvanbrua Best Time: 15:00
- Description:
- This is a 1.0 mi Bike Ride in South Bend, IN, United States. The Bike Ride has a total ascent of 9.45 ft and has a maximum elevation of 750.75 ft. This route was created by jvanbrua on 08/17/2007. View other Bike Rides that jvanbrua has done or find similar maps in Georgetown.
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[by Mel Wells] During her week as Literary Art's author-in-residence, Ruth Ozeki is spending several hours at Grant High School visiting students in their classes and discussing her work, both as a filmmaker and a novelist.
On Tuesday and Wednesday, Ozeki visited all five sections of Dr. Dreyer's Literature & Film classes. Students had studied her work in advance, specifically the film Halving the Bones, and were prepared with questions. But at the first of each class, Ozeki had students introduce themselves and talk briefly about what they enjoy the most about studying film and literature. She followed up with questions of her own for each student, and this prompted stories about how Ozeki had studied Hitchcock and filmmaking when she was a high school student, as well as discussions about adaptations and creative processes. Ozeki talked about her own creative journey, from studying Japanese and working as a bar hostess in Tokyo, to making low-budget horror films in New York City. "They're terrible," she told students, laughing. "But there was this new technology, VHS, so people could watch videos in their own homes for the first time and demand was high."
Ozeki started by making storyboards, but when the director realized he'd forgotten to hire an art director, he appointed Ozeki. "It was my first time on a set," she said, laughing, "so I learned by doing." She worked on the classics Mutant Hunt, Breeders, Necropolis and Robot Holocaust. "You know Mystery Science Theater?" she asked, and students nodded. "They're on there."
Ozeki went on to work for Japanese television, and began to edit films as well. "It was in the editing room that I learned how to tell stories," she said. She bought a super-8 camera and began filming scenes from Halving the Bones before she had a firm idea for the film. "In that way, I used found images," she said. The movie explores the reliability of memory and the truth of history.
When students asked about it, Ozeki talked about her Zen Buddhism and how valuable meditation is to doing creative work. "Over time, it builds a sense of confidence and fearlessness," she said. "You learn to be comfortable with who you are." She also answered questions about how her work as a filmmaker influenced her writing. "When writing, I enter a scene as though I have a camera," she said. "I look for a place to start, think about widening the frame, changing the angle, etc." Her hands operated an invisible camera. "The process is a very visual negotiation."
Ozeki is visiting students in the Japanese immersion classes before her lecture tonight at the Schnitz for Portland Arts & Lectures. Many thanks to the administration and teachers at Grant High School, especially Dr. Dreyer, as well as the students for being such attentive, involved participants in Ozeki's residency. And of course, a huge thanks to Ruth Ozeki for being a fantastic author-in-residence this week!
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text:
We speak with John Woods, President and Co-founder of the Ottawa School of Medieval Armed Combat, during a medieval festival in eastern Ontario. The Ottawa School of Medieval Armed Combat runs classes and events to teach medieval combat based on the writings of Fiore dei Liberi, an Italian fighting master from the second half of the fourteenth century.
For more information you can visit their website at www.ottawasword.com


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Why not learn more about Resources?
What Can You Get If You Are A Football Fanatic
Getting several mental advantages is what you can get when you are a football fanatic. The moment that you are a sports fan that there are also several physical changes that you will exeperince. A general happiness and a well being is what you will be able to feel whenever you are sports fanatic. The moment that you are sports fan that it is you that will also feel less lonely and less alienated. In this article, however, we will be talking about the different benefits that one can get wherever he will be a football fanatic.
A built in community is what you will get when you will be a fan. It is you that will have a good well-being the moment that you will feel that you are a part or affiliated with a group. Your social and physiological health will increase when you are a fan as you will be able to make connections with other people. Important for your social life is the connections that you will make to the people that you don't know or even get to see.
It is your well being that will boost with the help of community. There will be a significant decrease in the loneliness that you will feel once you will watch a game together with other fans. Not only that but feeling that you are a part of larger group will also bring you benefit in the long run. A longer connection and decrease alienation is what you will have when you are watching a game.
It is when you are a fan that you will also have common language with other fans as well. It is factors like barriers and division that's commonly present in any society but once you are a fan and a part of a community that you will be able to break these factors. It is when you will be rooting for the same team that you will have a common connection with the same fans as well. It is sports that can connect you more than any other factors.
The moment that you will be a fan that it is you that will also have a safe space. The moment that you will be a sports fanatic that it is here that you can show you the emotions that you have regardless of the sex that you have as you can cry, laugh, shout and all other emotions that you will be feeling. The moment that this one is done by them especially for men that they connect reduce the stress that they have. For their part, they will also be able to feel a sense of continuity.
And the last thing that you can also get is that you will be able to feel a sense of success. The moment that you or team wins that this is the feeling that you will get.


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If you are interested in hosting one of our mentor training conferences, contact us at... more >
Ratio Christi College Prep (RCCP) is a student apologetics alliance that seeks to train youth in apologetics, worldview, and evangelism. It seeks to equip the local body of Christ to help youth:
1) faithfully share and defend their faith;
2) be prepared for the culture war being waged on their faith; and
3) be skilled in civil engagement for the good of others.
The main method of accomplishing this is through RCCP equiping Christian adults to become mentor/teachers in their own community that lead a small group (10-15) of youth through apologetics, worldview, and evangelism training.
RCCP is a ministry of Ratio Christi, Inc.,Ratio Christi (Latin for 'The Reason of Christ') is a non-profit, tax exempt (501c3) educational and religious organization encouraging and strengthening the faith of Christian students and faculty around the world through the use of intellectual investigation and apologetics while sharing Christ's message and love to those that have yet to receive it.
Why is this needed? Find out more.
See much more about the overall Ratio Christi organization at the following links:


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Chef David Bishop teaching how to make sauted noodles. This is a great side disg for beef, pork and chicken.
With "My Queue" you can quickly save videos to watch later.
To add an item to your queue just click the sign next to the video.
Once you've added a video visit "My Queue" to start watching!


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A sword is considered by many seasoned anglers to be the toughest catch in the sea. Usually fighting from depths of 800 ft and beyond, landing a swordfish can be a brutal fight lasting over two hours. Carey manages to capture the splendor of the fish in his work of art.
The pendant is the perfect size for both men and women. Featuring Carey's signature hook executed in .925 Sterling Silver with optional 14 kt gold braid and highlights (ADDT. $ 50). Outstanding details, each piece is hand-poured and finished. This is a unique and rare gift that will be appreciated for a lifetime. Perfectly handcrafted in the US.
Available in two finishes
Sterling Silver $349.00
Weight: 7.2 dwt/0.38 oz
Measurement: 1 ¼ in X 7/8 in
Ships free insured (Cont US), signature required
To add a matching chain or choker to your purchase, click here


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Support » Fixing WordPress » Default navigation is giving a 404 error
Default navigation is giving a 404 error
My blog utilizes both categories and sub-categories. Posts are only associated with sub-categories, but I organize the sub-categories into categories. My Category base is "topic" and users are able to access any of the following pages:
When I use the default WordPress navigation with the default theme, the navigation for the first URL (http://www.domain.com/category) takes me to:
This URL give a 404 error. The other two URls work fine:
Does anyone know how to fix this? Is there a preferred method for reporting this bug?
I've experienced this bug as well. And just to include a bit more info, I'll just mention that I experience this while using the WP-PageNavi plugin. Here's a link to a post on the plugin support forum about this issue. Also, it occurs using the default theme. Any help with this would be great!
Anyone got anything on this? Is this a bug?Jeff Sherk
My permalink is set to Custom: /%category%/%post_id%, and I am using WP 2.7.1 with the WordPress Default theme. The problem occurs with or without using PageNavi 2.40, so the problem must with WordPress itself, not the plugin.
I tried installing this plugin as a fix, but it did not do anything:
When I go to a category page (let's call it 'stuff') like http://www.mysite.com/wordpress/stuff it correctly shows the posts for the first page of the category, and if you hover over the Older Entries links is says
but if you click it, you get a 404 Error.
I have posted a bug in the WordPress tracker, but if anybody knows of a solution it would be greatly appreciated.
ThanksJeff Sherk
I re-wrote the barefoot plugin for WP 2.7.1 and this fixed my problem… maybe it will help others as well. It also works with WP-PageNavi…
Plugin Name: Fix Paging in Category Listings
Plugin URI:
Description: Fixes a bug where next/previous page links are broken in category listings
Version: 0.6
Author: Doug Smith (modified by Jeff Sherk)
Author URI:
Modified on March 23, 2009 by Jeff Sherk for WordPress 2.7.1
Based on original plugin info listed below:
Plugin Name: Fix Paging in Category Listings
Plugin URI: http://www.thinkbarefoot.com
Description: Fixes a bug where next/previous links are broken in category by year/month listings
Version: 0.5
Author: Doug Smith
Author URI: http://www.thinkbarefoot.com
Copyright 2007 Doug Smith (email: firstname.lastname@example.com)
This program is free software; you can redistribute it and/or modify
it under the terms of the GNU General Public License as published by
the Free Software Foundation; either version 2 of the License, or
(at your option) any later version.
This program is distributed in the hope that it will be useful,
but WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
GNU General Public License for more details.
* Function to fix problem where next/previous buttons are broken on list
* of posts in a category when the custom permalink string is:
* /%category%/%year%/%monthnum%/%postname%/
* The problem is that with a url like this:
* /category/2007/10/page/2
* the 'page' looks like a post name, not the keyword "page"
* MODIFIED ON March 23, 2009 by Jeff Sherk
* Permalink structure was /%category%/%post_id% which looked like
* http://www.mysite.com/wordpress/news/27
* When attempting to go to another page, it looked like this
* http://www.mysite.com/wordpress/news/27/page/2
* but would generate a 404 error .
* This modification takes $query_string['category_name'] and chops the '/page'
* off the end. It also takes $query_string['paged'] and sets it equal to
* $query_string['p'], and then unsets $query_string['p'].
* 'p' is not needed, but 'paged' is needed in order to make it work correctly
function fix_page_in_category_from_query_string($query_string) {
//Check to see if the 'p' and 'category_name' are set in $query_string
if ( isset($query_string['category_name']) && isset($query_string['p']) ) {
$category_name = $query_string['category_name'];
$category_len = strlen($category_name);
//Check to see if the 'category_name' has '/page' on the end of it
if (substr($category_name, $category_len-5,5) == '/page') {
//Remove '/page' from the end of 'category_name'
$query_string['category_name'] = substr($query_string['category_name'],0,$category_len-5);
//Set 'paged' equal to the page you want to go to
$query_string['paged'] = $query_string['p'];
//Unset 'p' since we don't need it anymore because we set 'paged' instead
return $query_string;
add_filter('request', 'fix_page_in_category_from_query_string');
Hey jsherk,
This seems to be exactly the fix I'm looking for! However, I'm not sure how I should use your above code. Should I turn it into a plugin – if so, how? Should I include it in the header.php of the theme? Could you help me out?Jeff Sherk
Yes, you turn the above code into a plugin…
Copy the above code into a text file, and then save the text file, and rename it with a .php extension… let's call the file fix-category-pages.php
Now create a folder in the wp-content/plugins directory with the same name as the name of the file you created above (except no .php)… so let's call the folder fix-category-pages
So yo should now have this:
Now just go to your WP dashboard and go to the plugins section and activate the plugin and you should be good to go!
- The topic 'Default navigation is giving a 404 error' is closed to new replies.


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This course is the finest around with real parkland features . The greens are true/pure and fast constantly .The attention to detail is so impressive the courses are well maintained and have many picturebook holes .The driving range is state of the art with many features with bunkers, greens and a diamond cut fairway so it sets up like a Golf hole.The Academy course is newly built and a great test of golf this Is also maintained like the main course to high standard
Letchworth Golf Club
Letchworth Lane, Letchworth,Hertfordshire,SG6 3NQ, England
- holes:18
- par: 71 72
- length: 5.582 m 5.083 m
I want to play here! Enquire
- holes:18
- year:1905
- par: 71 72
- length: 5.582 m 5.083 m
- architect:Harry Vardon
golf course condition
customer service
greens condition
fairways condition
value for money
driving range
Practice area
Read more
Golf courses nearby
Hotels nearby
Reviews (33)
- Matthew towlerCaddy1 review
Great Course11/02/2017this review is the subjective opinion of a GOLFBOO user and not of GOLFBOO S.L
Average rating obtained byThis rating for Letchworth Golf Club. is the average of 31 ratings obtained from www.golfshake.comGreat course , very hot day , was hard getting around
Average rating obtained byThis rating for Letchworth Golf Club. is the average of 1 ratings obtained from www.ukgolfguide.comPlayed this course on a society day Friday 15.05.09 . I hope we return to this course in future years as I think it is an excellent society course . The staff were friendly , food excellent and the course perfect for society golf . It is a fair course if you hit the ball well you will score - hit a poor shot and there is trouble . I scored 34 points off 18 and finished 8th out of 36 players . Would have shot 36 points if not taken 4 shots to get out of the bunker on the last . Won with 41 points by a bandit off 16 .


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Despite industry support of a proposed rule raising the requisite assets investors need to buy into hedge funds, the Securities and Exchange Commission is now questioning whether more regulation may be needed.
"The rulemaking doesn't address the ongoing problem of the rapid growth of hedge funds and the lack of knowledge regulators have," said former SEC Chairman William Donaldson. "It is an increasing concern not only in the United States, but to regulators around the world."
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Doom Patrol #2 (Preview)
in Comic Previews Comment
- Story by
- J.M. DeMatteis and Keith Giffen
- Art by
- John Livesay, Kevin Maguire, and Matthew Clark
- Cover by
- Kevin Maguire and Matthew Clark
- Publisher
- DC Comics
It's a black hole, but it's not a black hole. If you're a Doom Patrol fan, that made perfect sense. And even if not, then you're in for one hell of a ride as the Doom Patrol face off against the most annoying alien this side of G'nort. Who's G'nort? What, do we have to spoon-feed this stuff to you?
Oh, and we send the Metal Men into space. Just because.


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Another day, another distinctly conspicuous shoe from the Jordan/Nike 2013 Black History Month Collection. While the Air Jordan 1 High BHM will catch eyes by way of its allover reflective build, the Jordan Spizike BHM Neon does so courtesy of an all volt (or volt-adjacent) tone over the upper, with black on the midfoot-speckling, outsole, elephant print and inner-lining, and "BHM" in blue on the tongue.
Stay tuned for further info, but a release next month certainly wouldn't shock.
Photos via Sole Up.


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Login/Create Account
Check Out the new Remax App
Ask About This Home
Large spacious remodeled North Cross Lake home on the canal. Plenty of room for entertaining. Three bedrooms with 2 full bath and one half bath downstairs. One bedroom with bath upstairs! Wood burning fireplace, New stainless appliances. 2 New HVAC units. New electrical, new plumbing. New roof. Large sun room. 2 car attached garage with a one car attached carport. Boat house included in sale but sold as is. Not included in square footage.


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The annual international Eurospoor model railway event will be staged at the Jaarbeurs in Utrecht, and includes many an excellent model railway layout in a large variety of gauges as well as an extensive marketplace. The Dutch HRCA will return for this venue in november 2017, with a 3-rail 0-Gauge layout 'British Tinplate Toy Trains' and a 3-rail Dublo layout. We would hereby cordially invite anyone visiting the Netherlands to come and pay us a visit.
Peter, Dutch HRCA
3-5 November 2017: Eurospoor
Any meetings or shows coming up. Mention date in topic title!
Post Reply
1 post • Page 1 of 1
Post Reply
1 post • Page 1 of 1


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Health Care Decision Analyst (HCDA) provides health care planners and marketers with an easy-to-use, cost-effective software tool for market data analysis and clinical risk adjustment. It uses client-server technology, allowing rapid access to a comprehensive array of public and internal proprietary databases. .
What makes HCDA unique? HCDA's menu-driven software includes a set of analytic modules that allow you to:
HCDA consists of six report modules, organizing standard reports into business decision-support areas.
Demographic Profile: Users can access a wealth of information about the underlying demographics in a market to support marketing and planning.
Patient Origin: Users can easily perform extremely detailed analyses with extraordinary flexibility to identify the geographic origin of patients from specified providers for any case type.
Market Share: This module provides insight into the competition and identifies areas for potential business expansion overall, by case type, payer or any other available field.
Demand Forecasting: Project resource demand based on expected shifts in population demographics to forecast future demand and need for services.


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whenever i go to enable adsense it tells me this account is dissaproved by google pls see for the problem
you will need to wait 6 month to get approved as you are from India.
That is so unfair
I don't think so. We ourselves are responsible for it. Most of the spammers, content thieves and sub standard content writers are from Asia.
Saleheensblog is right.
India and Asia have a much higher rate of spam, leading google Adsense to require a minimum of six months proof of high quality work before they'll accept your application for an account.
The quality of your hubs right now would be unlikely to meet Adsense approval standards, anyway.
They are very simple, written almost at a personal instead of a professional level, and are lacking in important grammar and punctuation such as capital letters at the beginning of sentences.
your hubs are too short, there are plenty of grammatical and spelling mistakes there also. to get approval you will also need to focus on your quality of writing.
you can try doccash, weanswer ........ for a quicker approval. Many Indians got a quicker approval writing there though I didn't try myself.
Best of luck
Salesheensblog--His English writing is pretty poor. Instead of giving him back doors to AdSense approval, maybe you could help him more by letting him know where he can ramp up his skills. I just have the feeling that if he continues to write like that and he does manage to get approved by AdSense, he'll get tossed eventually and lose AdSense forever. You've done a really good job of brushing up on your English, so maybe you could share your tips and tricks with him?
Thank you lrohner. You are just spot on. You know I myself didn't try the backdoor rather I tried to improve my English.
@saish few suggestion for you:
~Know that your English is very poor but don't be upset with your English.
~Start reading. Read hubs on your favorite topic, seo, keyword research, backlinking etc. By reading these you will not only be able to learn about useful online staffs but also learn how to present information in easy, simple and correct sentences.You don't need to read grammar books.
~Use MS word when you compose your content. It will diminish your error level a lot.
~Read forum posts and post your insightful comments there. Whenever you will try to post an informative comment you will be struck for sure but if you try to get a way out to express yourself you will certainly learn a lot. Don't post a spam comment or a irritating comment that [you have already done few times] adds nothing to the discussion.
~And the good news for you is this community is such great that they are more than happy to help you improving.
My English was way too poor but I have improved a lot recently with the help of respected fellow hubbers, reading a lot of hubs and being active in the forum. My first target was learning English not earning well. I am still learning and learning everyday. If you too do that you are sure to improve otherwise you will get busted.
Best of luck.
Here is another thought: Why would someone who actually got an Adsense account through such a service jeopardize their account by publicizing how they got it?
saleheensblog is speaking from experience. I, however, was approved about a week after I signed up and published a couple of hubs. Of course, I'm from Texas and Google probably figures that most Texans don't have sense enough to spam. Their best example of a Texan was George W. Bush. lol
by klaceyjsmith3 years ago
I am just learning all about everything on the internet, and I signed up for a Google AdSense account so that I could get paid for my articles. Well, I don't know what the heck I did, but they deleted my account...
by Julie McFarland4 years ago
I'd like to consider myself an asset to the hubpages community. Although I began relatively recently, I have over a dozen published hubs, and I even won the rising star award for one of them. I have applied...
by retireyoung23 months ago
I have been using Adsense for a few years now and trying to get my partner to join.I am curious as to how long it takes these days to get approved?Also does anyone have any tips for getting your account approved...
by Felishiya Fiamma6 months ago
Hi Hubbers, I checked my account yesterday on August 20, 2016 and to my surprise the Google Adsense has approved my application. It is hardly even two months, when I signed up.
by nutan.goel8 years ago
i mean only from blogs not from hubs(i make altogether1200-1400 dollars per month from all my blogs)what say u
by genebob3 years ago
While I link my Adsense account, it directs me to Google, which then asks if I want to accept the association, I say yes, then it directs me back to Hubpages, but it just loads a blank page, nothing comes up. I could...
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JUIC Training Group Results at the Northridge Open!
March 12th, 2012 Leave a comment Go to comments
Last weekend in Woodland Hills, CA was the Northridge Open presented by the Northridge Table Tennis Club at El Camino High School.Students of the JUIC Training Group (headed by Stellan & Angelita Bengtsson) had a great showing!
After a tough loss to former US Men's Singles Champion Jim Butler in the Men's Open, long time club member Earl Alto stayed focused and became the champion of the U2500 event! Also, Teddy Tran (who consistently drives down from Orange County every weekend specifically to train in Stellan's group) made it to the semi-finals of the Men's Open! And guess what? He's only 14 years old!


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Pumkins in the Pumpkin Patch Acrylic Painting
Explore new techniques in acrylic paint that allow blending and creative expression as we paint fall pumpkins in vivid tones or oranges.
Kids Place provides exceptional child care options for working families. As part of St. Louis Park Public Schools, we share the district's commitment to ensure all students are provided safe, nurturing environments in which to learn and grow.


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Once again, the Bishop Lynch TAPPS Academic team has claimed victory at the annual TAPPS Academic State Championship. The BL team scored an incredible 141.75 points, beating the second place team by nearly 30 points. . Under the outstanding leadership of moderator, Sister Cecilia Sehr, Bishop Lynch congratulates and thanks those teachers who served as coaches and guided the students throughout the year: Mr. Jeff Morrow, Mr. David Post, Ms. Doran Wofford, Mr. Joseph Inman, Ms. Annette Butler, Mr. John Athas, Mr. Rick Dunn, Dr. Mark Morris, Sr. Cecilia Sehr, and Mr. John Athas
Current Events & Issues - Jacob KingScience -- Ryan Smith Persuasive Speaking - Jacob King Biology - Ryan SmithLiterary Criticism -- Lauren Pauletti
Literary Criticism - Chelsea FirraCalculator - Kelly KwonSpelling - Lizette Hernandez TAPPS Math - Kelly Kwon
Calculator - Hwang Barrington Computer Science - Emily FitzharrisLiterary Criticism - Karen HessOriginal Oratory - Anthony FernandezReady Writing - Emily FitzharrisScience - Zach BennettSocial Studies - Anthony Fernandez
Computer Science - Victoria EhlingerCurrent Events & Issues - William JohnsonDuet Acting - Mason Walker & Katherine GroganMathematics - Zach BennettReady Writing - Karen Hess Spelling - Lucas Cavasso
Hwang Barrington - ScienceVictoria Ehlinger - CalculatorBen Steiner - Social StudiesMichael Varghese - Number Sense
Current Events & Issues - Caley ShannonDuet Acting - David Helms & Erin Little Mathematics - Dillon Lewis
Join us for the 2017 Homecoming celebrations!
Friday, October 13, 2017
Thank you for your support of Bishop Lynch!
>give now
Click below for current news.
Bishop Lynch High School | 9750 Ferguson Rd., Dallas, Texas 75228 | 214.324.3607 | FAX 214.324.3600 | Toll Free 888.835.3607


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Tribute: Rose Mary Matters
Rose Mary Matters died December 10
Rose Mary Matters died December 10
MyValleyTributes death records
Duane L. Granger died December 10
Luella M. Shaulis died December 10, 2016
Dr. Michael Sevilla from the Family Practice Center of Salem talked about some ways to keep up with your resolution this year.
A photo was taken from a Ranburne family's awareness Facebook page and made into a "laughing mockery joke."
Gloria Jean Butler passed away December 10
Helen Lucas, 102, passed away December 10
For those with students in Youngstown City Schools, the district's CEO is asking parents to complete a survey about how well you think the s…
At least 100 people were killed and hundreds injured in the quake, which also destroyed or damaged more than 11,000 buildings throughout Ind…
The latest weather forecast for Youngstown, Warren, Austintown, Boardman, Sharon, Lisbon and East Liverpool.
LeBron James scored 27 points to move into ninth place on the NBA scoring list.
The 2-year-old died when the van pinned her against a barn in September.
The suspect continued to drive for about 1.5 miles in the police SUV with gunshot wounds, officers said.
The latest weather forecast for Youngstown, Warren, Austintown, Boardman, Sharon, Lisbon and East Liverpool.
New Castle opened the 2016-2017 season in style grabbing a win over West Middlesex in the first day of the New Castle Tip-Off Classic.
Girard got their first win of the season Friday night topping Campbell Memorial 59-46 at Girard High School.
U.S. intelligence officials have accused Russia of hacking into Democratic officials' email accounts in an attempt to interfere with the pre…
The extended fight over the miners' benefits was intended to "highlight the seriousness of this issue," Sen. Chuck Schumer said.
The disastrous tally has been pushed to new heights by soaring opioid abuse.
McDonald Freshman Zach Rasile set a school record with 11 three pointers in a 123-45 win over Columbiana Friday.
Dakota Hobbs scored a team-high 17 points for Ursuline.
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text:
About KSDK
Mission StatementWe are community advocates and take pride in our commitment to inform and protect. We showcase the greatness of St. Louis and accentuate the remarkable people who live here. We celebrate St. Louis' rich heritage, promote the present and are passionate about the future.
About UsWe are not a TV station.
We are an information center that believes in connecting with our communities across all platforms with a passion for advocacy, pride in community service and above all, excellence in journalism.
Day in and day out, EVERYONE at KSDK is committed to inform and protect, to showcase the greatness of St. Louis and celebrate the remarkable people who live here.
Yes, our trophy case has 70 years of journalistic hardware, but that is NOT where we hang our hat. At KSDK, we are here for something bigger than ourselves – the opportunity to serve the greater good of St. Louis. We are part of the community, and they are part of us.
We are 5 On Your Side.
If the above made you feel inspired, excited, empathetic or any emotion that motivates you do to amazing things, apply now!
KSDK HistoryNewsChannel 5 has a rich broadcast heritage. We were known as KSD in the early years and were pioneered by the Pulitzer publishing family. When we signed on the air in February of 1947, KSD was just the seventh television station in the entire country! We were the only television station in St. Louis for the first six years of our operation. We have been affiliated with NBC from our very beginning. We were the first station in St. Louis to broadcast in color, the first to broadcast in stereo, and the first to broadcast HD. In 2006, we were just the eighth station in the entire nation to broadcast local news and programming in High Definition. During the mid-80's, KSDK was the broadcast home for the nationally syndicated Sally Jessy Raphael show. KSDK has won hundreds of regional Emmy awards, dozens of local and national Edward R. Murrow awards and two national Emmy awards. Our community initiative, A Place to Call Home has been recognized with a national Service to America award as well as being nominated for a national Emmy. On September 5th, 1995, KSDK was purchased by Gannett, Co. Inc. and to this day, NewsChannel 5 remains part of the TEGNA family. To learn more about TEGNA, visit www.tegna.com.
Click here to license our content:


text:
2006-2011 Harley FXD Dyna Series Clymer Repair Manual
Other products by Clymer
List Price : $51.95
Price : $41.56
You Save : $10.39 (20%)
1 New from $41.56
Price is accurate as of the date/time indicated. Prices and product availability are subject to change. Any price displayed on the Amazon website at the time of purchase will govern the sale of this product.
DescriptionClymer Product Repair / Service Manual This room includes mtliche s systems, maintenance and repair detailed photos and diagrams, the complete disassembly and assembly of the motorcycle application: 2006-2010 Harley FXD Dyna Super Glide Harley 2006-2011 FXDC Dyna Super Glide Custom Harley FXDL Dyna Low Rider (2006-2009, 2010-2011 HDI Japan only) Harley FXDWG Dyna Wide Glide (2006-2008, 2010-2011) 2006 35th Anniversary Harley Super Glide 2010 Harley CVO Dyna Fat FXD35 Bob FXDFSE2 2006-2011 Harley FXDB Street 2008-2011 Bob Fat Bob 2007 Harley-later-later Screamin 'Eagle Dyna FXDSE2 2008 Harley Screamin' Eagle Dyna 2009 Harley CVO Dyna Fat Bob FXDFSE Condition: New Quantity: 1


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Better Baked Ziti
1 box whole wheat or brown rice penne, cooked al dente
1.3 lb 93-99% lean ground turkey, browned
1 15-oz container fat free ricotta
1 15-oz container fat free cottage cheese
1 bag fat free shredded mozzerella, split in half
1.5-2 cans Furmano's crushed tomatoes w/ basil and oreg.
1 egg
Oregano, basil, garlic to taste
Dried parsely flakes
- Preheat oven to 375 degrees
- Mix all ingredients (besides parsley) in a large bowl.
- Pour into 13x9 baking dish. Sprinkle remainind half of shredded mozerella on top.
- Sprinkle with dried parsley.
- Cover with foil, and bake for about 450-55 minutes or until bubbling.
Pairs Well With
This baked ziti recipe is a little better for you than the full-fat stuff!
Disney's Tinker Bell "Secret of the Wings Cookbook"By BakeSpace Fairies
6 Recipes445 Downloads


text:
... year off. Very expensive and I fell a year ago and cracked my ribs. Not sure it's working for me
Osteoporosis - I've been on Aclasta for 6 years with the annual injections. I'm thinking of taking a
- Asked
- 11 Jul 2016 by Anne Magnuson
- Topics
- aclasta, osteoporosis, injection
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