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Clinical and Translational Allergy
The expression of CD123 can decrease with basophil activation: implications for the gating strategy of the basophil activation test
Alexandra F. Santos1,2,3,4,
Natalia Bécares1,2,
Alick Stephens1,2,
Victor Turcanu1,2 &
Gideon Lack1,2
Clinical and Translational Allergy volume 6, Article number: 11 (2016) Cite this article
Basophil activation test (BAT) reproduces IgE-mediated allergic reactions in vitro and has been used as a diagnostic test. Different markers can be used to identify basophils in whole blood and have implications for the outcome of the test. We aimed to assess changes in the expression of CD123 and HLA-DR following basophil activation and to select the best gating strategy for BAT using these markers.
BAT was performed in whole blood from 116 children. Peanut extract, anti-IgE, anti-FcεRI or formyl-methionyl-leucyl-phenylalanin (fMLP) was used for stimulation. Surface expression of CD123, HLA-DR, CD63 and CD203c was evaluated by flow cytometry.
In some cases, gating on CD123+/HLA-DR− led to the loss-to-analysis of basophils in conditions where basophils were activated. Adding CD203c as an identification marker restored the cell number. Basophils remained HLA-DR-negative with activation. CD123 expression decreased following stimulation with fMLP (n = 116, p < 0.001), anti-IgE (n = 104, p < 0.001) and peanut (n = 42, p < 0.001). The decrease in the mean fluorescence intensity of CD123 correlated with the up-regulation of basophil activation markers, CD63 (rs = −0.31, p < 0.001) and CD203c (rs = −0.35, p < 0.001). BAT to peanut gating basophils on CD203c+/CD123+/HLA-DR− reduced the false-negatives (1 vs. 5 %) and showed a higher diagnostic accuracy compared to using CD123+/HLA-DR− (97 vs. 91 %). CD203c+ appeared as an alternative gating strategy allowing two-colour BAT.
Basophils of a subset of patients down-regulate CD123 with activation. The use of CD203c before gating on CD123+/HLA-DR− cells or in isolation ensures the identification of the entire basophil population and accurate assessment of basophil activation, with important diagnostic implications.
The basophil activation test (BAT) is a flow cytometry-based assay that reproduces IgE-mediated allergic reactions in vitro. Following cell stimulation and activation, basophils undergo degranulation with the release of histamine, leukotrienes and cytokines, and up-regulate the expression of activation markers on their surface, such as CD63 and CD203c, which can be measured by flow cytometry [1, 2]. CD63 is a lysosomal-associated membrane protein (LAMP-3) and is not expressed on resting basophils but only after degranulation as the granules fuse with the plasma membrane. Its expression is bimodal, as only a subset of basophils express CD63, therefore it is usually represented as a proportion of positive cells (i.e. %CD63+ basophils). CD203c is constitutively and specifically expressed on the surface of resting basophils, which increases after basophil activation; thus, it can be used as an identification as well as an activation marker [3]. CD203c has been related to piecemeal degranulation and CD63 to anaphylactic degranulation [4, 5]. The BAT has been used primarily as a research tool and its application for clinical use in the diagnosis and monitoring of allergic diseases [6–10], namely following immuno-modulatory treatments such as allergen-specific immunotherapy [11–13] and omalizumab [14–16], is still in development. Our first allergen-specific (peanut) study that is clinically validated and based on double-blind-placebo-controlled-food-challenges (DBPCFC) initially established retrospectively diagnostic cut-off values for CD63 expression (n = 104) and verified this prospectively in a second population (n = 65) [6]. In this study [6], BAT showed 97 % accuracy in the diagnosis of peanut allergy. This enhanced diagnostic performance is in large part due to inclusion of CD203c in the gating strategy.
Different cell-surface markers may be preferred for the identification of basophils in whole blood. One of the commonly used is the combination of CD123 and HLA-DR [1, 2]. CD123 is the low affinity subunit of the IL-3 receptor and is highly expressed on plasmocytoid dendritic cells and basophils, and in low levels on monocytes, eosinophils, myeloid dendritic cells and hematologic progenitor cells. While eosinophils can be excluded by side-scatter, additional staining with anti-HLA-DR discriminates between HLA-DR-negative basophils and HLA-DR-positive dendritic cells and monocytes. To exclude hematologic progenitor cells, an additional marker specific for basophils such as CD203c could be used. In previous studies, the expression of CD123 and HLA-DR were shown to be stable with the atopic status of patients and following basophil activation [2, 17]. However, stimulation of the IL-3 receptor by IL-3 can increase the baseline expression of CD203c and possibly CD63 and maximise the up-regulation of CD63 upon basophil activation [4, 18]. Therefore, we hypothesized that CD123 expression could change in response to basophil activation. With increasing attention given to basophils in the coordination of adaptive immune responses and their possible role in antigen presentation [19–21], we considered that there could be an increase in the expression of HLA-DR by basophils following activation by allergen or other stimulants. Given the important implications for the gating strategy to be adopted for BAT in future studies, we sought to determine whether the expression of CD123 and HLA-DR remained unchanged with basophil activation and to select the best gating strategy using these markers.
Results of BAT to peanut performed in children attending our Pediatric Allergy clinic, aged from 5 months to 17 years, performed using the same methodology as part of two clinical studies [6, 10] were analyzed. Peanut allergy was diagnosed based on a positive oral peanut challenge or the combination of a recent clear history of one or more systemic reactions to peanut and a weal diameter on skin prick testing (SPT) ≥8 mm and/or serum peanut-specific IgE ≥15 KUA/l [22]. Peanut-tolerance was defined by a negative oral peanut challenge or the ability to eat an age-appropriate quantity of peanut regularly (as defined by a validated food-frequency questionnaire [23]) without developing any allergic symptoms. Ethical approval was obtained from the South East London Research Ethics Committee 2 and parents of all children signed written informed consent.
Whole blood basophil activation test
Heparinized whole blood (100 µl) was stimulated for 30 min at 37 °C with peanut extract (0.1; 1; 10; 100; 1000 and 10,000 ng/ml, ALK Abelló, Horsholm, Denmark) diluted in Rosewell Park Memorial Institute (RPMI, GIBCO, Paisley, UK). Polyclonal goat IgG anti-human IgE (1 µg/ml, Sigma-Aldrich, Poole, UK) and monoclonal mouse anti-human FcɛRI (2.5 µg/ml, Ebioscience, San Diego, CA, USA) were used as IgE-mediated positive controls. Formyl-methionyl-leucyl-phenylalanine (1 µM, fMLP, Sigma-Aldrich) was used as a non-IgE-mediated positive control (as it acts via a G-protein-coupled receptor (FPR-1) that activates MAPK pathways and phospholipase C bypassing part of the signalling pathway downstream the IgE receptor FcεRI). RPMI alone was used as a negative control. The reaction was stopped by adding cold ethylenediaminetetraacetic acid. Prior to erythrocyte lysis with BD Pharmlyse (BD Biosciences, San Jose, CA, USA), cells were stained with anti-CD123-fluorescein isothiocyanate (FITC) (Ebioscience), anti-CD203c-phyco-erythrin (PE), anti-HLA-DR-peridinin chlorophyll protein (PerCP) and anti-CD63-allophycocyanin (APC) (Biolegend, San Diego, CA, USA) for 30 min. In selected experiments, anti-CD14-PECy7, anti-CD3-pacific blue (PB), anti-CD19-PB, anti-CD41-PB and anti-CD56-PB (Biolegend) were additionally used. The surface expression of these markers was evaluated using FACS CantoII with FACSDiva software (BD Biosciences, San Jose, CA, USA). Data was analysed with FlowJo software version 7.6.1 (TreeStar, Ashland, OR, USA).
Basophil activation was expressed as a proportion of CD63+ basophils, corrected for the negative control, and as a ratio of the MFI of CD203c-PE of stimulated to unstimulated basophils, the stimulation index of CD203c (SI CD203c). The variation of CD123 was defined as the proportion of the difference between the MFI of CD123-FITC of the negative control and of the stimulated cells and the MFI of CD123-FITC of the negative control, and was calculated using the formula (MFI CD123-FITC of negative control − MFI CD123-FITC of stimulated cells)/MFI CD123-FITC of negative control. When assessing basophil activation induced by anti-IgE (n = 104), patients with non-responder basophils, i.e. basophils which did not respond to any IgE-mediated stimulants but only to fMLP, were excluded. When evaluating the response to peanut, peanut allergic patients with responding-basophils were considered (n = 42). In the data analysis, when only one concentration of peanut extract was used, 100 ng/ml was selected unless indicated otherwise.
Qualitative variables were represented as number of patients and percentage (taking into account the missing values) and groups were compared using the Fisher's exact test or Chi square test, as appropriate. Continuous variables were represented as median and range and were compared using the Mann–Whitney U test or Kruskall–Wallis test, as appropriate. Wilcoxon-signed rank test was used to compare samples before and after stimulation.
For receiver-operating characteristic (ROC) curve analysis, the performance of the average percentage of CD63-positive basophils at 10 and 100 ng/ml of peanut extract determined using different gating strategies was evaluated against the patients' allergic status to peanut, i.e. in relation to allergy versus tolerance.
All statistical analyses were performed with SPSS 20.0 for Windows. Significance was determined using a two-sided α level of 0.05.
Gating on basophils using CD123 and HLA-DR led to the loss of cells
Identifying basophils with CD123 and HLA-DR (Fig. 1a–d) led to the loss to analysis of cells particularly in conditions where basophils were activated. The baseline number of unstimulated basophils was variable (median = 1721, IQR = 1225–2184) but comparable between atopic and non atopic patients (p = 0.444) and between peanut allergic and peanut tolerant children (p = 0.739). Following basophil stimulation with fMLP (p = 0.012) and anti-IgE (p = 0.005), the number of basophils was significantly reduced compared to baseline (Table 1).
Basophils were identified in whole blood in the lymphocyte-monocyte area (a) as SSClow/CD123+/HLA-DR− (b, c) or as SSClow/CD203c+ (e) or as SSClow/CD203c+/CD123+/HLA-DR− (e, f) cells. CD63 expression was assessed in basophils identified as SSClow/CD123+/HLA-DR− (d) or SSClow/CD203c+/CD123+/HLA-DR− (g).
Table 1 Number of basophils (gated as SSClow/CD123+/HLA-DR− cells) and expression of CD123 on the surface of basophils (gated as SSClow/CD203c+ cells) as measured by MFI of CD123-FITC in different stimulation conditions
Of note, if only patients with non-responder basophils were considered (n = 12), the change in basophil number was significant after fMLP stimulation (p = 0.004) but not after anti-IgE stimulation (p = 0.099), suggesting that the reduction in cell number was dependent on basophil activation. Considering anti-IgE stimulation, 14 % of patients showed more than 25 % decrease in the number of basophils compared to the negative control (Fig. 2).
Variation in the number of identified basophils following anti-IgE stimulation compared to the negative control (n = 104). Variation was calculated as (number of identified basophils pre-stimulation − number of identified basophils post-stimulation)/number of identified basophils pre-stimulation
In 27 % of patients, this number decreased to below 1000 basophils (the minimum number of cells usually required for BAT). Selecting peanut allergic patients, a trend was seen toward a reduction in the basophil number after stimulation with 1000 ng/ml of peanut extract compared to the negative control (n = 42, p = 0.081). As the starting volume of blood, and thus the starting number of cells, was similar in all experimental conditions, we hypothesized that the expression of the identification markers, CD123 and/or HLA-DR, changed with basophil activation.
CD123 is down-regulated with basophil activation
To evaluate the changes in the expression of CD123 and HLA-DR on the surface of basophils, we used CD203c to gate on the basophil population (Fig. 1a–g). The baseline MFI of CD123-FITC was variable between patients (median = 1082, IQR = 98–1549) but comparable between atopic and non-atopic children (p = 0.153) and between peanut allergic and peanut tolerant patients (p = 0.826). Down-regulation of CD123 by basophils was seen following stimulation with fMLP (n = 116, p < 0.001), anti-IgE (n = 104, p < 0.001) and peanut extract (n = 42 peanut allergic patients, p < 0.001)—Table 1 and Fig. 3a. In 92.3 % of patients, anti-IgE stimulation led to a decrease in the MFI of CD123-FITC compared to the negative control: in 38.5 less than 25 % decrease, in 26.9 % between 25 and 50 % decrease, in 13.5 % between 50 and 75 % decrease and in 13.5 more than 75 % decrease (Fig. 4a). The down-regulation of CD123 expression on the surface of basophils stimulated by fMLP and by anti-IgE was correlated (rs = 0.723, p < 0.001), suggesting this phenomenon happened in the same patients with different stimulants. The decrease in CD123 expression with anti-IgE stimulation was similar between atopic and non atopic (p = 0.828) and between peanut allergic and peanut tolerant children (p = 0.431). The expression of CD123 was stable when basophils were not activated—for example, after stimulation with peanut in peanut tolerant patients (p = 0.658) or after stimulation with anti-IgE in non-responders' basophils (p = 0.083), while down-regulation was still observed in this subgroup after stimulation with fMLP (p = 0.006).
Changes in the MFI of CD123-FITC (a) and in the MFI of CD203c-PE (b), following stimulation with 100 ng/ml of peanut extract (n = 42 peanut allergic patients)
Down-regulation of CD123 expression with basophil activation. a Change in the MFI of CD123-FITC on the surface of basophils following anti-IgE stimulation. b Correlation between decrease in CD123 and up-regulation of CD63 on the surface of basophils
Basophils were HLA-DR-negative and were distinct from the CD123+ HLA-DR+ population of plasmacytoid dendritic cells. Following basophil activation, the HLA-DR expression did not increase and remained distinct from HLA-DR+ cells.
Decrease in CD123 is correlated with the up-regulation of CD63 and CD203c
Taken together the previous observations indicate that the down-regulation of CD123 by basophils is an activation-dependent phenomenon. A weak statistically significant correlation was observed between the decrease in the MFI of CD123 and the up-regulation of basophil activation markers after stimulation with anti-IgE (Fig. 4b) as measured by the stimulation index of CD203c (rs = −0.35, p < 0.001) or by the percentage of CD63-positive basophils (rs = −0.31, p < 0.001), suggesting that the basophils that down-regulate CD123 the most are also the ones that express more CD63 and CD203c. The correlations between changes in the MFI of CD123-FITC and in the percentage of CD63+ basophils or the SI of CD203c following stimulation with fMLP (−0.191, p = 0.04 and −0.223, p = 0.016, respectively) and following stimulation with peanut extract (−0.229, p = 0.145 and −0.31, p = 0.051, respectively) were less strong.
Additional use of CD203c prevented the loss-to-analysis of activated basophils
The down-regulation of CD123 with basophil activation has important implications in gating strategies that depend on CD123. Identifying basophils using CD123 and HLA-DR led to the loss-to-analysis of basophils, particularly of the ones with higher expression of the activation markers CD63 and CD203c, and thus leading to an underestimation of basophil activation (Table 2; Fig. 5a). Adding CD203c as an identification marker restored the cell number, regardless of the basophil activation status and allowed to include the basophils that were activated the most in the analysis, improving the outcome of the test (Table 2; Fig. 5b). The expression of CD203c remained stable or increased following basophil activation, allowing a good separation from the remaining blood cells (Fig. 3b).
Table 2 Comparison of different strategies to gate on basophils
Gating with CD123/HLA-DR led to the loss-to-analysis of basophils and underestimation of basophil activation. In a representative experiment, basophils were gated as SSClow/CD123bright/HLA-DR− (a) or SSClow/CD203c+/CD123+/HLA-DR− (b). The percentage of CD63+ basophils is represented in different conditions: negative control, peanut extract 100 ng/ml and peanut-dose–response curve. Note the bi-exponential display [38]
Selecting the optimal gating strategy using CD203c
In order to select the best gating strategy using the available markers, we compared the results of the BAT following the identification of basophils as SSClow/CD203c+ cells, as SSClow/CD123+/HLA-DR− cells or as SSClow/CD203c+/CD123+/HLA-DR−. To combine the three identification markers to gate on basophils, we first selected the SSClow/CD203c+ cells and then gated on the plot CD123/HLA-DR following the contour of the cell population of interest, including CD123low as well as CD123high cells, all HLA-DR− (Fig. 1f).
The gating strategy using CD203c+/CD123+/HLA-DR− proved superior to CD123+/HLA-DR− and similar to what was observed for gating on CD203c+ cells alone (Table 2). The combination of the three markers was superior to CD203c+ in the subgroup of peanut allergic patients improving the detection of basophil activation (Table 2, borderline non-significant p values for %CD63+ basophils, SI CD203c and MFI of CD203c) and in patients with a subset of CD203c+/HLA-DR+ cells thus avoiding contamination with HLA-DR+ cells. However, in the majority of patients, the gating strategies SSClow/CD203c+ and SSClow/CD203c+/CD123+/HLA-DR− were comparable. SSClow/CD203c+ could be used as an alternative gating strategy in a two-colour BAT.
In additional experiments (n = 10) using antibodies anti-CD14, anti-CD3, anti-CD19, anti-CD41 and in six of these experiments also anti-CD56, there was no contamination of the gating strategy using SSClow/CD203c+/CD123+/HLA-DR− with other immune cells nor did they express CD203c. Only in one patient, CD14+ cells were also CD203low and HLA-DR+. In the PB-positive (PB+) population, there was a minor expression of CD63, possibly by CD41+ platelets, but this population was gated out as it did neither express CD123 nor CD203c, and CD63 expression remained stable with activation (data not shown).
Gating strategy of BAT has important diagnostic implications
The diagnostic performance of BAT gating on SSClow/CD203c+/CD123+/HLA-DR− was superior to the one using SSClow/CD123+/HLA-DR− with a larger area under the ROC curve (Fig. 6). The optimal cut-off based on the ROC curve generated using the latter gating strategy resulted in a 91 % diagnostic accuracy with 5 % false-negatives and 3 % false positives (Table 3). Adopting the SSClow/CD203c+/CD123+/HLA-DR− gating strategy resulted in a diagnostic accuracy of 97 %, as we recently reported [6], with 1 % false-negatives and 2 % false-positives. Unusually, this methodological improvement resulted in both enhanced sensitivity and specificity of BAT in the diagnosis of peanut allergy. Figure 5 shows an example of a patient that would be considered false negative if gating was confined to CD123+/HLADR− cells. Furthermore, using this gating strategy, 15 % of patients showed <500 basophils in at least one condition and thus BAT would be uninterpretable.
ROC curve of the average %CD63+ basophils at 10 and 100 ng/ml of peanut extract using different basophil gating strategies: SSClow/CD203c+/CD123+/HLA-DR− (red), SSClow/CD123+/HLA-DR− (blue)
Table 3 Diagnostic accuracy of the basophil activation test to peanut using different gating strategies
BAT can be used to diagnose allergic disease and to study the underlying immunological mechanisms. The methodology of identification of basophils has important consequences for the outcome of the test. In this study, we showed that CD123 is down-regulated with basophil activation and gating strategies that depend solely on this marker lead to the loss-to-analysis of activated basophils and to the underestimation of basophil activation. Using CD203c in addition to CD123/HLA-DR or in isolation proved superior to gating on CD123/HLA-DR, reducing the number of false-negatives and false-positives and improving the diagnostic accuracy from 91 to 97 %. When BAT is used as an allergy test, the gating strategy adopted has diagnostic implications in the assessment of individual patients. Basophils have been identified with CD123 and HLA-DR in previous studies, using flow cytometry and other techniques [24], and CD123 expression was reported to be stable with the atopic status of the patient and following basophil activation, although no direct comparisons were made [2, 17, 25]. In our study, the MFI of CD123-FITC was comparable between atopic and non atopic patients, but there was a decrease in the MFI of CD123-FITC with basophil activation. This is in contrast to previous studies [2, 17, 25] and is probably related to differences in the study population (e.g. adults vs. children), in the disease models studied (e.g. patients with respiratory vs. food allergies) and in the study design (e.g. basophils stimulated with allergen in vivo vs. in vitro, which can have different kinetics). We do not believe compensation played a role in the observed changes as this phenomenon only happened in a subset of patients and in other patients the expression of CD123 was stable regardless of the increase in the expression of CD63 and CD203c. Furthermore, while the spectrum of CD203c-PE overlapped with CD123-FITC, the spectra of CD123-FITC and of CD63-APC did not overlap and we performed compensation before running the samples, used the same compensation matrix during acquisition of all samples for each patient and did not see any differences in compensation within the same experiment. Although the size and the granularity of basophils change with activation and degranulation [5, 26], these modifications do not lead to cell loss in the FSC/SSC gate and basophil numbers are stable with and without stimulation using identification markers such as CD203c. With respect to HLA-DR, basophils were HLA-DR-negative and kept distinct of HLA-DR-positive cells in all conditions. These findings are consistent with previous reports [17] and with recent studies that did not confirm a role for basophils in antigen-presentation in humans [27, 28].
The expression of CD123 being dependent on basophil activation raises the question as to whether CD123 can be used as a basophil activation marker. However, this phenomenon is significant in only a subset of patients and the dose–response with increasing concentrations of the stimulant is subtle; thus, CD123 does not seem to offer any advantage to the existing activation markers, such as CD63 and CD203c.
The combination of the three identification markers, CD203c/CD123/HLA-DR, proved superior to using CD123/HLA-DR. We compared the diagnostic performance of BAT to peanut identifying basophils as CD123+/HLA-DR− cells and as CD203c+/CD123+/HLA-DR− cells and the latter resulted in a greater area under the ROC curve (0.96 vs. 0.99) and improved diagnostic accuracy (91 vs. 97 %). Furthermore, if we took into consideration a minimum number of basophils as exclusion criteria, 15 % of patients would be inevaluable using CD123+/HLADR−. The consequences of the gating strategy adopted are clinically relevant as the purpose of the BAT is to diagnose peanut allergy in individual patients. An example is illustrated in Fig. 5, where with basophils gated as CD123+/HLA-DR−, BAT would be considered negative at the diagnostic concentration of 100 ng/ml but in fact it was clearly positive when basophils were selected using the three markers' strategy. This would be a false-negative with potential serious consequences, as it could lead to liberalization of peanut consumption with the risk of allergic reactions that are potentially severe. The loss-to-analysis of cells and/or the underestimation of basophil activation have important implications for the final outcome of the test and thus for the diagnosis of individual patients. The same applies to other clinical applications, such as monitoring of treatment, and to mechanistic experiments. The fact that we detected no contamination of the gating with other immune cells suggests that there would be no advantage in using a lineage negative antibody mixture to exclude other immune cells before gating on the CD203c+/CD123+/HLA-DR− cells as basophils.
Identifying basophils with CD203c alone lead to comparable outcome in terms of number of basophils and basophil activation markers to using CD203c+/CD123+/HLA-DR− and was also superior to using CD123+/HLA-DR−, as represented in Table 2. CD203c is a basophil specific marker in whole blood. Its constitutive expression is increased in patients with atopic eczema and food allergy [29–31], as previously described in terms of histamine release [32] suggesting it is a marker of underlying basophil activation, possibly reflecting ongoing piecemeal degranulation. This enhances the separation of CD203c+ basophils from the other blood cells in populations of highly atopic children [6]. The majority of patients in our study had eczema and other food allergies in addition to suspected peanut allergy and this represents a population where CD203c would be constitutively expressed at a higher level than in other children allowing a clear differentiation between CD203c+ basophils and other blood cells. The conjugation to the bright fluorochrome PE may have also contributed to the good identification of cells expressing CD203c. SSClow/CD203c+ is an alternative basophil identification strategy that has the advantage of serving also as an activation marker, enabling BAT to be performed as a two-colour (preferred) or even as a single colour BAT, which would make BAT easier and less expensive to perform.
This is the largest study looking at different strategies for identifying basophils using CD123 and its clinical implications and it is the first study to report the down-regulation of CD123 with basophil activation. The performance of BAT soon after blood collection and the use of live cells for flow cytometry on the same day contributed to the quality of the results. However, our study population was constituted mostly by highly atopic children enrolled in a study examining the use of BAT in the diagnosis of peanut allergy [6]. Therefore, our results may not apply to other populations, namely of non atopic patients or older patients being assessed for other conditions such as drug allergy. While assessing changes in basophil identification markers, we discovered that in a subset of patients basophils down-regulate CD123, the low affinity subunit of the IL-3 receptor, with basophil activation. This phenomenon seems patient-specific rather than specific for atopic or allergic status. In the patients where this is observed, the down-regulation happens only in conditions where basophils are activated and correlates with the degree of activation, as expressed by CD63 and CD203c. It is possible that the patients who showed higher down-regulation of CD123 after basophil activation are the ones with most severe clinical reactions but this remains to be confirmed. IL-3 is predominantly produced by T cells and is able to induce basophils to release histamine and up-regulate CD203c and CD63 in the absence of allergen [33, 34]. It can also act synergistically with allergen or other stimulants to increase basophil activation and histamine release [4, 18, 33]. The basophil intracellular pathways down-stream of the IL-3 and the IgE receptors seem indeed closely connected [34]. The response to this priming effect is variable between basophil donors and requires different concentrations of IL-3 [35]. Some research groups have used exogenous IL-3 to prime basophils in the BAT [4, 18, 36]. However, basophils secrete IL-3 themselves in response to IgE-mediated activation for autocrine priming, which has been suggested to be a possible mechanism underlying the hyper-reactive nature of the basophils of allergic patients [37]. We hypothesize that the down-regulation of CD123, which is part of the IL-3 receptor, could result from a basophil regulatory mechanism to avoid further cell activation, but this deserves further research.
Basophils down-regulate CD123 with activation in a subset of patients and this can have significant deleterious diagnostic implications. While performing the BAT, the use of gating strategies that depend solely on CD123 may lead to loss-to-analysis of basophils, particularly of the ones that highly express CD63 and CD203c, resulting in a false-negative outcome for the test. To overcome this limitation, additional use of CD203c, both identification and activation marker, prevents the loss-to-analysis of activated basophils and allows accurate assessment of basophil activation and, consequently, a more accurate diagnosis of allergy.
APC:
allophycocyanin
BAT:
basophil activation test
DBPCFC:
double-blind-placebo-controlled-food-challenge
FITC:
fluorescein isothiocyanate
fMLP:
formyl-methionyl-leucyl-phenylalanine
LAMP:
lysosomal-associated membrane protein
phycoerythrin
PerCP:
peridinin chlorophyll protein
ROC:
receiver-operating characteristic
RPMI:
Rosewell Park Memorial Institute medium
SI:
stimulation index
SPT:
skin prick test
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Ocmant A, Peignois Y, Mulier S, Hanssens L, Michils A, Schandene L. Flow cytometry for basophil activation markers: the measurement of CD203c up-regulation is as reliable as CD63 expression in the diagnosis of cat allergy. J Immunol Methods. 2007;320:40–8.
Wynn TA. Basophils trump dendritic cells as APCs for T(H)2 responses. Nat Immunol. 2009;10:679–81.
Tang H, Cao W, Kasturi SP, et al. The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling. Nat Immunol. 2010;11:608–17.
Charles N, Hardwick D, Daugas E, Illei GG, Rivera J. Basophils and the T helper 2 environment can promote the development of lupus nephritis. Nat Med. 2010;16:701–7.
Roberts G, Lack G. Diagnosing peanut allergy with skin prick and specific IgE testing. J Allergy Clin Immunol. 2005;115:1291–6.
Fox AT, Sasieni P, du Toit G, Syed H, Lack G. Household peanut consumption as a risk factor for the development of peanut allergy. J Allergy Clin Immunol. 2009;123:417–23.
Dijkstra D, Hennig C, Hansen G, Biller H, Krug N, Hohlfeld JM. Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge. Cytom A. 2014;85:580–7.
Chirumbolo S, Ortolani R, Vella A. CCR3 as a single selection marker compared to CD123/HLADR to isolate basophils in flow cytometry: some comments. Cytom A. 2011;79:102–6.
Dvorak AM. Ultrastructural studies of human basophils and mast cells. J Histochem Cytochem. 2005;53:1043–70.
Kitzmuller C, Nagl B, Deifl S, et al. Human blood basophils do not act as antigen-presenting cells for the major birch pollen allergen Bet v 1. Allergy. 2012;67:593–600.
Eckl-Dorna J, Ellinger A, Blatt K, et al. Basophils are not the key antigen-presenting cells in allergic patients. Allergy. 2012;67:601–8.
Ford LS, Bloom KA, Nowak-Wegrzyn AH, Shreffler WG, Masilamani M, Sampson HA. Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance. J Allergy Clin Immunol. 2013;131(180–6):e1–3.
Shreffler WG. Evaluation of basophil activation in food allergy: present and future applications. Curr Opin Allergy Clin Immunol. 2006;6:226–33.
Ford LS, Bloom KA, Nowak-Wegrzyn AH, Shreffler WG, Masilamani M, Sampson HA. Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance. J Allergy Clin Immunol. 2013;131:180-6.
Sampson HA, Broadbent KR, Bernhisel-Broadbent J. Spontaneous release of histamine from basophils and histamine-releasing factor in patients with atopic dermatitis and food hypersensitivity. N Engl J Med. 1989;321:228–32.
Okayama Y, Begishvili TB, Church MK. Comparison of mechanisms of IL-3 induced histamine release and IL-3 priming effect on human basophils. Clin Exp Allergy. 1993;23:901–10.
Verweij MM, Sabato V, Nullens S, et al. STAT5 in human basophils: IL-3 is required for its FcepsilonRI-mediated phosphorylation. Cytom B Clin Cytom. 2012;82:101–6.
Gentinetta T, Pecaric-Petkovic T, Wan D, et al. Individual IL-3 priming is crucial for consistent in vitro activation of donor basophils in patients with chronic urticaria. J Allergy Clin Immunol. 2011;128:1227e5–1234e5.
Chirumbolo S. The use of IL-3 in basophil activation tests is the real pitfall. Cytom B Clin Cytom. 2011;80:137–8 ; author reply 9.
Schroeder JT, Chichester KL, Bieneman AP. Human basophils secrete IL-3: evidence of autocrine priming for phenotypic and functional responses in allergic disease. J Immunol. 2009;182:2432–8.
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AFS and GL designed the study; AFS developed the basophil activation assay, performed the statistical analyses and wrote the first draft of the manuscript; AFS and NB performed the laboratory experiments; AS and VT provided laboratory assistance; GL critically reviewed the analyses and edited manuscript; all authors reviewed the manuscript and approved its final version. All authors read and approved the final manuscript.
This work was funded by the Medical Research Council (G0902018) and the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The clinical trials unit is supported by the National Peanut Board, Atlanta, Ga. The authors would like to thank Susanne Heck and Pj Chana (BRC Flow Cytometry Core, NIHR GSTT/KCL Comprehensive Biomedical Research Centre); all the members of the LEAP and the PAS Studies' teams, the Clinical Nurse Specialists at the Children's Allergy Service at St Thomas' Hospital and the study participants and their families. The Programme for Advanced Medical Education is sponsored by Fundação Calouste Gulbenkian, Fundação Champalimaud, Ministério da Saúde and Fundação para a Ciência e Tecnologia, Portugal.
A. F. Santos and her institution have received funding from the Medical Research Council (CRTF No. G0902018, MRC Centenary Early Career Award and MRC Clinician Scientist Fellowship MR/M008517/1), the Immune Tolerance Network/National Institute of Allergy and Infectious Diseases, United States (Grant Nos. ITN032AD and ITN049AD); and the National Peanut Board, United States; she has also received funding from the Gulbenkian Programme for Advanced Medical Education. G. Lack has received research support from the Immune Tolerance Network/National Institute of Allergy and Infectious Diseases, United States (Grant Nos. ITN032AD and ITN049AD); the Food Allergy Initiative, United States (Pronuts study); the National Peanut Board, United States (Project 26); the Food Standards Agency, United Kingdom (Grant Nos. T07049 and T07051); the Food Allergy and Anaphylaxis Network, United States; Medical Research Council & AsthmaUK Centre (Grant No. G1100090); Goldman Sachs Gives; and the Department of Health through the National Institute for Health Research Comprehensive Biomedical Research Centre award to the Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust; is on the DBV Technologies advisory board; is a voluntary scientific advisor for the Anaphylaxis Campaign and the National Peanut Board; has received lecture fees from Sodilac; Novartis, Switzerland; Nestl_e Nutrition; GlaxoSmithKline, United Kingdom; and the Serono Symposia International Foundation; and has stock/stock options in DBV Technologies. The rest of the authors declare that they have no relevant competing interests.
Division of Asthma, Allergy and Lung Biology, Department of Pediatric Allergy, St Thomas' Hospital, King's College London, 2nd Floor, Stairwell B, South Wing, Westminster Bridge Road, London, SE1 7EH, UK
Alexandra F. Santos, Natalia Bécares, Alick Stephens, Victor Turcanu & Gideon Lack
MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK
Immunoallergology Department, Coimbra University Hospital, Coimbra, Portugal
Alexandra F. Santos
Gulbenkian Programme for Advanced Medical Education, Lisbon, Portugal
Natalia Bécares
Alick Stephens
Victor Turcanu
Gideon Lack
Correspondence to Gideon Lack.
Santos, A.F., Bécares, N., Stephens, A. et al. The expression of CD123 can decrease with basophil activation: implications for the gating strategy of the basophil activation test. Clin Transl Allergy 6, 11 (2016). https://doi.org/10.1186/s13601-016-0100-4
Peanut allergy


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Featured Wedding: Edna & Ramon
The couple tied the knot in a simple yet lovely ceremony in a quaint church in Bacolod.
by Yayen Sulit | Dec 21, 2012
Edna and Ramon gathered their closest family and friends as they exchanged vows in a simple yet lovely ceremony in a church in Bacolod. Shades of green and white exuded a calming and refreshing vibe in this intimate affair that took place before daybreak one October morning.
Want to know more about Edna and Ramon's story? Click on any of the following links:
Read about their love story
Find out about their wedding preparations
Want your wedding to be featured on BridalBook? Click here to find out how.
Edna and Ramon have been working in the same company for several years before their romance blossomed in 2009. That year, the two were assigned to projects in Japan, where they got to know each other better.
Their first date, which comprised a series of activities, was on Valentine's Day. Unfortunately, the weather didn't cooperate, and so there was a change of plans. Instead of going on a romantic date, which was to include ice skating in one of Japan's scenic spots, Edna and Ramon ended up having dinner in an Italian restaurant.
A month after their first date, Ramon had to return to the Philippines to finish some errands. Constant communication through social networks kept their relationship on solid ground. Two years into their relationship, Ramon finally asked Edna to be his wife.
Wedding Preparations
Edna and Ramon tied the knot in an intimate ceremony at a quaint church in Bacolod. The event had the couple's close friends and family in attendance.
Different shades of green and white provided a serene and refreshing vibe to a laid-back affair that marked the couple's marriage. The lovely event featured simple yet elegant décor, which were mostly customized by the couple and the guests themselves. The bride shares, "We did a lot of DIY projects. What I'm very proud of is our wedding cord, since it is different from the usual. I took my inspiration from Kidada wraps and made it myself in seven hours."
In between the ceremony and reception, the couple had traditional Filipino favorites served to guests. "We had a cart of hot piyaya and Japanese hotcakes with hot chocolate, coffee, and water set up outside the church so the guests can grab a quick breakfast before going to the lunch reception," says Edna.
At the reception, the couple and their guests listened to the UP Singing Ambassadors' beautiful renditions of classic love songs. The wedding, with its simple and homey feel, was festive in more ways than one.
Phases and Faces Photo and Video Packages
Look Golden on Your Big Day With Makeup by Gold

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La prima edizione di Eurovizijos atranka (ufficialmente Eurovizijos dainų konkurso nacionalinė atranka) si è svolta dal 13 febbraio al 4 marzo 2010 e ha selezionato il rappresentante della Lituania all'Eurovision Song Contest 2010 a Oslo.

I vincitori sono stati gli InCulto con Eastern European Funk, che all'Eurovision si sono piazzati al 12º posto su 17 partecipanti con 44 punti totalizzati nella seconda semifinale, non riuscendo a qualificarsi per la finale.

Organizzazione
Nel dicembre 2009, l'emittente pubblica lituana LRT ha annunciato il ritiro del paese dall'Eurovision Song Contest 2010 per mancanza di fondi. Tuttavia, poche settimane dopo il paese è stato confermato nella lista dei partecipanti grazie all'internet provider Teo LT, che ha coperto le spese di partecipazione di  litas (circa €.

LRT ha optato per l'organizzazione di un programma di selezione per la sua undicesima partecipazione eurovisiva. Il format usato per l'edizione precedente, Lietuvos dainų daina, è stato scartato ed è stato rimpiazzato da Eurovizijos atranka, che rimarrà il metodo di selezione lituano per il festival europeo fino al 2019.

L'edizione 2010 si è articolata in più serate: tre semifinali, ciascuna con 11 o 12 partecipanti, da cui, in base al voto combinato di giuria e televoto, tre artisti per serata hanno avuto accesso alla finale (più altri tre scelti da una giuria esperta fra i non qualificati), e una serata finale con i 12 artisti qualificati. Il vincitore ha ricevuto un premio di  litas stanziati dall'Associazione lituana per la protezione del copyright (LATGA) per poter finanziare la propria partecipazione eurovisiva.

Partecipanti
Gli artisti interessati hanno avuto modo di inviare all'emittente le proprie proposte dal 6 al 24 gennaio 2010. Il successivo 1º febbraio sono state rese pubbliche le 36 canzoni accettate per prendere parte alle serate dal vivo, scese poi a 34 dopo i ritiri di Donata Virbilaitė e dei Vanilla Soud. Fra i partecipanti ci sono i 4FUN, vincitori della selezione del 2007, e Sasha Song, vincitore di Lietuvos dainų daina l'anno precedente.

Semifinali

Finale
La finale si è svolta il 4 marzo 2010 e ha visto competere i 12 artisti qualificati dalle semifinali. Donatas Montvydas ha vinto la terza semifinale, ma è stato squalificato dopo che è emerso che la sua Running Fast era stata pubblicata prima del 1º ottobre 2009, andando quindi contro le regole della manifestazione europea. È stato rimpiazzato da Amberlife, quarto classificato nella stessa semifinale.

Note

Voci correlate
 Lituania all'Eurovision Song Contest
 Eurovision Song Contest 2010

Festival musicali del 2010

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Retired Rabbi Spreads Passion for Social Action
July 11, 2012, New York, NY – Jewish Helping Hands today announced the launch of the Tikkun Olam grant program to help individuals and organizations domestically and abroad in their efforts to aid communities in need. The program offers potential awardees grants of up to $5,000 as well as partnership with Jewish Helping Hands (JHH). Such partnership could involve help with hands-on physical work and enjoying the benefit of the experience of the organization's board and leadership.
Jewish Helping Hands partners with local agencies within recipient populations, enabling individuals to bring about change in their own communities. The new grant program will aim to identify new projects similar to the ones Jewish Helping Hands has supported, including:
In Israel, Jewish Helping Hands (JHH) partnered with United Jewish Federation of MetroWest (NJ), the Koret Foundation, and the local government and Community Center of Rishon LeZion, a suburb of Tel Aviv, to establish a program to provide micro-loans and business training to Ethiopian immigrants to Israel. The program enabled participants to establish themselves and their families within Israeli society.
In Rwanda, JHH has worked with AVEGA, the Association of Genocide Widows, to support genocide widows and their families. The program enables women widowed during the 1994 genocide to find hope for the future, helping them to provide for themselves and their children through small financial grants and educational workshops.
In Cambodia, working through CambodiaSchools, Jewish Helping Hands has built a school in a village outside of Phnom Penh. With Arun Sothea, who at the age of four was orphaned in the Khmer Rouge reign of terror during the 1970s, JHH sponsors several dozen orphans in the village so that they are able to attend school.
Jewish Helping Hands is a New York City-based foundation created in 2006 by Rabbi Joel E. Soffin, Rabbi Emeritus of Temple Shalom in Succasunna, N.J., with the belief that a little bit of help can make a big difference in someone's life. The foundation continues the social action work that Rabbi Soffin initiated in more than 25 years as a congregational rabbi. Jewish Helping Hands reaches out to vulnerable populations, initiating projects to make a difference where help is most needed. In the United States and abroad, JHH projects provide ground-level financial and hands-on support. In each community where it works, local residents determine what would be most helpful.
For more information about the grant program, visit jewishhelpinghands.org/grants, or email JHH at grants@jewishhelpinghands.org.


Volkert provides on-site, technical support to the DDOT's ITS/TOA (Traffic Operations Administration) for on-going, multiple signal, safety, and ITS projects. Work involves assistance with project management, traffic signal systems operations, independent verification and validation, software and system integration, plan reviews, and general engineering for DDOT major initiatives and programs including citywide signal optimization, Capital Traffic Operation Platform (CapTop) Traffic Management System enhancements, Adaptive Signal Control System implementation, Vehicle Detection System installation, and the development and implementation of the ITS Master Plan.
Volkert's engineers review plans, schedules, and other deliverables prepared by other consulting firms and prepare comments. Deliverables include signal design and timing plans, traffic impact analyses, GPS data, and Synrchro and SimTraffic models.


The last thing that a company needs during an disaster is to wonder what to do next. Who do you call? Where are the backups and do they work? Do we have the capacity on the remaining systems? Who does what and when?
Proper planning and testing alleviates these questions, and many more in the event of disaster. Preparing and practicing a proper disaster recovery plan is paramount to getting back up and running when disaster strikes.
Typically, the reason people don't make disaster recovery plans is that they don't have the resources to do it. As the business owner, they're already doing more than one job. Just like anything else in business, formulating a proper recovery plan takes time.
To develop a realistic backup and disaster recovery plan, you must understand the potential hazards that could affect your business, as well as your business processes and technology needs. Once this is done, a plan can be made to ensure that you have enough resources and personnel to get the job done.
Get other people involved-ask TCSP technicians. Because backed-up data is a crucial part of every disaster recovery plan, you also need to consider how you're backing up your data. Backup methods and media have different longevity, storage requirements and other considerations.
What processes, components, and physical and human resources are required to run my business?
What is the likelihood of disaster, both natural and otherwise, for my area?
What other risks do I need to prepare for?
How long could I do without each IT component?
Do I have an alternative site where I could access my business data if something happens to my office?
Once you've written your plan, make sure everyone in your organization knows about it. Then, review it regularly. We recommend that you review your plan annually, a lot can change in a year.
Inventory your technology hardware and software.
Document your voice/telephone service, data circuit/ Internet service, web host/DNS registrar, and email service information.
Include the names and phone numbers of each of your service providers.
Include your account numbers, usernames, and passwords for those services.
Backup your critical business data locally and to an offsite location.
Verify and test your restore process regularly.
Document all of your essential business processes.
Create vendor/supplier and employee contact lists and update them monthly.
Identify two recovery sites, such as a hotel, a vendor's office, or even a relative's home.
Subscribe to a severe weather alerting system via text, smartphone, or email.
Maintain adequate disaster recovery supplies to support a family for three days.
Consider VOIP or a hosted PBX phone service.
Purchase a backup/satellite/mobile Internet service plan.
Review your insurance policy for adequate business loss coverage of your data.
Execute service-level agreements with key service providers, such as fuel, fleet services, and building repairs.
We specialize in preventive maintenance, software updates, and data backups to prevent downtime. TCSP offers on-site, on call, and fixed-cost contract IT solutions that are customized to fit each business.
Disaster can be moments away, even with new equipment. All of our clients are small and medium-sized businesses (SMB) so we advise that there be three "layers of protection", plus battery backups. Verify and test your backup and restore process regularly.
Real-life tests like this can often be more effective than a simple thought experiment. It's easy to overlook some of the implications of a disaster when you're just walking through it in your head. By actually running a test, by really shutting things down, you can discover issues in your plan that you might have never thought of. Test how quickly you can pull together key players. Involve everyone, and make sure each person has a role. Acknowledge that first-timers may be nervous. Take the lessons learned with you.
The total hourly cost of downtime is most likely a combination of lost sales, lost employee productivity, lost data, and lost customer confidence/satisfaction.


text:
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Philip G. Louis, Jr.,
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$110 Million Insurance Brokerage Acquisition
Represented acquired person in $110 million acquisition in the insurance broker industry.
Philip G. Louis, Jr., Robert J. Mahon
$81 Million Plastics Industry Acquisition
Represented the acquiring company in an $81 million acquisition in the plastics industry.
Philip G. Louis, Jr., Steven J. Foristal, Robert J. Mahon
$80 Million Financing
Represented closely held business operating in the gaming industry with respect to an $80 million credit facility established to fund major improvements.
$55 Million Sale of Advertising Company
$55 million sale of advertising company to public company. The asset sale transaction involved the sale of rights in over 400 locations in four states, the transfer of over 1,000 advertising contracts, and the sale of two commercial buildings. The team helped the client navigate various corporate governance issues and achieve its goal of selling the business.
Steven J. Foristal, Daniel J. Burke, Jr., Philip G. Louis, Jr., Patricia B. Beckerle, Brian C. Zaldivar, Caitlin Fagan
Multimillion-Dollar Sale of Family Pharmacy
Represented a family pharmacy that specialized in compounding, human fertility drugs and hormone replacement therapy. The buyer's tax structure required us to complete a tax-free reorganization of the client's business prior to closing. The transaction also involved complicated tax and dividend issues regarding the majority shareholder's redemption of shares in the holding company that owned the pharmacy prior to closing.
Philip G. Louis, Jr., Steven J. Foristal, Robert J. Browning
International Divestiture for Diversified Private Client
Represented diversified private company in a multimillion-dollar divestiture of its travel gear distribution business. This transaction involved lengthy negotiations with an overseas buyer and allowed our client to achieve its goal of exiting the travel gear business to further focus on its unrelated core business.
Philip G. Louis, Jr., Steven J. Foristal
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Represented acquiring and acquired persons in an $850 million reorganization in the pet food industry.
Multimillion-Dollar Deal in Client's First International Expansion
Represented a client that heat treats metal parts for general manufacturers, including those in the automotive and aircraft industries, in its first international expansion. The deal included providing assistance with a long term supply arrangement with an European equipment manufacturer as well as a long-term facility lease and construction agreements; retaining local counsel in Mexico; setting up foreign subsidiaries and adding a new U.S. subsidiary; as well as handling associated intellectual property, real estate and tax issues.
Philip G. Louis, Jr., Jennifer A. Schwesig, Hillary Bean
$111 Million Construction Industry Acquisition
Represented acquired persons in a $111 million acquisition in the construction industry involving industrial building, pipelines, and power and communication lines.
$300,000 Million Financing
Represented closely held business in structuring its credit arrangements through a credit facility with a consortium of U.S. financial institutions and handled the private placement of debt securities with an international insurance company and its affiliates.
East Coast Family Office Representation
Represented an East Coast family office operation in numerous investment-related matters, including investment in a nationwide consumer products enterprise; co-investment in a casual dining operation with locations throughout the Southwest; and and development of family office ownership and management structure.
Multibillion-Dollar Restructuring
Led a team in the complete internal ownership restructuring of seven related enterprises which manufacture and distribute retail products throughout the U.S. having an enterprise value of $3+ billion. Project included compliance with securities laws, three pre-merger filings with the Federal Trade Commission, 11 merger filings in four states, and complete restatements of organizational documents (articles of incorporation, bylaws and shareholders' agreement).
$300 Million Health Care Finance Acquisition
Represented a company in the health care finance industry acquired in a $300 million transaction.
Represented acquiring person in a $90 million acquisition in the electrical and electronic fabricated plastics industry.
$300 Million Medical Device Industry Acquisition
Represented a public company in the medical device industry acquired in a $300 million transaction.
$150 Million Consumer Food and Beverage Industry Acquisition
Represented acquired person in $150 million acquisition in the consumer food and beverage industry.
$300 Million Divestiture of Financial Services Company
Represented a private equity fund in a nearly $300 million divestiture of a financial services company specializing in the health care industry. This transaction involved lengthy negotiations with an NYSE-listed buyer expanding in the market, and allowed the fund to exceed is required rate of return on its investment.
Mark L. Stoneman, Philip G. Louis, Jr., Steven J. Foristal
Acquisitions in Health Care Field for Private Equity Fund
Represented a private equity fund in nine acquisitions of health care accounts receivables management and Medicaid eligibility companies with locations nationwide. These acquisitions involved related credit facility negotiations. As a result of these acquisitions, the private equity fund was able to achieve its roll-up strategy in the industry.
Department of Education Publishes Guidance – Limiting Bostock's Impact on Title IX
Agribusiness and Food Issues to Watch for 2021
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SCOTUS Issues Opinion on Possession of Property After a Bankruptcy Petition Filing
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Armstrong Teasdale Named 2020 Precision Partner Award Recipient by T-Kartor USA
Armstrong Teasdale has received the 2020 Precision Partner Award from client T-Kartor USA for outstanding achievement and a range of substantial contributions to the success of the company. The annual award recognizes a client...
Armstrong Teasdale Welcomes 13 Associates Nationwide
Consolidated Appropriations Act Brings Changes to PPP for Cooperatives
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TELUS is the largest telecommunications company in Western Canada, and the second largest in all of Canada. Krista Sheridan presented a case study of TELUS' journey to delivering the optimal, executive supported experience blueprint for their small business market segment.
They started asking"How can we improve" instead of just "how are we doing?"
They asked stakeholders, "What if we could help you be successful? What if we could help you understand metrics? Helped our customers stop going through a trial & error process." That got tremendous buy-in.
They constantly measured what worked and what didn't.
The team talked to stakeholders and customers, formally and informally, to find out what their issues were.
When speaking with the various stakeholders, they had to show them the WIIFM: what's in it for them. They explained why is loyalty important and how it contributes to the success of the company and all it's parts.
When the positive buzz started to happen, they spread it around to create energy and excitement about the project.
In addition to learning what made their internal teams tick, they also spent a great deal of time learning as much as they could about their customers. One innovated initiative was taking four days talking to customers ONLINE, then evaluating and prioritizing what was important to them.
Through that process, they learned how important the emotional connection is for their customers. They further realized how important it is for them to treat their customers as people.
Armed with a great deal of information, they rated each touchpoint (12 total) and ranked them in a "Triage Triangle" – which is a way to prioritize by how much impact a touchpoint has.
They were right about which touchpoints were important, but not how they were important or how to deliver.
They did not have to adjust their touchpoints to the maximum level in most cases.
They could actually increase customer satisfaction AND reduce costs. For instance, they found that 25% of interactions were desired to be online – a way to increase satisfaction for those that want that service which is less expensive for the company to manage.
Communicating their commitments and carefully managing customer's expectations throughout each interaction enhances the experience at little to no cost.
Borrow from marketers – WIIFM Rules!
Each stakeholder as his own needs and wants. Answer WIIFM to gain their support.
Customer experience improvement can be a long road – get them excited!
Build the Emotional Business Case.
Don't just think about dollars and cents.
Help stakeholders realize the outcomes of working with you.
Get others to help you sell your idea across the organization.
It's a marathon, not a sprint. A 3-legged race requires you to be in step with each stakeholder and go at the right pace.
Slow down, listen. Address concerns.
Focus on the right things, not everything.
Figure out what makes the most difference.
Realize that what's important to your customers may not always make sense to you.


The 10 Year Challenge continues to bring out more entries as the days go by.
It's been a long time since we saw such a trend take hold for so long but this 10-year challenge has really been trending for days and doesn't seem inclined to stop anytime soon.
At this point it's harder to find a celebrity yet to post their throwback pic, from Efia Odo to Lilwin to Moesha Boduong — they've all taken it.
But one post taken kudos on social media for being super inspirational is one posted by former CPP Presidential Candidate Ivor Greenstreet.
Greenstreet has shared a photo from 10 years ago when he still had use of his legs, before ending up in his current state.
"The Almighty God has been good to me too . #10yearchallenge. It ain't how hard u fall that counts, but how u get up #GRIT & #GRACE." he captioned the post which has generated so much engagement of social media.


text:
Central Witness in Trayvon Martin Trial Was Imposter, George Zimmerman Said in $100M Suit
By Luke Rosiak Published on December 5, 2019 •
The Trayvon Martin case was built on a fraud, with a key witness being swapped out with an imposter when the real witness wouldn't testify, George Zimmerman said in a lawsuit Wednesday.
The lawsuit says Martin was on the phone with his girlfriend, a vivacious 16-year-old named Brittany Diamond Eugene, when Zimmerman killed him on Feb. 26, 2012. At trial, prosecutors produced the plump, slow-spoken 18-year-old Rachel Jeantel as the girl who had crucial insight into his final moments by being on the phone with him.
The lawsuit says Eugene refused to provide the version of events used to build a narrative of racism at trial, so Jeantel, who reads at a fourth-grade level, was pressured into pretending to be "Diamond."
The lawsuit seeks $100 million and names both young women as defendants, plus Martin's parents, who it says were well aware of the swap.
Zimmerman's lawyer filed the lawsuit in state court in Florida and it also targets the state of Florida and its prosecutors, who allegedly initially falsely told the defense that Martin's cellphone was too damaged to extract its data, when it actually contained evidence damaging to their case. That includes not only evidence of the witness swap, but also texts showing Martin previously discussing gun sales and bragging of beating up a "snitch" and saying, "He aint bleed nuff 4 me, only his nosez." Prosecutors also ran out the clock by repeatedly ignoring the defense's entitlement to exculpatory evidence, the suit says.
It additionally names Benjamin Crump, the civil attorney for Martin's parents, who rode the case to fame and wrote a book about "genocide" of "colored people" despite allegedly playing a key role in a hoax.
The suit says:
Each and every one of them … instituted, ordered, commanded, conspired, and covered up the illegal substitution of Defendant Eugene, a legitimate phone witness to the days, hours and minutes before the death of Trayvon Martin with Defendant Jeantel, an imposter and fake witness, who told a made to order false storyline to prosecutors in order to cause Zimmerman to be arrested, tried, and convicted for second degree murder and sentenced to life in prison in a case that had already been investigated and closed by the Sanford, Florida police department, and after they had already exonerated Zimmerman, having concluded Zimmerman had acted in self-defense with no probable cause for arrest based on eye-witness testimony, physical evidence, and 911 audio recordings.
In the weeks after Zimmerman shot Martin, the death was met with little outcry because neighbor Jonathan Good told police he saw Martin mercilessly pummeling Zimmerman in his backyard.
That changed March 19, 2012, when Crump recorded an audio interview in which Martin's 16-year old girlfriend, "Diamond," provided a contradictory tale based on her being on the phone with him at the time of the incident. The tape precipitated the arrest of Zimmerman on April 11, 2012.
Phone records show Eugene and Martin spent hours on the phone with each other on a daily basis and exchanged loving and sexual texts, the lawsuit says.
After the shooting, Martin's girlfriend came to the home of Martin's mother, Sybrina Fulton, and provided a short letter memorializing her knowledge as the person on the phone at the time of his death. The letter was signed Diamond Eugene. Fulton did not tell prosecutors or the defense about the letter for nearly a year.
The lawsuit says Crump pressured Eugene to provide the audio statement that supported a narrative of racism, but that she refused to do the same in court.
Prosecutors initially had no contact with Eugene except through Fulton, but on April 2, 2012, they insisted on meeting her, so Fulton led them to her residence, it says. They knocked on the door and were referred to a different house, where Jeantel answered and claimed to be Diamond Eugene.
"Defendant Eugene could in no way be mistaken for Defendant Jeantel, who was 2 years older, 5 inches taller, and about 120 pounds heavier than Defendant Eugene. Defendant Fulton was alarmed and immediately called Defendant Eugene, who tweeted at about that same time at 6:27 PM 'Trayvon Martin Mom just called me' and at 6:32 PM 'She thought I was Trayvon Girlfriend, Asking Me Hella Questions,'" the lawsuit said, citing phone records and social media records.
Nevertheless, Fulton went back inside and sat next to Jeantel as she was interviewed, without telling prosecutors "what she knew, that Defendant Jeantel was not Defendant Eugene, that Defendant Jeantel was not Trayvon's girlfriend, that Defendant Jeantel was not the girl she had met with in her home on March 19, had spoken with 7 times, had texted with some 30 times, had driven back to her home at 2648 Flamingo Drive on March 19," it continued.
Jeantel was an 18-year-old ninth grader who read at a fourth-grade level, it says.
Martin's parents both lied repeatedly to cover up the swap, the lawsuit says.
Prosecutor Bernie "de la Rionda ignored the repeated false statements by Defendant Jeantel that he both knew and should have known to be false, including those which contradicted Defendant Eugene's phone records he had already obtained, and including Defendant Jeantel's statement that she was 18, even though Defendant de la Rionda knew from Defendant Crump's public statements that Defendant Eugene was 16," it said, adding:
Almost every time Defendant Jeantel made a statement he knew to be false, Defendant de la Rionda asked the question again and again in different ways until Defendant Jeantel's answer fit his narrative. By the end of the interview, Defendant Jeantel was emotionally exhausted and feeling guilty from her lying and told Defendant de la Rionda six times, almost shouting "I feel guilty" and "real guilty." When asked why she felt "real guilty," Defendant Jeantel then stated twice, shouting the second time "I ain't know about it!"
To explain the name on the letter and the changing age of the girlfriend, Jeantel testified that she initially gave a "fake name" and "fake age" and lied about why she wasn't at Martin's funeral.
The difference in the voices is stark between the initial call Crump released and the testimony provided by Jeantel, who spoke in a deep, slow mumble.
The lawsuit is based on the investigative work in a book and movie called The Trayvon Hoax by Joel Gilbert, a Hollywood director who has made entertainment films as well as conspiratorial conservative films.
The Trayvon Hoax, however, attributes its conclusions almost entirely to specific contents of cellphone data related to the criminal case, which Gilbert obtained under Florida's expansive open-records law, as well as copious social media postings by the young people involved.
Martin's dad Tracy Martin — who fathered six children with five women and spent time in prison for trafficking $1 million in cocaine — admitted to police that the cries of "help" on a 911 call came from Zimmerman, not his son, but later changed his story, the suit says.
Crump issued a statement on behalf of himself and the parents. "I have every confidence that this unfounded and reckless lawsuit will be revealed for what it is — another failed attempt to defend the indefensible and a shameless attempt to profit off the lives and grief of others," he said.
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Why the Left Ignores the Killing of a Pakistani Immigrant Uber Driver


Find your inner warrior with Sensei Beko Kaygee at the Zen Warrior retreat in Guernsey.
The channel island of Guernsey is known for its beautiful, quiet locations. That's why it's been chosen as the ideal spot for a three-day weekend of masterclasses led by international fitness and martial arts guru Sensei Beko Kaygee and local yoga company PowerYoga. Running from 24-26 March 2017, this retreat will offer you the chance to learn dynamic yoga, meditation and fitness skills, drawing on various techniques from around the world.
Sensei Beko Kaygee has been in the field of holistic health for over 22 years and adds invaluable knowledge to this one-off experience. The programme includes: Chi Kung (Qigong), a holistic series of exercises that have been passed down through generations in central China; Tribal Yoga, inspired by ancient Egyptian yoga and martial arts with rhythmic tribal movements; Capoeira Fitness, which combines dancing and combat to the beat of the Berimbau and African Drum; Tai Chi Chuan, incorporating a combination of gentle flowing exercises and some more physically demanding ones; and Dynamic Chi Yoga Flow, a programme that fuses healing Chi Kung exercises, postures, stretches and breathing techniques of yoga, and dynamic yet fluid movements from Tai Chi.
To make your trip to Guernsey even more appealing, Zen Warrior participants can benefit from special accommodation offers on the island. Moroccan-themed boutique hotel Ziggurat is offering rooms from £50 per night including breakfast. Cobo Bay Hotel, overlooking the beach on Guernsey's scenic west coast, is offering three nights priced at £198, including breakfast and chocolates, fruit and flowers in the room. St Pierre Park Hotel is offering three nights at £220 for single rooms and £270 for double or twin rooms including breakfast.
The retreat is priced at £100, which includes all eight classes (individual classes cost £15 or £20).


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redpajama_set_name: RedPajamaGithub


text:
Q: Flutter :How to update the path of the image i selected from the gallery automatically?:flutter Flutter
I used package image picker to selected image from galary and save the path of the image in firestore as String, But when the path of image change in the phone the image disapper from app and the path of image dont update in the firestore
What the Solution
Flutter

redpajama_set_name: RedPajamaStackExchange


text:
Thorn of the Rose
"O Scourge of the Sea! Though long you have stalked me, no more shall you withhold your truth from me…
"…O Scourge of the Sea! I see the curtain has parted, your true form at last revealed to me."
Keelath went out to look for the rinaani the next afternoon. He wasn't sure why, for he hadn't gone to see them since Mirium had left. When he reached their meadow, they were nowhere to be found.
Instead he sat and brooded, running over in his head ways to get more money. His father had gone over the ledgers with him that morning, demonstrating to him in no uncertain terms that they could not afford the full tribute. Despite this, Keelath was taken aback at his father's charity. Would he be angry when he learned Mirium's father was not some hero from the Kingslayer War, but an unimportant commoner?
"Military service pays well," Keelon had said at last. "It is what got us this land, after all. It's not what I would have wanted for you, for it is dirty work, too full of violence and pain for what I would wish on my sons. It would be a fitting way of making up the last of the tribute, however, and you are strong."
Keelath was considering that option again now. Though the wars had tapered off into simply going after the odd akor'mar bandit in the mountains, service would never be fully without danger. It was also take him away from Mirium, as the soldiers of Sun-on-the-Lake were often deployed for years at a time.
He considered other paths: manual labor, one of the crafts, even joining another theater troupe like Miri's. None of them paid so well.
As the sun slanted down over the trees, signaling the approach of nightfall, Keelath stood. He knew what he had to do, and he went to see his father, to ask after how he could take out a loan from the brokers in Yohon'nai, to be repaid at a later date through his service to the Council's armies.
Pages: Page 1, Page 2, Page 3, Page 4, Page 5, Page 6, Page 7, Page 8, Page 9, Page 10, Page 11, Page 12, Page 13, Page 14, Page 15, Page 16, Page 17, Page 18, Page 19
Author FoxFirePosted on June 28, 2022 December 15, 2022 Categories Long StoriesTags Sun-On-The-Lake, Avaliet, Keelon, Tyrric, Rosen, Keelath, Mirium
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When Terenure Rangers –the Club- collects annual membership subscriptions it collects data that helps it maintain its football activities and related health and safety and club development activities. The Club will only ever look for necessary information from members (and parents/guardians knowingly acting on behalf of a child under 18 years of age. Such information will most usually be player name, date of birth, contact number/email and health conditions.


Time is running out. It takes place on a world populated by deities heroes called Terminus. Guided Trail Rides. Fallen Fruit Magazine San Francisco, Austin Young) It was a 3 hour collaboration on Natoma Street, 3- 6pm with Sites Unseen , on July 29, California, The " Love" Edition is a zine made by Fallen Fruit ( David Allen Burns everyone who participated. Download fallen ride the sky. Pantheon: Rise of the Fallen is a group- focused MMORPG based on challenging gameplay and open world high fantasy.
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Saturn Sky Automobile Owner' s Manual. A trapped engineer and a desperate marauder scramble to fight an enemy with unspeakable power on its side. Visit to get the latest episodes and TV Airtimes.
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* FREE* shipping on qualifying offers. Fallen Fruit Magazine- LOVE What the world needs now is love sweet love. Unfortunately hard to navigate, this site aims to alleviate those issues , Bungie' s own Grimoire viewer can be bulky allow readers to. Easily navigate and read Bungie' s Destiny Grimoire cards.
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Transformers: The Ride 3D ( or simply Transformers: The Ride) is a 3D dark ride located at Universal Studios Singapore, Universal Studios Hollywood and Universal Studios Florida. The ride, based on the Transformers film franchise, was designed by Universal Creative, Oceaneering International, and Industrial Light & Magic. Each installation is reported to have cost US$ 100 million.
The Fallen is a fictional robot supervillain in the Transformers robot superhero franchise. According to Hasbro, he was formerly known as Megatronus, and was a multiversal singularity, meaning that while he exists across the multiverse, this is no longer the case because of an event called The Shrouding, where all multiversal singularities within the Hasbro- Takara multiversal now exist as. Fox News Jenna Lee " Happening Now" show host gives national attention to Fueled By The Fallen 9/ 11 Angel Cruisers on behalf of America' s Heroes.


The Outdoor Adventure Club's AIARE Level One Avalanche Class gives students a decision making framework to aid in planning and executing trips into avalanche terrain. The course also gives you practical experience on a full day ski tour that will help you make your own decisions when you travel in the backcountry after the class.
This three day long course is split fairly evenly between class and field time, where you'll develop knowledge of avalanches, and introduce a decision making framework to be utilized when planning and traveling in avalanche terrain. The last day is spent primarily in the field, on the perfect tour, planned and executed with the decision making framework introduced in the course.
You will practice companion rescue using an avalanche transceiver, probe and shovel during the class, but you'll also learn valuable information to help you avoid avalanches in the first place.
Course includes blue field book and student manual. Split boards and avalanche gear are available to rent.
Overnight accommodations are available in the town of Mineral, where the classroom sessions take place.
For more information and to sign up for this course, go to our website: www.outdooradventureclub.com/aiare-avalanche-classes-lassen-national-park or call 415-377-1195.


International entrepreneurship is a promising field to examine the dynamic aspect of social capital. International New Ventures (INVs) are companies that internationalize their operations rapidly and their nature is very dynamic. The purpose of this article is to examine the different roles of social capital, efficacy role, serendipity role and liability role in the international operations of seven Scottish and Greek SMEs. We argue that the different roles of social capital are not clearly demarcated in the current literature. We found that the efficacy role of social capital played a more important role in the initial stages of internationalization. However, the serendipity role of social capital was more important for companies with many structural holes that did not have the necessary network relationships to bridge them. All in all, we highlight that virtual social capital in the digital era is becoming more and more important.