Dendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.
Spontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.
In this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglandin (PG)E2 as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.
Cumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.
Dendritic cells (DCs) are bone marrow-derived leukocytes specialized in antigen presentation . They play an essential role in initiating and directing cellular and humoral immunity, including antitumor responses. Tumor production of immunosuppressive factors (cytokines, arachidonic acid metabolites, glycosphingolipids, polyamines) with detrimental effects on DC maturation and function can significantly prevent the establishment of effective antitumor immune responses . Recent evidence has indicated that induction of apoptosis in immune cells is yet another mechanism used by tumors to evade immune recognition . Indeed, several studies have demonstrated that DCs undergo apoptosis after interacting with cancer cells or tumor-derived factors in vitro [4–7]. However, these studies have used DCs generated in vitro following prolonged culture with cytokines and cytokine-driven activity may not reflect the functional status of DC populations circulating in vivo.
In vivo circulating blood DCs are identified by their high expression of HLA-DR and lack of specific lineage markers (CD3, CD14, CD19, CD20, CD56 and CD34) found on other leukocytes . DCs freshly isolated from blood offer the theoretical advantage of being in their natural state of differentiation, free from the influence of exogenous cytokines, more responsive and presumably capable of stimulating immune responses in a more physiological manner. Hence, there is active interest in using blood DCs as vectors for cancer immunotherapy, with preliminary reports confirming their clinical potential [9, 10].
Several studies, however, have demonstrated severe phenotypic and functional impairment of DCs in patients with breast cancer [11, 12]. Tumor-infiltrating DCs are neither mature nor activated [13, 14] and blood DCs express low levels of co-stimulatory molecules [11, 12] and IL-12  and exhibit an impaired capacity to stimulate T-cells [11, 12]. In this context, knowledge of the mechanisms responsible for tumor-induced DC defects in breast cancer is essential to overcome DC dysfunction and to harness their immunotherapeutic potential. Recent reports revealed spontaneous apoptosis of several subpopulations of peripheral blood mononuclear cells (PBMCs; T-cells, B-cells and monocytes) in patients with cancer [16–18]. Those findings together with the reported decreased DC function prompted us to assess the extent of spontaneous apoptosis in blood DCs from patients with breast cancer and to identify clinically available factors to protect blood DCs against tumor-induced apoptosis.
Thirteen female patients, 40 to 75 years of age, with histologically confirmed breast adenocarcinoma were enrolled in the study. All patients presented with early disease (stage I to II), were newly diagnosed and had received no prior cancer therapy. Staging was performed in accordance with the International Union Against Cancer, UICC TNM Classification . In addition, 15 healthy female donors, 24 to 73 years of age, volunteered for the study and served as controls. The Australian Red Cross Blood Service, Brisbane, provided buffy coats. The research ethics committees of both the clinical (Wesley Medical Centre and Royal Brisbane and Women Hospital) and scientific (Queensland Institute of Medical Research) institutions approved the study protocols.
The following monoclonal antibodies were used in this study: CD3, CD14, CD19, CD20, CD56, CD34, HLA-DR, CD80, CD86, tumor necrosis factor (TNF)-α and IgG1, IgG2a and IgG2b isotype controls from BD Pharmingen (BD Biosciences, San Jose, CA, USA); HLA-DR, CD40, CD83, CD19 and IgG1 isotype control from Beckman Coulter (Fullerton, CA, USA); and IL-10 and IL-12 from Caltag laboratories (Burlingame, CA, USA). All antibodies were used as the fluorescein isothiocyanate (FITC), phycoerythrin (PE), biotin, allophycocyanin (APC) or PE-Cy5 conjugate. The PE-conjugated Bcl-2 antibody reagent kit was purchased from BD Pharmingen. Complete media included RPMI 1640 supplemented with 10% fetal calf serum, penicillin (100 u/ml), streptomycin (100 μg/ml), L-glutamine (2 mM), HEPES (25 mM) and non-essential amino acids all purchased from Gibco Life Technologies (Gaithersburg, MD, USA). The combination of pro-inflammatory cytokines consisted of IL-1β (10 ng/ml), IL-6 (10 ng/ml) and TNF-α (10 ng/ml) obtained from R&D systems (Minneapolis, MN, USA) plus Prostaglandin E2 (PGE2, 1 μg/ml) from Sigma (St Louis, MI, USA). Double stranded RNA (poly I:C, 50 μg/ml)  was purchased from Sigma; IL-12 (100 ng/ml) was purchased from Mabtech (Stockholm, Sweden) and soluble human recombinant CD40-ligand (CD40L; 2 μg/ml) was kindly provided by Amgen (Seattle, WA, USA).
Venous blood obtained from patients and volunteers was collected in heparinized tubes. Samples were processed and PBMCs recovered by Ficoll-Hypaque centrifugation. To determine the proportion of apoptotic cells, Annexin-V binding and TUNEL assays were performed after blood collection. In all experiments, each patient was tested in parallel with at least one healthy volunteer. Annexin-V binding assays were performed using the Annexin-V kit (BD Pharmingen). Briefly, PBMCs were adjusted to 106 cells/ml and stained with a mixture of lineage markers (CD3, CD14, CD19, CD20, CD56, CD34) and HLA-DR for 20 minutes at 4°C. CD34 was added to the lineage mixture to exclude circulating hematopoietic stem cells. Cells were washed and resuspended in binding buffer prior to incubating with Annexin-V and 7-Aminoactinomycin D (7-AAD) for 15 minutes at room temperature. Samples were analyzed by fluorescence activated cell sorting (FACS) within one hour of staining. The TUNEL assay was performed using the Apo-BrDU-Red DNA fragmentation assay kit (BioVision, Mountain View, CA, USA). As above, PBMCs were adjusted to 106 cells/ml, stained for surface markers, fixed with 1% (w/v) paraformaldehyde and resuspended in ice-cold 70% ethanol for 30 minutes prior to incubation with DNA labeling and antibody solutions according to the manufacturer's instructions. Samples were analyzed within one hour of staining. In all experiments, 5 to 10 × 105 events were collected within the mononuclear cell gate. Data were acquired on a FACS Calibur flow cytometer and analyzed using CellQuest 3.1 (BD Bioscience), FloJo (TreeStar, San Carlos, CA, USA) or Summit (DakoCytomation, Fort Collins, CO, USA) software.
The breast cancer cell lines MCF7, MB435 and SKBR3 were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). The MA11 line was a generous gift of Dr Phil Rye (Norwegian Radium Hospital, Oslo, Norway). Tumor-derived supernatants (TDSN) were prepared by seeding 107 tumor cells in 20 ml of complete medium and incubating for 72 h. Before passage, culture supernatants were collected, centrifuged to remove cells, dialyzed against fresh medium (24 to 48 h at 4°C in the dark) to replenish nutrients and stored at -20°C. Dialysis membranes (membra-cell, Polylabo, Strasborg, France) with a molecular weight cut-off of 10,000 to 14,000 were used. For each experiment, PBMCs were cultured (107 cells/ml) for 24 h in the presence of 50% (v/v) TDSN or PBMC-conditioned supernatant (PBMC-SN) as a control. Cells were harvested, washed and apoptosis in lineage negative HLA-DR positive (Lineage (Lin)-HLA-DR+) cells determined using Annexin-V binding and TUNEL assays. For morphological analysis following culture with TDSN, viable (7-AAD negative) Lin-HLA-DR+ cells were sorted (99% purity) using a MoFlo Sorter (DakoCytomation), resuspended in complete medium and cyto-centrifuged. Histology was performed on cytospin preparations made by seeding 2 to 3 × 104 sorted cells onto a glass slide. These were air-dried and stained using May-Grunwald-Giemsa.
Four-color flow cytometry was used to analyze the phenotype and cytokine secretion of Lin-HLA-DR+ cells. Briefly, PBMCs were cultured (107 cells/ml) in 6-well plates for 18 to 36 h in complete medium in the presence of inflammatory cytokines (a cytokine cocktail (CC) containing IL-1β (10 ng/ml), IL-6 (10 ng/ml), TNF-α (10 ng/ml) plus PGE2 (1 μg/ml)), poly I:C (50 μg/ml) or CD40L (2 μg/ml) and subsequently stained for flow cytometric analysis. Doses and incubation times were optimized in preliminary experiments. For cytokine secretion, 107 PBMCs were cultured with the CC, poly I:C or CD40L (in addition to IFN-γ and IL-1β) in the presence of brefeldin-A (10 μg/ml; Sigma). Cells were stained for surface markers, fixed with 1% w/v paraformaldehyde and stained with cytokine-specific monoclonal antibodies (TNF-α, IL-10 and IL-12) in 0.2% w/v saponin/PBS at 4°C overnight. For determination of Bcl-2 expression, 106 PBMCs were stained for surface markers (CD3, CD14, CD19, CD20, CD56, CD34 and HLA-DR), fixed with 1% w/v paraformaldehyde and stained with anti-Bcl-2 or isotype control in 0.2% w/v saponin/PBS at 4°C overnight. In all experiments, 5 to 10 × 105 events were collected within the mononuclear cell gate. Data were acquired on a FACS Calibur flow cytometer and analyzed using CellQuest 3.1 (BD Bioscience), FloJo (TreeStar) or Summit (Cytomation) software.
Comparisons of samples to establish statistical significance were determined by the two tailed Students' t-test or one way analysis of variance (ANOVA) followed by Bonferroni's comparison test. Results were considered to be statistically significant when the p-value was <0.05.
In accordance with the published literature [11–14], we also confirmed that there is significant DC dysfunction in patients with breast cancer prior to therapy (Additional file 1). Given that apoptotic DCs are ineffective at inducing immunity, here we examined the presence of apoptotic DCs obtained from patients with breast cancer (stage I to II; n = 13) compared to age-matched healthy females (n = 15). In order to include all cells undergoing apoptosis, gating was set as described in Fig. 1a. This strategy of double gating confirmed the viability of all cells while eliminating acellular debris. Blood DCs were identified as Lin-HLA-DR+ cells and apoptosis was assessed using Annexin-V and TUNEL assays. The minimal proportion of spontaneously apoptotic blood DCs in healthy volunteers was significantly increased in patients with breast cancer (p < 0.05 and p < 0.01, respectively) (Fig. 1b,c).
Spontaneous apoptosis of blood dendritic cells (DCs) in patients with breast cancer. Peripheral blood mononuclear cells isolated from patients with breast cancer (stage I to II; n = 13) and age-matched healthy volunteers (n = 15) were analyzed for apoptosis by flow cytometry. (a)In order to include all apoptotic cells and avoid debris, gating was set to include only 7-AAD negative events (R1) which were then visualized and re-gated on forward (FSC) and side scatter (SSC) characteristics (R2). Within this mononuclear cell population, blood DCs were identified as lineage negative HLA-DR positive cells (R3). Representative dot plots are shown. (b)Apoptosis in blood DCs from patients (black) and healthy volunteers (grey) was determined by two different methods, Annexin-V binding and TUNEL assays. In all experiments, each patient was tested in parallel with at least one healthy volunteer. Representative histograms are shown. (c)Summary of apoptosis data. The proportions of Annexin-V+ and TUNEL+ blood DCs estimated for all patients (black) and healthy volunteers (grey) are shown accordingly. Error bars indicate the standard error of the mean. Statistically significant differences between controls and patients are indicated as: *, p < 0.05; **, p < 0.01.
Given that significantly less apoptosis was observed in samples from healthy volunteers, we hypothesized that tumor products were responsible for the elevated proportion of apoptotic blood DCs in patients with breast cancer. To more accurately examine this hypothesis, we used an in vitro model of blood DC culture in the absence of exogenous cytokines . For this purpose, PBMCs were incubated in the presence of TDSN and apoptosis measured in Lin-HLA-DR+ cells. Incubation with PBMC-SN served as a control. All supernatants were filtered and dialyzed against fresh medium prior to use. This approach excluded the possibility of apoptosis induced by nutrient depletion. Analysis of Annexin-V binding and TUNEL assays in different cultures revealed minimal apoptosis on fresh samples and less than 10% apoptosis following culture with PBMC-SN (Fig. 2a,b). On the other hand, incubation with TDSN resulted in a significant (p < 0.05) increase in the proportion of apoptotic Lin-HLA-DR+ cells. Moreover, morphological evaluation frequently demonstrated features of apoptotic death in cultures containing TDSN (Fig. 2c). These data suggest that factors derived from breast cancer cell lines induce apoptotic death of Lin-HLA-DR+ cells.
Apoptosis in blood dendritic cells (DCs) is induced by breast tumor-derived supernatants. (a)Apoptosis in blood DCs was determined by Annexin-V binding and TUNEL assays following culture (24 h) in the presence of 50% (v/v) peripheral blood mononuclear cell (PBMC)-derived supernatant (PBMC-SN) or tumor-derived supernatants (TDSN; MCF7, MB435, MA11 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. (b)Summary of apoptosis data. The proportions of Annexin-V+ and TUNEL+ blood DCs represent the average ± standard error of the mean of five independent experiments for which statistically significant differences compared to PBMC-SN are shown: *, p < 0.05; **, p < 0.01; ***, p < 0.001. (c)For morphological analyses (original magnification times 1,000), 50 cells were assessed in three separate fields. In contrast to PBMC-SN, cultures incubated with TDSN (MA11) frequently demonstrated features of apoptotic death in blood DCs (reduction in cell volume and nuclear size, cytoplasmic blebbing, cell membrane convolutions and formation of apoptotic bodies). Three independent experiments with similar findings were performed.
It has recently been demonstrated that protection of DCs from apoptosis can improve antitumor immunity in vivo . Therefore, the identification of factors that protect blood DCs against tumor-induced apoptosis could enhance their potential for immunotherapy. Analysis of Annexin-V binding revealed that pre-incubation with a CC did not decrease apoptosis in blood DCs whereas poly I:C stimulation induced a modest (not significant) reduction in apoptosis (Fig. 3). In contrast, incubation with CD40L demonstrated a significant (p < 0.05) decrease of TDSN-induced apoptosis to levels seen in control cultures. These data show that CD40 stimulation leads to increased resistance of blood DCs to TDSN-induced apoptosis.
CD40 stimulation protects blood dendritic cells (DCs) against tumor-derived supernatant (TDSN)-induced apoptosis. TDSN-induced apoptosis in blood DCs was determined by Annexin-V binding following incubation (24 h) in the presence or absence of: (a)a combination of inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglanding (PG)E2 as a cytokine cocktail (CC)); (b)synthetic double-stranded RNA (poly I:C); or (c)CD40 ligand (CD40L) prior to culture (24 h) with 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11, MB435, MCF7 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. Five independent experiments were performed for which p-values are indicated.
Due to the differential effects of inflammatory mediators on DC apoptosis, we assessed the functional maturation induced on Lin-HLA-DR+ cells following stimulation with a CC, poly I:C or CD40L. All maturation stimuli induced significant increases in the expression of HLA-DR and co-stimulatory molecules (CD40, CD80, CD83 and CD86) (Fig. 4a). The cytokine secretion profile, however, was markedly different between stimuli. While the CC induced minimal cytokine secretion, poly I:C induced moderate secretion of TNF-α as well as IL-12. On the other hand, CD40 ligation induced modest secretion of TNF-α and robust production of IL-12. Given that IL-12 has been reported to protect in vitro-derived DCs from apoptosis induced by co-culture with prostate cancer cells , we examined whether IL-12 could protect blood DCs from apoptosis induced by breast tumor-derived supernatants. Exogenous IL-12 significantly (p < 0.05) reduced blood DC apoptosis (Fig. 4b), indicating that IL-12 also has a protective role against tumor-induced apoptosis.
Protective effect of IL-12 on tumor-derived supernatant (TDSN)-induced apoptosis. (a)Expression of HLA-DR and co-stimulatory molecules (CD40, CD80, CD83 and CD86) as well as secretion of cytokines (tumor necrosis factor (TNF)-α, IL-10 and IL-12) was determined for blood DCs following stimulation (24 h) with a cytokine cocktail (CC), synthetic double-stranded RNA (poly I:C) or CD40 ligand (CD40L) as described in Materials and methods. Histograms indicate expression in the absence (shaded) or presence (non-shaded) of stimulation. Numbers indicate delta mean fluorescence intensity (ΔMFI, stimulated cells minus unstimulated cells) and are representative of five independent experiments. (b)TDSN-induced apoptosis in blood DCs was determined by Annexin-V binding following incubation (24 h) in the presence or absence of exogenous IL-12 prior to culture (24 h) with 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11, MB435, MCF7 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. Five independent experiments were performed for which the p-value is indicated.
Given that members of the Bcl-2 family of proteins are involved in the regulation of tumor-induced apoptosis in DCs derived in vitro , we examined whether protection from TDSN-induced apoptosis could be mediated by modulation of the expression of the anti-apoptotic protein Bcl-2. We found that incubation of cells with TDSN, but not PBMC-SN, resulted in significant (p < 0.05) down-regulation of Bcl-2 expression in blood DCs (Fig. 5a,b). Interestingly, pre-incubation of cells with the CC or poly I:C did not significantly change the reduction of Bcl-2 expression caused by tumor supernatants (Fig. 5b). In contrast, pre-incubation with IL-12 or CD40L resulted in sustained expression of Bcl-2, abrogating the suppressive effect induced by the TDSN (Fig. 5b). In fact, comparable levels of Bcl-2 expression were observed on fresh samples or following culture with CD40L (Fig. 5b). These data suggest loss of Bcl-2 is involved in blood DC apoptosis induced by TDSN and that protection from TDSN-induced apoptosis (as shown for CD40L and IL-12) involves sustained expression of this molecule.
Effect of tumor-derived supernatant (TDSN) and stimulation on expression of Bcl-2. (a)Expression of Bcl-2 in blood dendritic cells (DCs) was determined: ex vivo (Fresh); following culture (24 h) in the presence of 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11); or following stimulation (24 h) with a cytokine cocktail (CC), synthetic double-stranded RNA (poly I:C), IL-12 or CD40 ligand (CD40L) prior (24 h) to culture with 50% (v/v) TDSN. Representative histograms (filled) are shown with numbers indicating the mean fluorescence intensity (MFI) of Bcl-2 expression. The unfilled histogram shows staining with isotype control. (b)Summary of Bcl-2 expression data. Results show the average ± standard error of the mean of five independent experiments for which statistically significant differences compared to fresh cells (*, p < 0.05; ***, p < 0.001) or after incubation with TDSN (##, p < 0.01; ###, p < 0.001) are indicated.
In this study, we document the presence of a significantly higher percentage of apoptotic blood DCs in patients with breast cancer compared to healthy volunteers, suggesting that in these patients, a higher proportion of blood DCs are programmed in vivo to undergo apoptosis. This phenomenon appeared to be related to their cancer as all patients were newly diagnosed (no prior therapy) and no comparable apoptosis was observed in blood DCs from healthy volunteers. Although previous reports have described increased apoptosis in tumor-infiltrating DCs in patients with melanoma and ovarian cancer , our findings clearly indicate that the inhibitory influence of the tumor extends far beyond the tumor microenvironment. In keeping with this, previous studies [16, 17] have described spontaneous apoptosis of several mononuclear cell subsets (T and B lymphocytes, NK cells and monocytes) in blood of patients with different types of cancer, suggesting a rather generalized phenomenon. This, however, is the first description of spontaneous apoptosis in DCs from the peripheral blood of patients with cancer.
The physiological and clinical significance of blood DC apoptosis in patients with cancer is of substantial interest. Circulating DCs are essential for adequate immunity given that they continually replenish the pool of tissue-residing DCs and play a critical role in shaping immune responses in vivo . Indeed, most circulating DCs appear to be en route from the bone marrow to peripheral and lymphoid tissues or from non-lymphoid tissues to the regional lymph nodes and spleen [24, 25]. Given that apoptotic cells are rapidly cleared from the circulation by the reticulo-endothelial system, our observation of a higher fraction of blood DCs undergoing apoptosis in patients with breast cancer suggests increased turnover of these cells in vivo. If this assumption is correct, continual efforts to replace the pool of blood DCs from bone marrow would impose chronic stress on the immune system of breast cancer patients, resulting in a relative paucity of DCs in the circulation [15, 26] as well as a failure to effectively replenish DCs that infiltrate breast tumor tissue [13, 14] or in the ability of DC to migrate to lymphoid organs  for the initiation of T-cell immunity. Accordingly, in patients with operable breast carcinoma, blood DC numbers are significantly reduced over prolonged periods of time (approximately 48 weeks post surgery) independently of other blood cell counts (monocytes, neutrophils, platelets), suggesting diminished availability of DC precursors in these patients (A Pinzon-Charry et al, unpublished observations). Moreover, in a cohort of 35 patients with early (stage I and II) and advanced (stage IV) breast cancer (stage I, n = 17; stage II, n = 10; stage IV, n = 8), we found that reduction in blood DC numbers correlated with disease progression . The resulting immune dysfunction would lead to reduced antitumor immunity  and, thus, tumor progression.
The increased pro-apoptotic effect of tumor supernatants demonstrated in this work may have a differential effect on the various DC subsets. In a separate study, we have carefully examined the DC compartment for various immune functions. We identified a population of Lin-HLADR+ cells that is CD11c and CD123 negative that appears to be particularly resistant to apoptosis induced by supernatants derived from breast (MB231, MA11, MB435, SKBR3 and MCF7) as well as colon (LOVO) cancer cell lines. In contrast, Lin-HLA-DR+CD11c+CD123+ DCs consistently undergo increased levels of apoptosis under the same conditions .
We therefore set out to directly confirm the role of tumor products in the induction of blood DC apoptosis in breast cancer. We found that supernatants derived from several breast cancer lines significantly reduced blood DC survival as assessed by Annexin-V, TUNEL and morphological analyses. Our findings on blood DCs confirm previous studies on in vitro generated monocyte-derived DCs wherein tumor products (IL-10, prostanoids, gangliosides or ceramides) induced marked levels of apoptosis [4, 5, 29]. Moreover, in view of the increased level of apoptosis in circulating DCs in patients with cancer described here, it is tempting to speculate that pro-apoptotic tumor products that regularly gain access to the peripheral circulation at high concentrations, such as IL-10 and gangliosides [30, 31], could potentially impair viability of blood DCs in vivo .
From an immunotherapy perspective, our results are relevant in two ways. Firstly, because blood DCs have been proposed for use in cancer immunotherapy . Blood DCs offer the theoretical advantage of being in their natural state of differentiation and presumably capable of stimulating immune responses in a more physiological manner. Apoptotic DCs, however, are ineffective at inducing immunity , which may explain, at least in part, the failure of blood DCs from breast cancer patients to capture antigens and generate adequate T-cell responses as described by us (Additional file 1) and others [11, 12]. Secondly, because therapeutic DCs would be subject to suppression when re-introduced into patients and, thus, re-exposed to pro-apoptotic products derived from the tumor. In this regard, our results demonstrate that the addition of specific maturation stimuli can protect blood DCs from tumor-induced apoptosis, thus facilitating their survival and potential effectiveness.
Indeed, by comparing a range of stimuli available for clinical use, including inflammatory cytokines (CC), poly I:C and soluble CD40L, we found that the CC and poly I:C induced robust phenotypic maturation, but failed to protect blood DCs from apoptosis. These results imply that the upregulation of maturation and costimulatory molecules may have only minimal effect on DC survival. In contrast, CD40 stimulation induced strong phenotypic maturation, in addition to augmented IL-12 secretion and protected blood DCs from TDSN-induced apoptosis through sustained expression of the anti-apoptotic molecule Bcl-2. Similarly, exogenous IL-12 protected blood DCs from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-induced protection could be mediated, at least in part, through IL-12 secretion.
Together with Toll-like receptor (TLR) interactions, DC survival induced by CD40L appears to be mediated by the activation of NF-κB transcription factor proteins . In a mouse model, the beneficial effect of CD40 ligation has been related to the function of the anti-apoptotic protein Bcl-2, which counter-balances the apoptotic property of various DC maturation stimuli . Our data support and advance this idea in that we identify IL-12 as playing a similar protective role. Further studies on the mechanisms involved in this process will provide better understanding of the homeostatic function of Bcl-2 in DC survival. Interestingly, TRANCE/RANKL, another modulator of NF-κB, has been implicated in prolonged DC survival , particularly after stimulation with CD40L . A recent report correlated a high expression of RANKL in breast tumor cells with a decreased metastatic (bone) phenotype . Considering our data, it is tempting to speculate that the joint effect of RANKL with CD40L leading to prolonged DC survival may prevent tumor growth; an additional argument for the potential benefit of CD40L linked with DC immunotherapy for breast cancer.
In models in which DCs are generated in vitro, CD40 stimulation can induce increased DC survival and IL-12 secretion , thus promoting IFN-γ production by T-helper cells  as well as tumor-specific cytotoxic responses . These data strongly support the use of CD40L conditioning for DCs in cancer immunotherapy. Hitherto, one study has shown the potential of CD40L as an efficient stimulator of professional APC under clinically applicable conditions  and two ongoing cancer trials with CD40L-conditioned DCs  are awaiting completion. Our results demonstrate that ex vivo conditioning of blood DCs with CD40L can protect them from tumor-induced apoptosis and, thus, further support this approach.
Our data demonstrate that in the peripheral circulation, more blood DCs are undergoing apoptosis in patients with breast cancer than in healthy donors. Given that no significant apoptosis was detected in samples from healthy donors, we propose that these cells are programmed in vivo to undergo apoptosis by a mechanism related to the presence of tumor products. Indeed, supernatants from breast cancer cell lines induce significant apoptosis of blood DCs in vitro. Moreover, we show that loss of expression of the anti-apoptotic molecule Bcl-2 is involved in apoptosis induced by breast cancer cell line supernatants and demonstrate that exogenous conditioning with CD40L (and IL-12) protects blood DCs from apoptosis through sustained expression of Bcl-2. Cumulatively, our findings support the use of exogenous conditioning of DCs to ensure their survival and, thus, may prove crucial in improving the efficacy of DC-based immunotherapies for cancer.
This work was funded by the National Breast Cancer Foundation, Australia. APC had been supported by the University of Queensland International Postgraduate Research and the Paul Mackay Bolton Cancer Research Scholarships. The authors are grateful to Grace Chojnowski and Paula Hall for technical assistance with FACS and MoFlo-sorting. We also thank the Australian Red Cross Blood Service and Sullivan Nicolaides Pathology Laboratories for blood samples and logistic assistance. We are grateful to Amgen for kindly providing CD40L and mostly to our patients and volunteers without whom this study would not have been possible.
AP-C and JAL conceived and designed the study. AP-C and TM performed the laboratory studies. AP-C, CF and JAL enrolled the patients. AP-C, MAM, CS and JAL analyzed the data. All authors contributed to writing the paper.


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NYC magician Oz Pearlman wins Long Island Marathon
Men's marathon winner Oz Pearlman, of Manhattan, crosses the finish line on Sunday, May 3, 2015 at Eisenhower Park in East Meadow. Credit: Joseph D. Sullivan
By LAURA ALBANESE laura.albanese@newsday.com @AlbaneseLaura Updated May 3, 2015 8:43 PM
All smoke, no mirrors: The marathon magician made the competition disappear.
Oz Pearlman, a New York City-based magician and mentalist, led a pack of about 960 runners to win the Long Island Marathon on a sun-soaked day Sunday-- a year after coming in second place in the race and two weeks after competing in the Boston Marathon.
Pearlman, 32, who crossed the finish line at Eisenhower Park in East Meadow in 2 hours, 25 minutes, 25 seconds, beat out second-place finisher Jerry Pannullo by more than 20 minutes. Pannullo, 45, of East Islip, came in at 2:46:51 and Brooklyn's Viktor Chistyakov, 36, was third at 2:53:22.
Look up LI Marathon results and photos
It's no wonder the gap was so big between Pearlman and the rest of the pack -- at times, this magician seemed to all but saw the course in half. He's won about 15 marathons since he first started competing in 2004, he said, and halfway through this one, he was close to the leaders of those running the half marathon.
"I just went fast," he said. "[It was] hot, hot. Definitely the heat was a factor."
Pearlman said his first marathon was the Philadelphia Marathon. He ran it in a little over 3:30:00 and it made him miserable, he said. So he did it again.
"It was a brutal experience -- there was some crying, some walking," he said. "There was no crying today . . . I was like, a glutton for punishment. I said, I'm doing another one and so it began. It's an addiction."
This passion took him and his fellow runners from the starting point on Charles Lindbergh Boulevard, past Mitchel Athletic Complex and through the grueling stretch up Wantagh Parkway.
Race director Jason Lipset estimated that, including half marathoners and 10K runners, close to 6,000 runners participated overall.
Kelly Gillen, 32, of Manhattan, led the women in 3:03:57. A Manhasset native who recently received her doctorate in breast cancer research from Cornell University, she said she has been running in and around Eisenhower Park for years. Gillen ran for Manhasset Middle School before moving to Westchester and her first races were in this park, she said.
Magician survives heat, wins LI Marathon
"I was in fourth place at Mile 12 and I just tried to find the next girl in front of me and pass her, and [then] the next," said Gillen, who also won in 2013. "I had family at different points in the course and they surprised me and that gave me a little extra energy . . . I thought they'd only be at the finish line."
Gillen was followed by Jessica Ramsay, 37, of Hampton Bays, who finished in 3:08:57. Sayville's Nadine Moors was third in 3:12:35.
Ramsay, an art teacher in the Sachem school district, ran her first marathon since having her youngest daughter in 2010.
"I've been running a lot with the stroller," she said. "It was nice to just go out today and run without a stroller."
Both Pearlman and Ramsay have competed in more marathons than they can remember, they said.
And though there are few things that beat the high of running through the finish line, a little flair is nice, too. That last counts double when, like Pearlman, you're a professional showman.
When he crossed the final threshold, he held his hands over his head in the manner common to marathon winners everywhere . . . except one hand suddenly clutched a fanned deck of cards.
Were they up his sleeve? (Well, no; Pearlman ran shirtless.) So where did they come from, anyway?
"Oh, I can't tell you," he said, grinning. "It's a trade secret."
By LAURA ALBANESE laura.albanese@newsday.com	@AlbaneseLaura
Laura Albanese is a general assignment sports reporter; she began at Newsday in 2007 as an intern.
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redpajama_set_name: RedPajamaCommonCrawl


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Бе́тти Э́двардс (; род. 1926, Сан-Франциско, США) — американский преподаватель искусства, доктор наук, основоположник методики правополушарного рисования, автор бестселлеров «Художник внутри вас» и «Раскройте в себе художника».

Биография
Родилась в 1926 году в городе Сан-Франциско штата Калифорния.

Поступила в Калифорнийский государственный университет на факультет искусств, который окончила в 1947 году со степенью бакалавра искусства. Там же, в 1976 году получила степень магистра и доктора наук в области искусства, образования и психологии.

Обучала слушателей в Высшей школе «Венис» в Лос-Анджелесе, затем в местном колледже. С 1987 и по 1991 год работала преподавателем в Калифорнийском университете, обучая студентов рисунку, живописи, истории искусств, арт-подготовке преподавателей и теории цвета. После выхода на пенсию основала центр образовательных программ, изучающих полушария головного мозга.

В разные годы деятельности проводила профессиональные семинары по всему миру, а также занималась бизнес-консультированием крупных национальных и международных корпораций по вопросам решения различных творческих проблем.

Книга «Откройте в себе художника» стала бестселлером, выпущенным на разных языках мира включая английский, французский, испанский, немецкий, польский, венгерский, русский и японский языки. Сегодня на её основе преподаются уроки изобразительного искусства во многих школах мира, а также многочисленные курсы по рисованию и пластике для взрослых.

Один из её учеников Стив Лестер живёт и работает в Москве, Санкт-Петербурге и Нижнем Новгороде, обучая по методу Бетти Эдвардс скульптурной пластике и правополушарному рисованию.

Теория правополушарного рисования
Концепция метода основана на работах Роджера Сперри касающихся функциональной специализации полушарий головного мозга. Благодаря физическому разделению мозга на два полушария он использует два способа переработки реальности. Один из них использует аналитический и вербальный режим мышления — им перерабатываются речь, звуки, математические вычисления и алгоритмы. Второй использует образный и перцепционный режим — он отвечает за восприятие цвета, сравнение размеров и перспективы предметов, видя их целиком «как есть». Впоследствии, в работах Бетти эти режимы получили названия «Л-режима» и «П-режима» соответственно.

Суть метода правополушарного рисования заключается во временном подавлении работы левого полушария и передачу ведущей роли в рисовании правому, более пригодному для этой деятельности. При работе игнорируются мнения из памяти о том, как объект должен выглядеть. Вместо этого художник «видит объект на самом деле» — сравнивая его размер в целом, размер отдельных элементов, отношение пространства, света и тени и объединении всего этого в единую картину. Для этого используется ряд упражнений, облегчающих переход начинающего художника в «П» режим.

Иногда метод правополушарного рисования ошибочно путают с рисованием преимущественно левой рукой и рисованием только перевёрнутых предметов. В действительности, левши и правши могут использовать наиболее развитую из своих рук, а рисование перевёрнутых картин является только тренировочными занятиями, проходящими на первоначальных этапах обучения.

Согласно методу Бетти Эдвард рисование состоит из 5 базовых навыков, и двух тренировочных.

Базовые навыки:
 Восприятие краёв
 Восприятие пространства
 Восприятие соотношений
 Восприятие света и тени
 Восприятие целостного образа или гештальта

Тренировочные навыки:
 Рисование по памяти
 Рисование при помощи воображения

Метод Эдвардс, впервые опубликованный в 1979 году быстро стал революционным и получив положительную оценку среди признанных художников и преподавателей, которые немедленно взяли его на вооружение.

Семья
У Бетти Эдвардс есть сын, дочь и двое внуков. В настоящее время она живёт и работает в Сан-Диего в Калифорнии. В её интересы, помимо живописи входят садоводство, кулинария и чтение.

Книги
 «Художник внутри вас» (1979), переиздания в 1989, 1999 и 2008 годах.
 «Откройте в себе художника» (2000), переиздания в 2004 и 2009 годах.
 «Цвет: искусство смешивания цветов» (2004).

См. также
 Негативное пространство

Примечания

Ссылки
 Официальный сайт Бетти Эдвардс
 Официальная группа на Facebook

Родившиеся в Сан-Франциско
Художники США

redpajama_set_name: RedPajamaWikipedia


text:
TRUMP-TIONARY
The real definitions of "globalist" and "nationalist"
A nationalist in Texas.
Image: AP Photo/Evan Vucci
Ana Campoy
Donald Trump had a vocabulary lesson for the thousands of supporters who gathered at a Houston rally on Monday (Oct. 22.) for Ted Cruz, Texas's candidate for the US Senate. He defined two terms: globalist and nationalist.
America is winning again. America is respected again because we are putting America first. We're putting America first. It hasn't happened in a lot of decades. We're putting them first. We're taking care of ourselves for a change folks… But radical Democrats want to turn back the clock… for the rule of corrupt, power-hungry, globalists. You know what a globalist is? You know what a globalist is? A globalist is a person that wants the globe to do well, frankly, not caring about our country so much.
And you know what, we can't have that. You know they have a word. It sort of became old-fashioned. It's called a nationalist, and I say really, we're not supposed to use that word. You know what I am? I'm a nationalist, okay? I'm a nationalist. Nationalist. Nothing wrong. Use that word. Use that word.
The crowd responded with an enthusiastic chant of "USA, USA, USA, USA, USA, USA."
But there's a lot more behind those words than the simple definitions provided by Trump. Here's a more in-depth look at their meaning.
On the surface, the term could be taken as a stand-in for "globalization supporter." Its origin, however, is more problematic.
It can be tracked back the Second World War. Though it was first used by a historian to describe Adolf Hitler's expansionist ambitions, "globalist" soon started acquiring anti-Semitic connotations, according to The Atlantic. For example, in 1943, an isolationist Republican politician used"globalist" to describe colleagues who were advocating for taking in European refugees; his speech was later published in full by a group that sought to "preserve America as a Christian Nation being conscious of the fact that there is a highly organized campaign to substitute Jewish tradition for Christian tradition."
The terms retains some of its ant-Semitic roots, experts say. Here's former Ku Klux Klan leader David Duke, responding to a tweet by a Trump critic:
"Globalist" is also often used to describe George Soros, a figure despised by both white supremacists and the alt-right. He is Jewish.
Like all dog-whistling, the term is intentionally vague. However, the word's history is intertwined with conspiracy theories that have accused Jews of plotting to take over the world since the early 1900s. (Whether Trump is familiar with that history or not, he and other current and former members of his cabinet have called Jewish members of the team, such as former National Economic Council director Gary Cohn and senior advisor Jared Kushner, globalists.)
Alt-right commentators such as Alex Jones—who calls himself "Globalist Enemy Number One"—have expanded the globalist definition to rail against LGTBI rights, Hollywood, China, and pretty much anyone they don't agree with.
"Nationalist"
The standard definition of nationalism is "loyalty and devotion to a nation." It sounds like patriotism, which is "love for or devotion to one's country," per Merriam Webster.
But the definition of nationalism also includes "exalting one nation above all others and placing primary emphasis on promotion of its culture and interests as opposed to those of other nations or supranational groups." This exclusionary aspect is not shared by patriotism.
Nationalism takes a more discriminatory tone when Trump positions nationalism as the opposite of the anti-Semitic "globalist." Add "white" to "nationalism," and you get the name of a movement that openly promotes the idea that one race is superior to others.
After taking a deep dive into what the term means politically—including by reading George Orwell's Notes on Nationalism—the Cato Institute's Alex Nowrasteth explained the difference between the two:
Patriotism is love of country while nationalism is love of country combined with dislike of other countries, their peoples, or their cultures. Nationalism also extends to dislike of fellow citizens who are different, which is why nationalists frequently support nation-building campaigns of government schooling to assimilate citizens to a state-determined norm, national languages, and other means of creating ethnic, religious, or other forms of uniformity.
Nowrasteth's also found that nationalism can lead well beyond discrimination—to imperialism, for example, and mass killings. After communist regimes, nationalist governments like Chiang Kai-shek's Nationalist government in China and the Young Turk's movement in Turkey have killed the most people, according to his research.


After studying this publication, you'll come to appreciate the facility of occasion at paintings and in lifestyles. via turning in your GPS (Gratitude, Play, and Surprise), you are going to interact staff with a view to result in better productiveness and group functionality. Use this e-book as a source to convey extra pleasure, spontaneity, enjoyable, and playfulness for your office. prepare to create a happier, more fit office as you have a good time all that's good!
Six years on from Queensland's tragic 'inland tsunami', this re-creation of The Torrent reconnects with the survivors on the center of the disaster. On January 10, 2011, after weeks of heavy rain and as floodwaters started to weigh down a lot of southeast Queensland, a 'wall of water' hit Toowoomba and the Lockyer Valley.
The athlete is a secret to many and the adventure athletes come upon contain a couple of advanced occasions that through the years may end up in limitless good fortune in and out of doors of the wearing surroundings. in spite of the fact that being an athlete additionally brings on advanced concerns and calls for a different set of private improvement prone particularly constructed and meant for the athlete.
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Genuine mountain landscape right outside the door, South Tyrolean hospitality inside the hotel.
We, the hosts of the 4-star Wellbeing Hotel Watles, want to guarantee a relaxing holiday in a stylish and cosy atmosphere. Your stay amidst the splendid nature in a beautiful panoramic setting will become an unforgettable experience!
The family-operated Panorama Hotel in Vinschgau awaits you with South Tyrolean hospitality in the newly designed reception area. Enjoy our culinary delights in the sun-flooded panoramic dining room and in the cosy Tyrolean, wood-panelled "Stube" room. You will be spoiled with regional delights with international influence.
The tastefully furnished rooms & suites, some of which have been recently renovated, combine modern and alpine design. Our spacious and modern family suites with separate parents and children's room offer plenty of space for recreation for the whole family.
The Watles Wellness Hotel offers relaxation for body and soul on more than 1000 m²: you will be able to relax in the spacious indoor pool with massage benches, in the sauna area, in the light-flooded relaxation areas or in the big garden with rattan furniture.
We also look after your beauty! In the spacious beauty area with 6 treatment rooms you will be spoiled at the highest level with natural products. We also offer a professional and guided sports and activity programme amidst an unspoilt natural landscape.
Enjoy your stay in our hotel. We are looking forward to welcoming you!


Although the panel is the most important piece of electrical equipment in your home, it is often overlooked. Panels are like the brain of your home -they help ensure the safe operation of electrical throughout your home. They are built to be reliable and last. But even panels get old and worn out.
Regular maintenance on your panel can help the panel last longer, prevent future problems and quickly address any issues that may arise. Replacing a panel is quite the undertaking – but for older homes a panel upgrade may be necessary to ensure the safety of your home. Having the proper protection such as grounding and arc fault breakers will make your home electrical even safer.
See the link below for tips, guidelines, and safety concerns about panels.


text:
[![NPM version][npm-image]][npm-url]
[![Build status][travis-image]][travis-url]
[![Test coverage][coveralls-image]][coveralls-url]
[![Dependency Status][david-image]][david-url]
[![License][license-image]][license-url]
[![Downloads][downloads-image]][downloads-url]

Compose generator and async/await middleware like koa.
Allows to use both generator functions and async/await functions.

```js
var compose = require('composition');

var stack = [];

// generator functions
stack.push(function* (next) {
  yield next;
});

// regular functions that return anything
// but they should be promises!
stack.push(function (next) {
  return Promise.resolve(true);
});

// async/await functions
stack.push(async function (next) {
  return await Promise.resolve(true);
});

// compose it into a function
var fn = compose(stack);

// this function returns a promise
fn.call(this).then(function (val) {
  console.log(val);
}).catch(function (err) {
  console.error(err.stack);
  process.exit(1);
})
```

[gitter-image]: https://badges.gitter.im/thenables/composition.png
[gitter-url]: https://gitter.im/thenables/composition
[npm-image]: https://img.shields.io/npm/v/composition.svg?style=flat-square
[npm-url]: https://npmjs.org/package/composition
[github-tag]: http://img.shields.io/github/tag/thenables/composition.svg?style=flat-square
[github-url]: https://github.com/thenables/composition/tags
[travis-image]: https://img.shields.io/travis/thenables/composition.svg?style=flat-square
[travis-url]: https://travis-ci.org/thenables/composition
[coveralls-image]: https://img.shields.io/coveralls/thenables/composition.svg?style=flat-square
[coveralls-url]: https://coveralls.io/r/thenables/composition
[david-image]: http://img.shields.io/david/thenables/composition.svg?style=flat-square
[david-url]: https://david-dm.org/thenables/composition
[license-image]: http://img.shields.io/npm/l/composition.svg?style=flat-square
[license-url]: LICENSE
[downloads-image]: http://img.shields.io/npm/dm/composition.svg?style=flat-square
[downloads-url]: https://npmjs.org/package/composition

redpajama_set_name: RedPajamaGithub


This is a lovely neighborhood. The houses are well kept and diverse. Most houses have character and have both front and back yards and sidewalks are clean. Residents maintain their properties. This is a great area for walking, there are long, safe flat walks or you can go hiking from your home to Glen Park Canyon (an Urban wilderness). Access to transit is excellent. There is easy access to Muni J-Church, BART (Glen Park, or Balboa stations) and many Muni bus lines. If you drive there is easy access to the 280 freeway, as well as major cross-town routes (Ocean, Alemany, Bosworth, San Jose, Geneva). Neighborhood shopping and Restaurants require some walking, Mission street, Glen Park and Ocean Ave are all within 10 min. There are 2 Safeway stores and a Whole Foods store within 2 miles. Stonestown Mall / Farmer's Market is only a 10 min drive. Schools in the area include Glen Park, & Monroe elementary, and Balboa High School, and Lick Wilmerding private high school. Recreation is available, aside from walking, Balboa park is close and includes an indoor swimming pool, basketball courts, tennis courts, skate park, baseball fields, Boxer Stadium, and several new playgrounds.
"Okay City College Adjacent Hood"
Alright, you shouldn't really think of Mission Terrace as anything like the Mission. In fact, I think that it doesn't actually even border the Mission District, being separated by part of Bernal Heights. This neighborhood is not exactly beautiful, but it does have a certain quality to it that some might find attractive. It is made up of Spanish Revival style bungalows. They are fairly nicely kept and judging by the luxury cars parked outside, this neighborhood must have some high end sections.
So what should we make of this neighborhood?
Rents—The rents are fairly varied in this neighborhood—which is a little strange because from the outside the small lots and Mission style can look fairly homogenous. Most homes however go for about $850/mo. You can definitely find some for twice that amount.
Crime—Crime is only moderate here and there were no murders here last year.
Parks and Recreation—One of the nice things about this neighborhood is the presence of Balboa Park.
Education—The neighborhood is also to the east of City College of San Francisco, perhaps the best community college in the Bay Area. The local school, Balboa High is actually considered a fairly strong school although when you look at their scores on standardized tests they are strangely all over the board with some areas coming in the bottom quarter while others reach up into the top (and a fair number in between as well).
Restaurants—Like with its neighbor, the Excelsior District, restaurants are mostly along Mission and are mostly un-impressive. These are the kind of places you go to when you are too lazy or tired to go anywhere else.
Some of the lower rent prices in this neighborhood can be explained by the nearby college, though I don't think it is very common for students to move near community colleges in the same way they do for UCs or private universities like Stanford.
Overall, a little boring but definitely worth a look if you want to keep rents low or are a student in the area.
Mission Terrace lies close to the San Francisco-Daly City border line. There isn't anything really special about Mission Terrace, there are only homes and more homes in this neighborhood. For one, transportation may be relatively simple because there are several entrances and exits to the 280 freeway. But living right next to that brings disadvantages to living here. The noise is one, living here you will never be able to escape the buzz that comes from the 280 freeway, it's like what you see in movies when people live right next to train tracks, there will always be the expectation of loud annoying noises. I'm also quite sure that living next to the 280 freeway will also bring traffic jams every now and then. Many freeways and highways in San Francisco tend to jam up during rush hours in the morning and at night. MUNI transportation isn't that bad, there are several stops within Mission Terrace. There is also the Balboa Park BART station nearby. Mission Terrace is also surrounded by a plethora of schools, you have three high schools near the Balboa Park BART station; they are Lick Wilmerding High School, Leadership High School, and Balboa High School. There is also the City College of San Francisco on the other side of the 280 freeway. Residents of this neighborhood should expect a plethora of students and cars going through during the mornings and during after school times.
lynnet2 There isn't any freeway buzz in Mission Terrace proper (streets within the pillars), this would be South of Cotter and North of Onondaga. I was concerned about noise before I bought my house so I walked the neighborhood (San Jose Avenue and looping back up Cayuga). The freeway is dips BELOW the neighborhood for about a mile. As I was walking North along San Jose I could start to hear noise past Cotter Street. As I turned into (East) the hood the noise would vanish.
Big tip - it gets windy here about 4 pm BUT my friend has a house on the East side of the street (Otsego) and her deck overlooking her backyard is fantastic! Her house is a wind block. We sit out there and sip wine till the sun goes down. My house is on the South side, and although I have a nice deck off my rear living room, sometimes it is too windy to sit and enjoy in the evening. Mornings are fine - I go out to drink my tea and read the paper.
Mission Terrace is a great place to be if you want to both get into the heart of the city quickly and also have somewhat of a suburban feel in your world. On the west side, there's also easy access to BART at the Balboa Park Station.
Mission Terrace is also situations close to the ALC Lifecycle kick off route along Geneva coming up from the Cow Palace. It certainly is a cultural wonder to experience the energy of the ALC ride kickoff, and I highly recommend it -- you'll have to look up the date and time for this year at AidsLifecycle.org. As a rider, I can only say the traffic jam we bicyclists created was a thrill, but the cheers from the side of the road was and even more impressive as an expression of supportive.
"Times are changing in Mission Terrace"
Much of the Mission district survived the 1906 earthquake and fire, and refugees, primarily Italians and Irish, flooded in from across the city. They were joined by Mexican immigrants from the 1910 Mexican Revolution and Central Americans fleeing political oppression. Wave after wave of primarily Latino immigrants arrived in the ensuing years, melding the area into a rich and vibrant cultural community. It is with this history that we have Mission Terrace.
There are rich neighborhood murals in Balmy Alley, the Cesar Chavez School, the 24th Street Bart Station and numerous other locations. Working class social causes, art and music thrive in the Mission area today.
The latest wave of immigrants to Mission Terrace are the young, affluent, high-tech set, and easily lured by burritos and bungalows with potential. This new scene is creating quite a stir in the community and the gentrification debate roars on.
But everything old is new again. Ironically the least desirable neighborhoods in the '50s and '60s (Noe Valley, Glen Park and especially Bernal) are now the most desirable. Mission Terrace is finally getting the Glen Park spillover (Glen Park having gotten the Noe Valley spillover around 2000) Did you know Noe Valley is now 80% white?
It's hard to know the make up of Mission Terrace as the census data is only broken down to zip codes and Mission Terrace is just a small piece of the 94112. I do know that since 2004 five houses on my block have sold to thirty-something white families and one to a thirty-something Asian family. Of the selling families one was Mexican (foreclosed), one was from El Salvador (died) one was black (retired to Washington), one moved to Mill Valley (you guessed it - white), one family was Filipino and the last family was white. Yup better buy shades cause we're definitely gettin whiter here.


text:
Q: Firestore multiple compound indexes I have a filter component that can filter based on multiple fields. All of the fields are using the equality operator, and I have sorting based on "created" field.
Something along the lines of
let queryBuilder = firebase.firestore()
    .collection('myCollection')
    .doc(docId)
    .collection('myInnerCollection')
    .orderBy('created', 'desc');

const {field1, field2, field3} = filter;
if (field1) {
    queryBuilder = queryBuilder.where('field1', '==', field1);
}
if (field2) {
    queryBuilder = queryBuilder.where('field2', '==', field2);
}
if (field3) {
    queryBuilder = queryBuilder.where('field3', '==', field3);
}

For every field I'm required to create a compound index with 'created' field. i.e:
index field1(+) created(-)
index field2(+) created(-)
index field3(+) created(-)

Once I do that, every combination of filtering works. So my question is, if I know that field1 is always populated should I create just two compounded indexes:
index field1(+) field2(+) created(-)
index field1(+) field3(+) created(-)

Or having the 3 indexes is also ok?
Is there any performance/storage penalty of using each one? if so, what is the penalty (assuming I have 100K records in my collection)?

redpajama_set_name: RedPajamaStackExchange


text:
Tag: leeds festival
Leeds Festivals 2017 Review
Posted on 23rd October 2017
The sun was shining at this year's Leeds Festival. We could end our review right there considering the deluge suffered by past events, but let's push past the obvious – unnaturally good weather aside, this was an event defined by a stellar line-up from some of the most exciting artists and acts on the planet, and it lived up to its potential and then some.
A secret set by Queens of the Stone Age kicked off Friday night, and they were expertly followed by The Blossoms – it was a big slot, but they never faltered. Charlemagne and Blown Rose went down a treat, as everyone expect they would, and sets were livened up by covers of "There is a Light and it Never Goes Out" by The Smiths and "Imagine" by John Lennon. We fully expect to see these guys headlining sometime soon.
Liam Gallagher bought predictable Britpop swagger, and the set was discretely peppered with songs from his former band alongside new hits from the man himself.
Muse headlined Friday night, and it was hard for anticipation to get any higher. This is a band infamous for phenomenal live shows, and their explosive set surpassed all expectations with a fantastic mix of new songs and past back catalogue.
Saturday saw indie band Bastille excelling in their herculean task of warming up for blockbusters Kasabian. When the Leicester lads did finally take to the stage, their fantastic setlist seemed to work perfectly whether you were a Kasabian virgin or a diehard fan. There were also a few nice surprises, including covers of Daft Punk and Nirvana alongside the band's own superhits, such as Shoot the Runner and Treat.
Sunday also belonged to the main stage. Nu-metallers Korn blasted through their set, and their blissful unawareness that this wasn't quite the right crowd for them somehow helped make them seem perfectly placed, especially when finishing with such a legendary metal anthem as Freak on a Leash
Major Lazer came next to perform with surprising energy considering the array of songs they packed into their set, but it was festival closer Eminem that was on everyone's minds. Hits like "Stan", "The Way I Am" and "Like Toy Soldiers" have lost none of their charm, and a huge set of classics ended the festival perfectly.
Leeds Festival 2017 Dates, Line Up, Tickets and Map
Leeds Festival organisers have just announced early bird tickets for the 2017 event. Leeds Festival 2017 will be taking place at Bramham Park from 25th – 27th August, and you can snag your weekend pass right away starting at just £213. If you're hoping that Leeds Fest will be the first thing you mark on your 2017 calendar come Christmas time, you're not alone. The 2016 event just wrapped up to general acclaim.
Of course, Leeds will never want for attendees. With 12 stages and a whole host of acts to grace them each year, the festival has become a rite of passage for anyone in the North who likes music. The likes of Oasis, Green Day, and the Foo Fighters have topped the line-up over the years, and the headliners continue to be drawn from rock's A-List.
Never say that Leeds doesn't roll with the changes though. One of this year's most popular acts was Disclosure, representing the ascension of hip-hop and dance with the younger generation. Other stand-out acts included Little Simz, Anderson, and Bugzy Malone, and there were plenty of big name bands to steal the show.
Blossoms were chief among them. They've just launched an incredibly successful debut album, so these guys have really been turning things up across the festival circuit this summer. Coming to Leeds on Sunday afternoon, they entertained a capacity crowd with tracks such as Honey Sweet, Charlemagne, and At Most A Kiss.
Anyone who checked out Blossoms were surely in attendance for The 1975, who topped the bill on Friday night and cranked out single after single from I Like It When You Sleep, for You Are So Beautiful yet So Unaware of It.
Attendees were able to transition from young up-and-comers to legends of music thanks to the festival's diverse line-up. The Red Hot Chili Peppers and Nas both put on phenomenal shows in front of immense crowds. Nas blended a selection of classics with fresh new tracks, while the LA funk rock icons offered generations of hits to a field of fans who all seemed to know every word by heart.
Leeds 2017 is expected to draw the same fusion of old and new, and you can get there via coach, TransPennine Express, car, or by train and shuttle. Make sure you grab your pass early for the best deal.


The One You can Know and Trust with Your Heating & Air Conditioning.
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It is an absolutely beautiful fall day here in south Florida. Tropical Storm Nicole washed through on Wednesday and left us with some clean, crisp weather to enjoy. Hopefully it will persist through the weekend!
This week has been a great week for finding great reads. A great read to me is a book that has a wide appeal, strikes a chord with every reader, and has a timeless quality. Even a book as diverse as "The Girl Who Played with Fire" may not fit the "timeless" category, but it certainly appeals to anyone who has ever rooted for the underdog.
"The Good Earth," "The Red Tent," and "Olive Kitteridge" start the list. "The Help" is a deeply moving novel that is nothing short of wonderous. Definitely an all-time favorite of mine. "The Girl Who Played with Fire," a mystery with a very dark side, is intriguing and impossible to put down. If you've read "The Girl with the Dragon Tattoo," you probably already have this second book from the trilogy. For readers of the fascinating memoir "Glass Castle" by Jeanette Walls, Belleyre Books has "Half Broke Horses," the new true-life novel by Walls that tells the story of her larger than life grandmother. (Belleyre also has a copy of "Glass Castle.") Next on the list is "The Corrections," an Oprah's Book Club selection by Jonathan Franzen. His new book, "Freedom," is topping the best-seller list and has also been selected for Oprah's Book Club. Finally, "The Book Thief," a book I find hard to keep on the shelves, has just come in. Most of these great selections are in affordable, portable paperback. Please email me (belleyrebooks@bellsouth.net) with questions or comments!


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Liquordy-split: Alcohol delivery company reports 1,000% increase in visits due to coronavirus
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By Joe Ives
April 13 2020, 11.30
Alcohol delivery company Booze Up has reported a 1,000% rise in visits and 300% increase in sales following the coronavirus outbreak.
The company, founded in 2008, delivers wine, beer, spirits and cigarettes to customers in London, Surrey, Middlesex and Kent.
On March 25 it was announced licensed alcohol shops would be listed amongst essential business, and more and more people have been willing to pay premium prices to get their tipples delivered.
Ashleigh Fletcher, 41, founder and co-director of Booze Up said: "It's been very very interesting seeing how people have reacted.
"Initially seeing people run for toilet roll, then for food, and then finally run for alcohol."
ASHLEIGH FLETCHER: "It's been very very interesting seeing how people have reacted"
Customers have been bulk buying alcohol and up to 50 packets of cigarettes, with the biggest single order coming in at £2,034.
Booze Up has now started opening at 6 pm, three hours earlier than usual, just to keep up with demand.
With orders booming, Mr Fletcher has been working 18 hour days for the past 3 weeks.
He empathised with other business owners who were struggling due to COVID-19 but admitted the crisis had made many people more aware of alcohol delivery services.
Mr Fletcher said: "If we are gonna go out of business we're probably going to be the very last because of the nature of our business.
"We deal with all walks of life from the average Joe to the royal family."
Many of Booze Up's customers are in isolation and the company has to adapt its age checks, asking people to bring ID to their windows to verify that they are over 18.
At the same time, drivers now have to wear masks and gloves when out on orders.
Mr Fletcher has said he has received an increased level of contact from wholesalers no longer able to sell to pubs, bars and clubs.
Despite the company which owns Corona reporting £132million in losses, Booze Up has seen a 200% increase in demand for the beer.
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Managing Editor of Reporter and Reporter Online
Cheryl Magness is managing editor of Reporter and Reporter Online and manager of LCMS News. She has written for a variety of publications, including The Federalist, Touchstone and The Lutheran Witness, and is a contributor to the book He Restores My Soul from Emmanuel Press. She has degrees in English and music and enjoys playing piano and spending time with her husband and three children.
'Joy:fully Serving': Convention volunteers pack gift baskets for families in need
More than 50 volunteers prepared 275 baskets in four categories: mom care, baby care, spiritual care and household.
Registration still open for LEA convocation
The convocation is set for Oct. 10–12 in St. Louis under the theme "Opening Eyes, Minds, & Hearts."
Two resolutions from the 2019 LCMS convention — Res. 11–04A ("To Affirm the Common Humanity of All People and Ethnicities") and Res. 11–05A ("To Encourage Responsible Citizenship and Compassion Toward Neighbors Who Are Immigrants Among Us") — are available in both English and Spanish. See both versions below.
Walking together: Delegates work and worship in Tampa
The 67th Regular Convention of The Lutheran Church—Missouri Synod was held July 20-25 in Tampa, Fla., under the theme "Joy:fully Lutheran."
'Hatred did not succeed': Lutherans respond to mass shootings with Gospel comfort
The shootings occurred on Aug. 3 in El Paso, Texas, and on Aug. 4 in Dayton, Ohio.
'Defending Life' conference set for Sept. 6–7 in California
The conference is funded in part by a grant from the LCMS Office of National Mission.
Friedrich accepts call to Concordia, St. Paul
The Rev. Dr. Brian Friedrich, president of Concordia University, Nebraska, has accepted a call to become the tenth president of Concordia University, St. Paul, Minn., effective Jan. 1.
Delegates passed all 15 resolutions presented by the three committees on July 25.
Delegates elect members to national mission, international mission and other Synod boards at the 2019 Synod convention in Tampa, Fla.
Delegates pass resolutions on National Witness, Life Together and Retention
The resolutions affirm deaconess and DCE ministry, encourage active witness, call for continued vigilance in child protection, and encourage lifelong catechesis.
LCMS convention passes four National Witness resolutions by wide margins
The resolutions addressed church planting, shared congregational ministry, campus ministry and the legacy of Rosa J. Young.
Follow convention news wherever you are
Here are all the ways to stay informed about the 67th Regular Convention of The Lutheran Church—Missouri Synod, set for July 20–25 in Tampa, Fla.
Five more issues of 'Today's Business' — containing the schedule for each day, substitute or revised resolutions, wording for proposed amendments, and other official announcements and information — will be published at the convention.
Call Day: Seminary students receive calls, learn placements
A total of 201 students received their first calls to pastoral and diaconal ministry and their assignments as vicars and deaconess interns during four services held on the two seminary campuses April 29–30 and May 1.
Volunteer for 'Project Restore' in Nebraska
Good Shepherd Lutheran Church in Fremont, Neb., is seeking volunteers for the ongoing recovery effort, which is expected to take at least a year.
CTCR report looks at 'Christians and Social Media'
The report seeks to consider how Christians can "best use — or, if necessary, avoid the use of — this technology to glorify our God and Savior, build up the Body of Christ and extend His kingdom in the world."


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Legislative annexation an 'unusual' option for Mobile
Posted by Dale Liesch | Jan 6, 2022 | Latest, News | 0 |
If a local annexation push fails in the city of Mobile for a second time, one political operative believes the state Legislature could hold the key to expanding the municipality's borders. However, local legislators aren't so sure.
Mayor Sandy Stimpson, in 2019, pushed to annex about 13,000 new residents into the city from a portion of Mobile County. When it came time for the Mobile City Council to vote to allow a referendum on the issue, members of the body defeated the initiative along racial lines. The plan did not receive supermajority support, as the four White members of the council voted for it and the three Black members voted against it.
Since then, Stimpson has been positive about the move's chances when it is reintroduced to a council featuring four new faces. In previous interviews, Stimpson has said annexation of a portion of West Mobile would put Mobile over 200,000 in population and open the door to millions of dollars in additional federal grants.
The city's population stands at about 184,000, as of the 2020 Census, and Mobile has fallen to the fourth largest city in the state.
Jon Gray, a partner in Strategy Public Relations, said if he were Stimpson he would work through the Legislature to push annexation and use the Republican supermajority to his advantage.
Gray said that while most local bills start with a sign-off from a local legislative delegation, which seems unlikely in this case, legally speaking they don't have to. Instead, Gray said, a Republican lawmaker could write an annexation bill that would be voted on by the entire body and avoid the local delegation altogether.
"The Legislature could put forward a bill to call for an election to allow folks to decide to annex in," Gray said. "That way they could get around the local delegation."
Even if the bill were to go nowhere in the state House and Senate, Gray believes it would be good leverage for the mayor to use when the debate comes before the City Council.
"He could be in a situation where he says 'make a deal, or we go to the Legislature,'" Gray said.
But current and former legislators don't see Gray's hypothetical as a viable possibility.
Beth Lyons, a former legislator turned Mobile attorney and lobbyist, said the move would be legally possible, but unusual.
"Doing something like that without the acquiescence of the local delegation; that wouldn't be done," she said. "Procedurally, it would be legal."
Republican State Sen. Chris Elliott, who has been a part of a number of annexations in Baldwin County, said if he were a Republican lawmaker in the Mobile delegation, he wouldn't push the issue.
"I know the city of Mobile needs to grow, but it's a bad precedent," Elliott said. "One of the good things about the Legislature is that you've got to work with these people the next day. There's mutual respect."
State Rep. Chris Pringle, a member of the Mobile delegation, said the move would "cause quite a stir."
"It could be tried," he said. "I don't know how successful it would be."
Any attempt at annexation through the Legislature would also get tied up in court, Pringle predicted, as he referenced a letter sent to Stimpson and members of the City Council by the Southern Poverty Law Center warning caution against annexation that diluted Black voting power.
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I have a weird thing about making centerpieces - or I guess you could call them Christmas arrangements (but I like making them for all the seasons). I love using different vase fillers, twinkle lights, branch lights, greens, Christmas balls, branches and other picks.
You should see all the glitter in the guest room... ha! It looks like Christmas has exploded in there between the decor, glitter and shopping bags.
Taking my decorating up a notch this year and getting some lights outside. (finally!) I picked up some greens to wrap around the porch and plan on stringing some lights in there too! Hoping that it turns out!
I also am making this planter to put by the door! Filling this pot with some dirt and using light up LED branches, a log, some glittery branches and I want to find some green pine branches! (thinking a trip to the woods OR to the dumpster at Home Depot will be my supply of those!
I'm still so in love with this JOY light up sign that I got last year! I think JOY is my favorite Christmas word... Do you do stockings at your house? We always had stockings growing up, but they never had anything in them. My boyfriend's family has ALWAYS done stockings and it's something they make a big deal about! And it's super fun - so we have been exchanging stockings the past few years too!
Here are the twinkle lights and the LED candles I picked up at Michael's! Can't wait to get creative. Filling mason jars with the twinkle lights is super simple yet pretty.
Christmas is even making it's way into the man cave! The deer even got decked out! And I decided to light up the window ledge too! Can't wait to sit down here with the fireplace and holiday lights on!
Even the mule deer is decked out!
Alright I'm sure you all are sick of my home decor now.. ha!
We are hosting my family for Christmas and also B's family that is coming over New Year's weekend! Time to start menu planning! Going to make a menu for my side of the family first, since it's a smaller group. Here is what I have planned for the 14 of us!
I need your input though! Does anyone have any good recipes for a christmas punch or cider? either alcoholic or not! Also any tips for an amazing crudite tray? Thinking assorted nuts, meats, hummus, cheese, etc!