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Research ArticleImmunologyOncology Free access | 10.1172/JCI90499
Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer
Nicholas A. Taylor,1 Sarah C. Vick,1,2 Michael D. Iglesia,3 W. June Brickey,1,2 Bentley R. Midkiff,1 Karen P. McKinnon,1,2 Shannon Reisdorf,1 Carey K. Anders,1,4 Lisa A. Carey,1,4 Joel S. Parker,1,3 Charles M. Perou,1,3 Benjamin G. Vincent,1,4,5 and Jonathan S. Serody1,2,4
1Lineberger Comprehensive Cancer Center,
2Department of Microbiology and Immunology,
3Department of Genetics,
4Department of Medicine, and
5Curriculum in Bioinformatics and Computational Biology, University of North Carolina (UNC), Chapel Hill, North Carolina, USA.
Address correspondence to: Jonathan Serody, Lineberger Comprehensive Cancer Center, 450 West Drive, CB7295, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Phone: 919.445.4175; Email: jonathan_serody@med.unc.edu.
Authorship note: N.A. Taylor and S.C. Vick are co–first authors. B.G. Vincent and J.S. Serody are co–senior authors.
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First published August 21, 2017 - More info
Published in Volume 127, Issue 9 on September 1, 2017
J Clin Invest. 2017;127(9):3472–3483. https://doi.org/10.1172/JCI90499.
Copyright © 2017, American Society for Clinical Investigation
First published August 21, 2017 - Version history
Received: September 6, 2016; Accepted: July 11, 2017
Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.
Breast cancer is the leading cancer diagnosis and second-leading cause of cancer death for women in the United States (1). Patient prognosis is determined largely by stratification based on age, lymph node involvement, tumor grade, and tumor cell-surface expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (2–7). Early diagnosis and targeted therapies, both hormonal and immunologic, are critical for the effective treatment of ER-, PR-, and HER2-positive disease. Given the lack of targeted therapies for patients whose tumors do not express ER, PR, and HER2, termed triple-negative breast cancers, the survival benefit from recent therapeutic advances has eluded this group of patients. This is especially important for clinicians and patients, as triple-negative tumors present with higher grade at diagnosis and an increased incidence for disease relapse, early metastasis, and death (8–11).
Transcriptome profiling has defined 5 intrinsic molecular subtypes of breast cancer: luminal A, luminal B, HER2 enriched, basal-like, and claudin-low (12, 13). The basal-like and claudin-low subtypes are distinguished by increased expression of genes associated with tumor cell proliferation and adaptive and innate immunity (14, 15). Claudin-low tumors are further separated from basal-like tumors by their decreased expression of cell-cell adhesion claudin proteins, increased expression of genes associated with epidermal-to-mesenchymal transition, and stem cell–like characteristics (16). Importantly, basal-like and claudin-low tumors make up the majority of triple-negative tumors and have a significantly poorer prognosis than luminal and HER2-enriched tumors (17).
In addition to tumor-intrinsic genetic features, tumor growth is dependent on tumor-extrinsic factors, which include the tumor stroma and the local immune response (14, 18). Tumor-infiltrating lymphocytes (TILs)can have divergent effects in the tumor microenvironment (TME). Whereas the presence of multiple immune cell types may herald productive antitumor immunity (15), TILs may also contribute to tumor progression through immune suppression or the production of soluble factors that regulate cell proliferation, migration, and angiogenesis (19).
While the presence of TILs is associated with an improvement in the survival of patients with multiple different tumor subtypes, this has not been found for specific subtypes of breast cancer. The luminal A subtype, which exhibits no appreciable immune infiltration, carries the best prognosis, whereas the basal-like and claudin-low subtypes exhibit a marked immune infiltrate with worse clinical outcomes (20). In this study, we sought to understand the mechanism by which immune infiltration is associated with poor clinical outcomes for patients with claudin-low tumors.
We show that patients with claudin-low tumors have an increased number of CD4+ T cells with focal increases in Tregs in the TME compared with other intrinsic subtypes. To understand the mechanism for this, the p53–/– T11 and T12 genetically engineered mouse models (GEMMs) were used, which closely recapitulated the cell-intrinsic and -extrinsic characteristics of human claudin-low breast cancer. In human and mouse studies, gene expression profiling revealed increased expression of immune genes and Treg-associated genes in the claudin-low subtype. Importantly, checkpoint inhibition was found to be effective for claudin-low tumors only when combined with stringent Treg depletion. Taken together, these experiments highlight the role of Tregs in suppressing the immune response in claudin-low breast tumors and provide an understanding of the differential response to immune checkpoint inhibition in breast cancer.
Claudin-low tumors show increased expression of immune genes relative to other intrinsic subtypes of breast cancer. The original microarray used to classify the intrinsic subtypes of breast cancer included over 1,700 genes (21, 22), with an emphasis on cell-intrinsic transcriptional characteristics. We were interested in determining whether there were cell-extrinsic transcriptional differences that could be used to classify breast cancer subtypes. For this analysis, we evaluated the expression of 102 immune genes that encompass downstream effector molecules, chemokines and chemokine receptors, immune response signal mediators, cell-specific markers, and markers of oxidative stress, termed the intrinsic immune genes (IIGs) (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI90499DS1). Gene expression profiling by microarray analysis was done on 337 breast tumors as previously described (23). Unsupervised hierarchical clustering of UNC337 by only those genes in the IIG list divided patients into 3 clusters: low-expressing luminal, moderate-expressing HER2 enriched, and high-expressing basal-like and claudin-low tumors (Figure 1A). By gene expression signature quantification, HER2-expressing tumors had significantly lower immune gene expression than claudin-low tumors (P < 0.05) (Figure 1B). Furthermore, claudin-low tumors showed significantly increased expression of a gene signature associated with Treg activity (P = 0.001, data not shown).
Human tumors exhibit differential immune gene expression. (A) Unsupervised hierarchical clustering of breast cancer samples from UNC337 by IIG list. (B) Overall expression of the immune gene signature by each subtype. (C) Representative H&E staining of human subtype–specific breast cancer. Original magnification, ×5. (D) Representative IF staining of human tumors for CD4 (red) and FoxP3 (green). Original magnification, ×20. (E) Whole-slide scoring of CD4+ infiltrate for each subtype. (F) Unsupervised clustering by the IIGs in GEMMs (n = 59). Statistical significance determined by Kruskal-Wallis test, with Dunn's post-test for multiple comparisons. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.
To confirm these findings, we performed transcriptome evaluation using the IIG classifier from a previous published breast cancer database (Molecular Taxonomy of Breast Cancer International Consortium [METABRIC]; http://www.cbioportal.org/study?id=brca_metabric#summary), as the stringency filter for tumor purity eliminated the inclusion of claudin-low tumors from the TCGA database (https://cancergenome.nih.gov/). Unsupervised hierarchical clustering of the METABRIC data by only those genes in the IIG list revealed that clinical breast cancer samples divided into 3 clusters: low-expressing luminal, moderate-expressing HER2 enriched, and high-expressing basal-like and claudin-low tumors similar to what was shown in the UNC337 data (Supplemental Figure 1). The claudin-low subtype had a significantly increased immune gene expression compared with the other subtypes in the METABRIC data set (P < 0.0001).
Claudin-low breast tumors show increased infiltration by CD4+ cells. In order to determine whether IIG expression was associated with differences in Th cells, Tregs, or cytotoxic T cells (Tc cells), patient biopsy samples were studied by histology and immunofluorescence (IF). Figure 1C shows H&E stains of the greatest area of immune infiltration of a representative luminal A and claudin-low tumor. Tumor tissue slides were analyzed for Tc cells (CD8+), Th cells (CD4+FoxP3-), and Tregs (CD4+FoxP3+). Representative images of CD4 and FoxP3 IF staining from the highlighted areas shown in Figure 1C demonstrate CD4+FoxP3+ cell infiltration into the tumor (Figure 1D). Claudin-low tumors recruited significantly more CD4+ T cells to the tumor site than luminal A tumors (P = 0.002; Figure 1E), with areas of focally increased numbers of Tregs (Figure 1D). No differences in the number of CD8+ T cells were found across the different intrinsic subtypes (data not shown). These data indicate the heterogeneity of the immune response in breast cancer subtypes, with claudin-low tumors having a significantly increased T cell infiltrate (P < 0.01) with focally increased numbers of Tregs. In contrast, luminal tumors showed an extremely limited number of CD8+ and/or CD4+ T cells.
Claudin-low GEMMs closely recapitulate human claudin-low breast cancer. Given the difficulties with standardizing tumor latency using clinical samples and the extreme difficulty evaluating the TME at the onset of tumor formation, we sought an in vivo method to better evaluate mechanisms for immune infiltration in claudin-low breast cancer. For this analysis, we used GEMMs of the claudin-low subtype, which have been found to be genotypically similar to human claudin-low tumors (24). Unsupervised clustering of the GEM models using the IIG list (Figure 1F) recreated the clustering profile seen in human samples with very modest expression of immune genes in 2250 tumors (luminal A) compared with very high expression in T11 tumors (claudin-low). We generated a cell line from T11 tumors to standardize claudin-low tumor growth in vivo. Gene expression profiling of these tumors showed that the parental T11 tumor and tumors derived from injection of the T11 cell line clustered tightly together within the claudin-low subtype, demonstrating that the T11 cell line is genetically quite similar to T11 tumor cells (Supplemental Figure 2).
Claudin-low tumors recruit increased numbers of immune cells to the tumor. Tumors representing the subtypes luminal A, HER2/Neu, and claudin-low were studied in GEMMs to evaluate the immune response during tumor progression. In order to evaluate both early and late time points, tumors were harvested at approximately 20 mm2 (Figure 2, A–D) or 100 mm2 (Figure 2, E–H), respectively, and analyzed by flow cytometry to determine the composition of TILs over time. FACS analysis work flows are shown in Supplemental Figure 3, and representative FACS plots are shown in Supplemental Figure 4. The total number of immune cells was significantly increased in T11 claudin-low tumors (Figure 2, A–H). Specifically, the number, but not the frequency, of CD4+FoxP3– T cells was increased in the TME in both 20-mm2 and 100-mm2 tumors (Figure 2, A and E). Interestingly, we found an increase in the number of Tregs both at 20 mm2 and 100 mm2 in the T11 claudin-low model. There was a statistically significant increase in the number of CD19+ B cells in mice, with 20 mm2 T11 claudin-low compared with Neu tumors, and a significant increase in B cells in mice with 100 mm2 T11 tumors compared with luminal A tumors (Figure 2, C and G). Additionally, we found very few CD8+ T cells in the TME of mice with either luminal A– or Neu-expressing tumors. There was a statistically significant increase in the number and frequency of CD8+ T cells in mice with 20-mm2 and 100-mm2 claudin-low tumors (Figure 2, B and F). There was an inverse correlation between the presence of Tregs and diminished number of both CD8+ T cells and CD19+ B cells (Figure 2, I and J). To confirm our results were not specific to the T11 cell line, we utilized an additional claudin-low tumor cell termed T12 to examine immune infiltration into the TME at late-stage tumor growth. We found an increase in Tregs in mice with 100 mm2 tumors in the T12 model, although the increase was not significant. We investigated the activation status of the cells migrating into the TME by looking at naive and memory T cell populations (Supplemental Figure 5). Compared with luminal A– and Neu-expressing tumors, the T11 and T12 claudin-low tumors had increased effector memory cells in both the CD4+FoxP3– and CD8+ T cell populations. (Supplemental Figure 5, B and D). Thus, these data indicated that there was a significant difference in the population of immune cells in the TME in mouse luminal A, Neu-expressing, or claudin-low tumors. Additionally, our data suggest that the early presence of Tregs was associated with diminished numbers of CD8+ T cells and B cells in mice with claudin-low tumors.
T11 and T12 (claudin-low) tumors recruit elevated numbers of immune cells to the tumor site. WT mice were injected with 1 × 106 2250 tumor cells or 1 × 104 T11 or T12 cells. Neu-N mice were injected with 5 × 104 NT2 cells. Tumors were harvested at 20 mm2 (2250, n = 9; NT2, n = 10; T11, n = 12) (A–D) or 100 mm2 (2250, n = 10; NT2, n = 5; T11, n = 10; T12, n = 6) (E–H), digested, enriched for lymphocytes, and analyzed by FACS. (A) CD4+ TILs, 20 mm2. (B) CD8+ TILs, 20 mm2. (C) CD19+ TILs, 20 mm2 (2250, n = 4). (D) CD4+FoxP3+ TILs, 20 mm2. (E) CD4+ TILs, 100 mm2. (F) CD8+ TILs, 100 mm2. (G) CD19+ TILs, 100 mm2. (H) CD4+FoxP3+ TILs, 100 mm2. (I) Correlation between the percentage of CD8+ T cells and FoxP3+CD4+ T cells in mice with 20 mm2 T11 tumors. n = 16 mice. (J) Correlation between CD19+ B cells and FoxP3+CD4+ T cells in T11 mice with 20 mm2 tumors. n = 16 (taken from the same group as evaluated in I). Statistical significance determined by Kruskal-Wallis test with Dunn's post-test for multiple comparisons. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Tregs in mice with tumors show enhanced suppression and chemokine production. Next, we evaluated whether the Tregs isolated from tumor-bearing mice were efficient at suppressing T cell responses. As shown (Figure 3, A and B), Tregs were capable of suppressing T cell proliferation in a mixed lymphocyte reaction assay. At high ratios of Treg/T effector cells (Teffs), Tregs were more effective when isolated from mice with 100-mm2 compared with 20-mm2 tumors. Thus, tumor-bearing mice have functional Tregs that display enhanced suppression in mice with larger tumors.
FoxP3+ cells from T11 tumor–bearing mice are functionally suppressive. FoxP3GFP mice were injected with 5 × 104 T11 cells. GFP+FoxP3+ splenocytes were sorted from tumor-bearing mice at (A) 20 mm2 or (B) 100 mm2 and plated with irradiated allogeneic stimulator cells and different ratios of WT Teffs for 3 days. Teffs were then FACS analyzed for dilution of proliferation dye. n = 3 mice at each tumor size. Data are presented as mean ± SEM.
To determine what mediators were responsible for recruiting Tregs to the claudin-low tumor site, we compared gene expression differences between Neu-expressing and claudin-low tumors (Figure 4A). T11 tumors expressed significantly higher levels of multiple chemokines, with CXCL12 being 22-fold higher in these tumors compared with Neu-expressing tumors (95% CI: 17.1 – 28.3; P < 0.001). ELISA of tumor homogenate for CXCL12 confirmed the array findings, with claudin-low tumors expressing higher levels of CXCL12 protein than Neu-overexpressing tumors (Figure 4B). Elevated levels of CXCL12 protein were also found in the serum of claudin-low tumor-bearing mice (Figure 4C). Tregs that infiltrate T11 tumors demonstrated higher expression levels of CXCR4, a receptor for CXCL12, compared with conventional CD4+ T cells (P = 0.03) or CD8+ T cells (P = 0.003; Figure 4, D and E).
T11 tumors express high levels of CXCL12. (A) Whole-tumor RNA was isolated and analyzed using the PAMM-011 RT2 Profiler PCR Array. Gene expression is presented relative to NT2 tumor controls. (B) Tumors were harvested on day 21 PTI, homogenized, and analyzed by CXCL12 ELISA (n = 3–4 per group). (C) Serum was harvested on day 21 PTI and analyzed for CXCL12 ELISA (n = 3 per group). (D) Representative overlay of CXCR4 expression by CD4+FoxP3+ Tregs (Treg CD4), CD4+FoxP3– T cells (Non-Treg CD4), and CD8+ T cells. (E) MFI values for the expression of CXCR4 from TILs isolated from mice with 20 mm2 T11 tumors (n = 6). Statistical significance was determined by Student's t test (B and C) or Mann-Whitney U test (E). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
To evaluate whether the CXCL12/CXCR4 axis was critical to the recruitment of Tregs in mice with claudin-low tumors, mice were treated with the CXCR4 inhibitor AMD3100. There was a trend toward decreased Treg numbers in AMD3100-treated mice (Figure 5A), which was greater when comparing the number of FoxP3+PD-1+ Tregs, where PD-1 indicates programmed death receptor 1 (P = 0.07) (Figure 5B). To further evaluate whether the CXCL12/CXCR4 axis was critical to the recruitment of Tregs, shRNA knockdown of CXCL12 was achieved by lentiviral expression of CXCL12-targeted shRNA into T11 tumor cells. (Figure 5, C and D). We generated 2 cell lines, one with a 55% knockdown of CXCL12 (KD1) and one with an 85% knockdown (KD5). There was a nonsignificant reduction in both Tregs and PD-1+ Tregs in the TME using the more robust knockdown of CXCL12 at day 13 post tumor implantation (PTI) (Figure 5, E and F). Thus, these data indicate that inhibition of the CXCL12/CXCR4 axis in vivo had a modest but not significant effect on Treg accumulation in claudin-low tumors.
Role of CXCL12/CXCR4 pathway in Treg infiltration into claudin-low tumors. (A and B) WT mice were implanted on day –2 with osmotic pumps loaded with PBS or 10 mg AMD3100 in PBS (n = 7 per group) and challenged with 1 × 104 T11 cells. Tumors were harvested on day 12 PTI and FACS analyzed. (A) Percentage and number of CD4+FoxP3+ TILs. (B) Percentage and number of CD4+FoxP3+PD-1+ TILs. (C) T11 cell line was transfected with lentiviral-expressing CXCL12-targeted shRNA, and (D) CXCL12 knockdown was confirmed using CXCL12 ELISA. (E and F) WT mice were injected with 1 × 104 cells of WT, scramble, or knockdown (KD) lines (n = 5 per group). Tumors were harvested on day 13 PTI and FACS analyzed. (E) Percentage and number of CD4+FoxP3+ TILs. (F) Percentage and number of CD4+FoxP3+PD-1+ TILs. Statistical significance was determined by Mann-Whitney U test. Data are presented as mean ± SEM.
Checkpoint inhibition therapy does not alter T11 tumor growth. Because of the increased number of immune cells infiltrating the tumor, we hypothesized that the T11 claudin-low model would be responsive to checkpoint inhibition therapy with blocking antibodies against the inhibitory receptors PD-1 and cytotoxic T lymphocyte–associated protein 4 (CTLA-4). Surprisingly, checkpoint inhibition therapy conferred no benefit on tumor growth or survival in the T11 claudin-low model (Figure 6, E and F), despite expression of PD-1 on CD8+ T cells (Figure 6A). This result was confirmed in the T12 claudin-low model (Supplemental Figure 6). However, and unexpectedly, FACS analysis of the immune cells infiltrating the tumor showed that Tregs had the highest levels of PD-1 and CTLA-4 expression, as measured by FACS mean fluorescence intensity (MFI) (Figure 6, A–D).
Tumor-infiltrating Tregs express increased levels of PD-1 and CTLA-4; however, PD-1 and CTLA-4 inhibition does not increase survival in T11 tumor–bearing mice. (A) Representative FACS overlay and (B) total MFI values of PD-1 expression by T11 TILs at 20 mm2. n = 4. (C) Representative FACS overlay and (D) total MFI values of CTLA-4 expression by T11 TILs at 20 mm2. n = 5. (E) Growth curves of T11 tumor–bearing mice receiving anti–PD-1 and anti–CTLA-4. (F) Survival analysis of data presented in E (n = 5 for each group). Statistical significance was determined by Mann-Whitney U test. Data are presented as mean ± SEM. *P < 0.05.
Depletion of Tregs delays T11 tumor growth. The inverse correlation between the presence of Tregs and the absence of CD8+ T cells and CD19+ B cells in the TME (Figure 2, I and J) led us to hypothesize that approaches that target Tregs would enhance the antitumor immune response. To deplete Tregs, we made use of mice in which the diphtheria toxin (DT) receptor had been knocked in to the FoxP3 locus (DEREG mice) and treated with DT to specifically deplete Tregs in vivo. DEREG mice given DT exhibited a significant delay in tumor growth and a nonsignificant improvement in survival (Figure 7, A and B), with all mice eventually succumbing to either autoimmunity or tumor. As an alternative nongenetic approach to treatment, tumor-bearing WT mice received a single treatment of low-dose (100 mg/kg) cyclophosphamide (Cy) to selectively deplete Tregs (25). As with DT treatment of DEREG mice, a single injection of low-dose Cy significantly delayed tumor growth (Figure 7, A and B), but again, all mice eventually succumbed to death upon tumor administration. While Treg numbers were decreased compared with those in untreated T11 tumors after Cy, they had increased to above baseline by the time tumors reached 20 mm2 (Figure 7C). Thus, Treg depletion alone appeared to modestly affect tumor growth.
Treg depletion delays tumor growth in the absence of adoptive transfer. WT untreated, Cy-treated, and DEREG mice were injected with 1 × 104 T11 cells (n = 5 per group). Cy-treated mice received 100 mg/kg Cy on day +2. DEREG mice received 1 μg DT on days –1, 0, +6, and +7. (A) Growth curves. (B) Survival. There was a significant difference in survival between both Treg-depleted groups compared with the untreated control group (P < 0.05; log-rank test), but not between treated groups. (C) Treg numbers following a single dose of Cy were reduced compared with those in untreated T11 tumors. Statistical significance was determined by Mann-Whitney U test. Data are presented as mean ± SEM. ***P < 0.001; ****P < 0.0001.
Depletion of Tregs plus checkpoint inhibition improved survival and increased cytokine production by CD8+ T cells. While depleting Tregs delayed tumor growth, we evaluated whether addition of checkpoint inhibition therapy would enhance the benefit. Given the finding that blocking CXCR4 had a modest effect on the recruitment of Tregs in T11 tumors, we initially evaluated whether combining checkpoint inhibition with AMD3100 would have an impact on tumor growth. There were no synergistic effects between CXCR4 inhibition and checkpoint inhibition, as we found no difference in tumor growth using combined checkpoint inhibition and control treatment or AMD3100 (Supplemental Figure 7, A and B). Mice receiving AMD3100 had a reduction in Tregs 12 days after the onset of treatment (Supplemental Figure 7, C and D), but by day 16, the reduction in infiltrating immune cells was no longer specific to Tregs, as we saw a reduction in CD4+ and CD8+ T cells (Supplemental Figure 7, E and F). When combined with checkpoint inhibition, all cell subsets were significantly reduced in the TME (Supplemental Figure 7, C–E). As a more rigorous test, Tregs were depleted using DT treatment in DEREG mice in combination with anti–CTLA-4 and anti–PD-1 monoclonal antibody treatment. Tregs were depleted from approximately 20% to 5% after DT treatment (Figure 8F). T11 tumor–bearing mice were followed for 25 days PTI until tumor growth reached 170 mm2 or mice developed significant autoimmunity from Treg depletion. Treg depletion in combination with anti–PD-1 and anti–CTLA-4 therapy resulted in decreased tumor growth and significantly improved survival (P = 0.03) (Figure 8, A and B). Unfortunately, because mice treated with DT developed severe autoimmunity, we were unable to extend the tumor growth curves past 25 days PTI per institutional IACUC guidelines. We then sought to determine whether delayed tumor growth was correlated with increased cytokine production by T cells. We analyzed IFN-γ production by CD8+ T cells in both the tumor and the spleen by FACS as a measure of productive CD8+ T cell responses. While this evaluation was limited greatly by the significant occurrence of autoimmunity in mice treated with DT, there was a nonsignificant increase in the percentage of CD8+ T cells that generated IFN-γ, but not in total CD8+ T cells, in mice treated with DT that also received checkpoint inhibition therapy (Figure 8, C–E).
Treg depletion with anti–PD-1 and anti–CTLA-4 immune checkpoint inhibition significantly delays T11 tumor growth. WT or FoxP3-DTR mice were injected with 1 × 104 T11 cells. DEREG mice received 1 μg DT on days –1, 0, 6, and 7 PTI. DT + PD-1 + CTLA-4 mice received 1 μg DT on days –1, 0, 6, and 7 PTI and anti–PD-1 and anti–CTLA-4 antibody on day –1, then every other day for the duration of the experiment. Tumor growth evaluation was terminated at day 25 PTI due to autoimmune manifestations and tumor growth reaching an end point of 170 mm2. (A) Mice depleted of Tregs and receiving anti–PD-1 and anti–CTLA-4 (n = 3) had a significant survival benefit compared with untreated mice (n = 4) or those treated with anti–PD-1 and anti–CTLA-4 alone (n = 3) (CLTLA4 + PD-1 vs. DT + CLTLA4 + PD-1: P = 0.03; log-rank test). (B) Individual replicates of tumor growth curves. (C–E) Mice treated with anti–PD-1 and anti–CTLA-4 alone (Treg+) (n = 3) or anti–PD-1 and anti–CTLA-4 with DT (Treg–) (n = 3). Mice were sacrificed 25 days PTI and FACS analyzed for percentage of CD8+ T cells and IFN-γ production by CD8+ T cells in tumor and spleen. (C) Representative FACS plot of IFN-γ production by CD8+ T cells from tumor or spleen of tumor-bearing mice. (D) Percentage of CD8+ T cells and IFN-γ production by CD8+ T cells from tumor. (E) Percentage CD8+ T cells and IFN-γ production by CD8+ T cells in spleen. (F) Percentage and total number of CD4+FoxP3+ Tregs from tumors in mice treated with anti–PD-1 and anti–CTLA-4 alone (Treg+) (n = 3) or anti–PD-1 and anti–CTLA-4 with DT (Treg–) (n = 3). Analysis of T cell TILs was not possible after day 25 in DT-treated mice due to severe autoimmunity necessitating termination of the experiment at that time. Data are presented as mean ± SEM.
To circumvent the issues with autoimmunity in mice given DT treatment prior to tumor implantation, we treated a small group of mice with DT PTI. While we were limited again in the ability to generate significant numbers of DEREG mice to evaluate, there was prolonged survival until day 40 for half of the mice given DT therapy PTI (Supplemental Figure 8). Because of the autoimmunity associated with the use of DEREG mice, we sought a pharmacological approach to suppress Treg function in vivo. Previous work has shown that selective inhibition of the p110δ isoform of PIK3 using a small molecule inhibitor delayed 4T1 tumor growth in mice and reduced Treg suppression (26). We used the small molecule inhibitor PI-3065 in addition to checkpoint inhibition in our T11 claudin-low model. Treatment with PI-3065 was associated with a statistically significant improvement in survival, with a period of tumor stability in half of the treated mice (Supplemental Figure 9, A and B). However, we were not able to mediate tumor regression or affect overall survival. Treatment with PI-3065 and checkpoint therapy did significantly reduce the number of Tregs infiltrating into the tumor (Supplemental Figure 8C). However, it also led to a reduction in the number of CD4+ FoxP3-negative T cells accumulating in the TME.
Triple-negative breast cancer has the worst prognosis of the histologic subtypes due to presenting at higher grade at diagnosis, increased recurrence risk following treatment, and shortened time to metastasis (27). The paradoxical observation that the molecular subtypes comprising these tumors also exhibit high levels of immune cell infiltration suggests that these infiltrating cells may have a deleterious impact on patient outcome. Our study supports this conclusion for claudin-low breast cancer. Clustering by the IIG alone was sufficient to separate triple-negative basal-like and claudin-low tumors from luminal tumors and HER2-enriched tumors. This finding was not due to aberrant gene expression by tumor cells, but to increased immune cell infiltration into the tumor, especially CD4+ T cells. These results are in contrast with those in prior studies where numbers of TILs were found to be favorably prognostic in breast cancer (5). Importantly, these studies did not stratify breast tumors by molecular subtype, highlighting that the significance of the immune infiltrate may vary widely by subtype. The use of mouse models that mimic human tumor biology is critical for finding pathways important for tumor pathogenesis and potential. The GEMMs utilized by our laboratory are derived from and studied in immunocompetent mice that recapitulate tumor biology at the genetic and whole tumor level. As expected by similarity to the human patient samples, murine tumors exhibited substantial differences in immune cell infiltration based on intrinsic subtype.
Recently, there has been intense interest in biomarkers of response to checkpoint inhibitor therapy. One stratification that has been proposed for patients with melanoma is immune-excluded versus immune-infiltrated tumors, with data suggesting that checkpoint inhibition is not effective in immune-excluded tumors (28). Our data would suggest that a third group of tumors, those that are actively immune suppressed, may also not respond to checkpoint inhibition. The presence of Tregs as a biomarker for antitumor response has been confusing, with data indicating both negative and positive clinical benefits (29–31). This may be due to the critical issue of timing in the evaluation of Tregs from the TME. In claudin-low murine tumors, Tregs made up as much as 60% of all CD4+ cells in the tumor and were present in substantial numbers prior to the expansion of other adaptive immune cells. These cells were both functional and durable, with continued presence as the tumors grew. In many of the T11 and T12 tumors, Tregs dominated the immune response, which was associated with diminished numbers of CD19+ B cells and CD8+ T cells. Thus, there may be a critical early period when tumors can recruit sufficient numbers of Tregs to abrogate a functional adaptive immune response.
We sought to determine the mechanism that accounts for the preferential recruitment of Tregs to claudin-low tumors. CXCL12, a chemokine critical for the homing of hematopoietic stem cells (32), was expressed in mice 22-fold more with claudin-low compared with Neu tumors. Further, Tregs that infiltrated claudin-low tumors expressed high levels of CXCR4, one of the receptors for CXCL12. Inhibition of CXCR4 with AMD3100 demonstrated a modest role for this axis in Treg recruitment. AMD3100-treated mice demonstrated a trend for decreased Treg numbers in the tumor compared with controls, which contained elevated PD-1+ Tregs. However, the modest decrease in the recruitment of Tregs found using AMD3100 was not sufficient to augment the antitumor immune response using checkpoint inhibitor therapy, in part because exposure to AMD3100 also diminished the recruitment of CD4+ non-Tregs and CD8+ T cells to the TME. Similarly, selective inhibition of p110δ isoform of PI3K using a small molecule inhibitor plus checkpoint inhibition reduced the number of Tregs, which was associated with a significant (P < 0.05) improvement in tumor growth compared with that in control mice receiving checkpoint inhibition alone. Unfortunately, this combination did not result in tumor regression or enhanced overall survival. Our data suggest that robust and specific Treg depletion is necessary to engender an effective antitumor response. This level of depletion in our mouse studies was associated with profound autoimmunity. Thus, it is not clear whether prolonged, robust Treg depletion in patients could be tolerated to enhance checkpoint inhibitor therapy for the treatment of claudin-low tumors.
The function of PD-1 on the surface of Tregs is not entirely known (33, 34). PD-1hi Tregs in the TME in glioma patients have been found to be exhausted, yet capable of generating proinflammatory cytokines, such as IFN-γ (35). This suggests that one mechanism for the impaired function of anti–PD-1 antibody therapy is the presence of PD-1–expressing Tregs that can (a) bind antibody if PD-1 is preferentially expressed on these cells and (b) become activated in the presence of anti–PD-1 antibody. Our data requiring depletion of Tregs to mediate the antitumor activity of anti–PD-1 antibody therapy would be consistent with this mechanism.
In summary, we have found that claudin-low tumors are highly enriched with Tregs. Enhancing the immune response to these tumors by depleting Tregs in addition to immune checkpoint inhibition impaired tumor growth and prolonged survival, but was insufficient to mediate tumor regression. Generation of CXCL12 by the tumor cells played a modest role in the recruitment of Tregs to the tumor site. These studies suggest that future clinical trials for patients with triple-negative breast cancer should target the immunosuppressive environment generated by the tumor in combination with checkpoint inhibition.
Mice and cell lines. BALB/cJ and C57BL/6J (B6) females and C.B6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax (DEREG) females were purchased from The Jackson Laboratory. Neu-N mice were purchased from Charles River Laboratory. Female mice (8 to 14 weeks) were used for all experiments. Rat Neu-expressing NT2 cells (HER2/Neu) and the 2250 (luminal A), 2225 (basal-like), and T11 (claudin-low) tumor models have been described (36–38). T12 cells were prepared by harvesting a T12 tumor from a tumor-bearing mouse, followed by manual digestion with razor blades and chemical digestion with Liberase TM (Roche) and DNase I (Sigma-Aldrich). Digestion was stopped by the addition of EDTA. The 2250 cells were prepared by harvesting a 2250 tumor from a tumor-bearing mouse, followed by manual digestion with razor blades and chemical digestion as described above. BALB/c mice were injected with 1 × 106 2250 cells (luminal A) in Matrigel HC low-growth factor, 1 × 104 or 1 × 105 T12 (claudin-low) cells in Matrigel HC low-growth factor, or 1 × 104 T11 (claudin-low) cells in PBS. Neu-N mice were injected with 5 × 104 NT2 cells (Neu) in PBS. Tumors were orthotopically transplanted by intradermal injection into a mammary fat pad and measured as previously described (11). Blood and serum samples were harvested by cardiac puncture at the termination of the experiment.
Human breast tumor microarray data sets. All human tumor and normal tissue samples were obtained from fresh-frozen invasive breast carcinomas that were profiled, as described previously, using oligo microarrays (Agilent Technologies) (18, 19). We used the microarray data set previously published as the UNC337 data set in the NCBI's Gene Expression Omnibus (GEO GSE18229) (4). All microarray and patient clinical data are available in the University of North Carolina Microarray Database (ref. 20; https://genome.unc.edu/cgi-bin/SMD/publication/viewPublication.pl?pub_no=81). The probes for all analyses were filtered by requiring the lowest normalized intensity values in both sample and control to be greater than 10. The normalized log2 ratios cyanine-5 (Cy5) dye intensity for experimental sample/cyanine-3 (Cy3) dye intensity for reference of probes mapping to the same gene (EntrezGene ID as defined by the manufacturer) were averaged to generate independent expression estimates. The METABRIC data set contains 1,981 samples derived from breast tumors of patients participating in the METABRIC trial (39). Gene expression data were acquired by Illumina HT-12.
Mouse breast tumor microarray data sets. All mouse samples from fresh-frozen invasive breast carcinomas were analyzed as described previously, using Agilent mouse oligo microarrays (18). Data normalization and preprocessing were identical to those described for the UNC337 data set. We used 2 samples obtained from the combined data set previously published (GEO GSE3165, GSE8516, GSE9343, GSE14457, GSE15263, GSE17916, GSE27101, and GSE42640) (21). The remaining 57 samples represent newly obtained tumor samples using methods approved by IACUC guidelines. Total RNA was purified from 20 to 30 mg of mouse mammary tumor using the QIAGEN RNeasy Mini Kit following the manufacturer's protocols. RNA quantity and quality were determined using the Nanodrop spectrophotometer and Agilent Bioanalyzer, respectively. Total RNA was reverse transcribed and labeled with Cy5 dye for experimental samples and Cy3 dye for mouse reference samples using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (18). Equal quantities of labeled mouse reference RNA and tumor RNA were cohybridized overnight to Agilent microarrays, washed, and scanned; signal intensities were then determined.
Microarray data analysis. Hierarchical clustering of the UNC337 data set to group samples by intrinsic subtype was performed using the intrinsic gene list as described. (22). Centroid linkage hierarchical clustering was performed using Cluster v3.0 (23). Student's t tests for gene expression data were performed using R (24).
H&E and IF analyses of patient samples. Staining was performed on tumor-infiltrating lymphocytes in 85 breast cancer whole tissue sections (WTS) obtained from UNC hospitals surgical pathology archive. IHC and dual IF on WTS with immune infiltrates were performed with the Bond fully automated slide-staining system using the Bond Polymer Refine Detection Kit (DS9800, Leica Microsystems). Slides were deparaffinized in Bond Dewax Solution (AR9222) and hydrated in Bond Wash Solution (AR9590). Antigen retrieval was done at 100°C either in Bond Epitope Retrieval Solution 1, pH 6.0 (AR9661), or in Solution 2, pH 9.0 (AR9640). IHC slides were visualized with DAB and counterstained with hematoxylin using Bond Polymer Refine Detection. Stained slides were dehydrated. IF slides were counterstained with Hoechst 33258 (Invitrogen) and mounted with ProLong Gold antifade reagent (P36934, Life Technologies). Tumor tissue slides (n = 10 per subtype) were stained for CD8, CD4, and FoxP3 and analyzed for coexpression at the cellular level using TissueStudio on-slide cytometry to measure tumor infiltration by Tc cells (CD8+), Th cells (CD4+FoxP3-), and Tregs (CD4+FoxP3+). All samples were evaluated by pathologists blinded to the tumor subtype.
Imaging and digital image analysis. H&E and IF–stained slides (CD4, CD8, CD19, and FoxP3) were digitally imaged at 20× magnification using the Aperio ScanScopes XT and FL (Aperio Technologies). The hematoxylin and DAB OD parameters were adjusted for both algorithms. The nuclear segmentation factor and the weak positive threshold were tuned for the cytoplasmic v2 and the color deconvolution v9 algorithms, respectively. High-resolution acquisition (20× objective) of the stained slides in the DAPI, Cy3, and Cy5 channels was performed in the Aperio ScanScope FL (Aperio Technologies). Cell nuclei were visualized in the DAPI channel; CD8, CD19, and FoxP3 were visualized in the Cy3 channel; CD4 was visualized in the Cy5 channel. To determine the number of cells coexpressing FoxP3/CD4, slides were analyzed using Definiens Tissue Studio image analysis software (Architect XD v 2.0.4, Tissue Studio v 3.5).
Antibodies and flow cytometry reagents: human antibodies. Mouse monoclonal antibodies against human CD4 (clone 4B12), CD8 (clone 4B11), and CD19 (clone BT51E) were purchased from Leica Microsystems. FoxP3 (clone 236A/E7) was purchased from Abcam. CD279 (PD-1, clone J105) was purchased from eBioscience.
Antibodies and flow cytometry reagents: mouse antibodies. Flow cytometry monoclonal antibodies against murine CD4 (GK1.5 or RM4-5), CD8 (53-6.7), PD-1 (J43 or RMP1-30), CD45 (30-F11), CD62L (MEL-14), and CXCR4 (2B11) were purchased from eBioscience. CD19 (6D5) and CD44 (IM7) were purchased from BioLegend. Intracellular antibodies FoxP3 (FJK-16s), TNF (MP6-XT22), IFN-γ (XMG1.2), and Helios (22F6) were purchased from eBioscience. Cell viability was determined using Blue or Aqua Fluorescence Reactive Dye (Life Technologies).
Flow cytometry. Cells were surface stained, fixed, and permeabilized overnight using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), and intracellular staining was performed the following day according to the manufacturer's instructions. Data were acquired using MACSQuant (Miltenyi Biotec), BD Canto, or LSR II (BD Biosciences). Acquired data were analyzed using FlowJo Flow Cytometry Analysis Software (Tree Star Inc.).
Isolation of murine TILs. Murine tumors were resected and digested in Liberase TM (Roche), DNase I (Sigma-Aldrich), hyaluronidase (Sigma-Aldrich), and collagenase XI (Sigma-Aldrich), as previously described (25). Single-cell suspensions were enriched for lymphocytes by isolating cells at the interface of 44% Percoll (Sigma-Aldrich) in media and Lympholyte-M (Cedarlane) gradient. Isolated cells were stimulated for 4 hours in PMA/ionomycin prior to intracellular cytokine staining.
In vivo cell inhibition and depletion. Monoclonal antibodies used for in vivo antibody inhibition and depletion were purchased from BioXCell or obtained from the laboratory of Bruce Blazar (University of Minnesota, Minneapolis, Minnesota, USA) (anti–PD-1 mAb). Mice undergoing immune checkpoint inhibition received i.p. injection of 100 μg anti–CTLA-4 (9D9), 200 μg anti–PD-1 (J43), or isotype antibody on day –1 PTI and then every other day throughout the experiment (40, 41).
Mice undergoing Treg depletion (DEREG mice) received i.p. injections of 1 μg DT in PBS on days –1, +1, +6, and +7 (27). Mice undergoing specific inhibition of p110δ PI3K were given drug (75 mg/kg PI-3065, once daily) or vehicle (0.5% methylcellulose with 0.2% Tween 80) by oral gavage from day +1 through the duration of the experiment (26).
Treg-suppression assay. For the Treg-suppression assays, we evaluated splenic Tregs. FoxP3+GFP+ cells were sorted using a MoFlo Cell Sorter (Beckman Coulter). Stimulator cells were isolated from WT B6 splenocytes following CD90 microbead depletion (Miltenyi Biotec) and irradiation at 2,100 centigray (cGy). Responder cells were isolated from WT BALB/c mice using the Cedarlane T Recovery Column Kit. Isolated cells were then B220 and CD25 depleted using phycoerythrin-conjugated (PE-conjugated) antibodies and anti-PE magnetic bead sorting (Miltenyi Biotec). Responder cells were stained with the Cell Proliferation Dye eFluor 670 (eBioscience) and plated at varying Treg/Teff ratios. Cells were cocultured for 3 days, stained, and FACS analyzed.
Real-time PCR array. Whole tumor RNA was isolated using the RNEasy Kit (QIAGEN) and reverse transcribed. Real-time PCR was performed on the ABI 7300 (Applied Biosystems Inc.), using Master Mix from the Mouse Inflammatory Cytokines and Receptors (PAMM-011) RT2 Profiler PCR Array System (QIAGEN). Ct values were determined by ABI software. Data analysis was performed using the web-based RT2 Profiler PCR Array Data Analysis version 3.5 (QIAGEN).
AMD3100 and Cy. Alzet osmotic pumps, model 2002 (Alza), were loaded with 10 mg AMD3100 (Tocris) dissolved in PBS. Control mice received pumps loaded with PBS without AMD3100 (28). Pumps were subcutaneously implanted dorsally on day –2. Mice were then challenged with 1 × 104 T11 cells, and tumors were harvested 12 days later at approximately 20 mm2 in accordance with IACUC guidelines. For low-dose Cy studies, a single dose of 100 mg/kg Cy was given PTI. This dose was sufficient to deplete 60% of the Tregs for approximately 8 days with rebound increases above baseline at day 12 after therapy (data not shown).
CXCL12 knockdown. T11 cells were treated with 4 μg/ml Polybrene and cultured overnight with Mission shRNA Lentiviral Transduction Particles (Sigma-Aldrich) containing shRNA specific for CXCL12 or a nontarget control (scramble) at an MOI of 10. Lentiviral particles were removed the next morning and new medium was added. The following day, cells were cultured in medium containing 6 μg /ml puromycin and expanded to select for transduced cells. Cells were tested for CXCL12 production by real-time PCR and ELISA. Two different knockdown cell lines with different levels of knockdown of CXCL12, as evaluated by ELISA assay, were characterized. Knockdown of CXCL12 had no impact on the growth of T11 cells in vitro (data not shown).
Statistics. Data are presented as mean ± SEM. Statistical differences were determined using Student's t test, Mann-Whitney U test, or Kruskal-Wallis test, with Dunn's post-test for multiple comparisons where appropriate. Survival data are presented using Kaplan-Meier survival analysis. Survival results were analyzed using the log-rank test. Statistical analyses were conducted using GraphPad Prism 6 software, unless otherwise noted. Results were considered statistically significant at P ≤ 0.05.
Study approval. All animal experiments were conducted in accordance with protocols approved by the UNC IACUC. All human tumor and normal tissue samples were collected using IRB-approved protocols.
NAT and SCV designed and performed experiments, analyzed data, and wrote the manuscript. MDI and BRM performed experiments and analyzed data. WJB, KPM, SR, and JSP analyzed data. CKA and LAC provided clinical samples for analysis. BGV designed experiments, analyzed data, and wrote and edited the manuscript. CMP and JSS conceived of the project, analyzed data, and edited the manuscript.
The UNC Flow Cytometry Core Facility is supported in part by a P30 CA016086 Cancer Center Core Support grant to the UNC Lineberger Comprehensive Cancer Center. This work is supported by Breast Cancer SPORE (P50CA058223 CMP to JSS), the UNC Oncology Clinical Translational Research Training Program (5K12CA120780 to BGV), and the University Cancer Research Fund (to JSS and BGV).
Conflict of interest: The authors have declared that no conflict of interest exists.
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Version 1 (August 21, 2017): Electronic publication
Version 2 (September 1, 2017): Print issue publication


Clawback is a general term referring to title conditions used to safeguard the financial interests of an authority.
Clawback can be imposed through a charge or a legal condition being placed on the asset transfer. Conditions can range from a need to consult before disposal, to reimburse any grant or discount given, or to provide the local authority with a share in any increase in value on disposal or due to any other material change in circumstance (generally a change in use in planning terms) relating to the property. When a legal condition is placed on an asset transfer it is often referred to as an 'economic development burden'.
they can relate to the delivery of outcomes or to the asset itself.
It is important that any use of clawback provisions complies with Government guidance i.e. is "flexible and imaginative and should not create barriers to wider policy objectives" (HM Treasury, 2006). A list of factors to be considered in drafting clawback conditions, and guide to options for structuring them, can be found in HM Treasury's 2006 'Guide to Protecting Public Investments' (see below).
If a charge is to be imposed, the mechanism used is a standard security (see below), a statutory form of charge introduced in Scotland by the Conveyancing and Feudal Reform (Scotland) Act 1970 ("the 1970 Act") and the only means of securing a debt over land/heritable property in Scotland.
Conditions can be imposed that give the local authority first right of refusal over the property should the community organisation look to dispose of it for any reason (usually before it is marketed to anyone else). These are commonly known as 'pre-emption rights' and can be structured to take effect, for example, when planning permission is applied for.
Another example is that the local authority may place restrictions on changes in use of the property (in planning terms). These should be defined explicitly in terms of what the property can be used for (permitted uses) rather than what it can't.
Restrictions around how the asset is used and run on a day-to-day basis are unlikely to be appropriate and may undermine the achievement of policy objectives behind the transfer.


The 2010 Shanghai World Expo ended on October 31, 2010 and it hit a record of 73.08 million visitors during the six-month period.
The number of visitors exceeded the previous record of 64.21 million visitors attending the 1970 Osaka World Expo.
According to Expo's organizers, the Expo zone handled an average of 400,000 visitors per day. The highest number of daily visitors was 1.03 million on October 16. In addition, the number of overseas tourists exceeded 3.5 million.
The government had invested CNY28.6 billion in the Expo and the event has brought in CNY80 billion in direct tourism revenue.
The 2012 World Expo will be held in Yeosu city of South Korean.


Mission description: A raven arrived early in the morning from a small village within our boarders requesting some help with a group of bandits raiding their village, you are charged with finding these bandits and dealing with them however you see fit.
Mission details: The Village is located near to the border of the country and is raided almost every other day by a group of 5 bandits all are low level to where a genin can handle them in a fight and only wield a kunai. mission is repeatable due to multiple bandit groups being able to raid the small village and the small village can be different. when confronted the leader will send in the first to fight when he loses he will send in the second, after which the last two will jump in and the leader will try to escape while you're distracted, do not let him get away for he will go get another group of bandits and come back resulting in a failure. if he is captured the leader will attempt to cut you in on a deal, whether they are all caught or all killed mission will be complete, if one gets away mission failure.
Other: only the leader of the bandits actually talks all the others only make noises like whooping and hollering.
Chiaki had her assignment, a band of bandits had attacked a nearby village and she was needed. She gulped down her morning oolong tea and stuffed her feet into her shoes. She didn't have any ninja gear so she opted for the most mobile of her clothing. She wore a plaid yellow skirt and tight fitting collared button down shirt which was pink. Under her skirt were black leggings and as she tied her brown shoes she already started to gather her long hair. Perhaps she would have to cut it short one day, but today was not that day. The bandits wouldn't be there if she didn't hurry.
She dressed hurriedly, gave her home a once over, and nodded. The dishes would have to wait till she got home. Slamming the front door she dead-bolted it and jumped down the first three steps, taking the rest as a racehorse would, making about as much noise. One of her neighbors opened their door and shouted something incoherently as she bolted down the street, she was to meet whoever was helping her on this mission at the city gates and she didn't want to miss them. She came to a halt, already out of breath and looked around. There wasn't anyone here yet, but she knew that if she waited long enough someone would come down the street with the very same paper that she had. She tightened her forehead protector, a leaf emblazoned on it and the protective part around the back of her neck, it fit snugly like a choker as she waited in the cool morning. She wondered if perhaps she should have dressed warmer as she hopped from foot to foot trying to keep warm.
Perhaps the person was going to be late for their rendezvous or... A shudder of dread ran down her spine. Perhaps this person wasn't going to show at all. perhaps this was going to be a solo mission and they forgot to say that. She buried her nose back in the scroll reading it again. No, no no no. This couldn't be a solo mission right? She would be completely consumed by the reading, and unaware of her surroundings.
Fuyuko slowly raised herself from the bed, rubbing her eyes as she heard the sound of a bird pecking against the window. The bird was rather persistent and wouldn't stop. It was only when Fuyuko actually saw what kind of bird it was that it made sense that the pecking wouldn't stop. It was a messenger bird here to deliver a message to either herself or Takao. Sluggishly she made her way over to window, opened it up, petted the bird briefly before removing the message that was in tube on its back. With the message delivered it quickly took off again.
Fuyuko stared after the bird for a bit before she diverted her attention back to the scroll. It was addressed to her so she broke the seal and opened up the message. It was a notification of her being sent out on a mission. A simple mission, a village on the border of the country was a requesting aid as it had been harassed by a group of bandits for a while now and was no longer able to defend itself from them. She skimmed over the majority of the mission details after the initial bit, she already knew what she had to do, but wanted to make sure there were no extra details. Sure enough at the end of the mission details she was paired up with someone else. They were to meet up at the southern gate of the village before their departure.
Fuyuko rolled up the scroll and tossed it onto the desk. Both of her hands rubbing her eyes before they were dragged down her face. She disliked having to be awoken from her sleep, but duty called and that was ultimately still more important to her. This didn't mean she moaned about it internally, if she had known yesterday she would've gone to bed earlier and gotten more sleep. She let out a deep sigh before she rushed herself into her mission gear. Her hair tied in a ponytail and her forehead protector proudly strapped around the place the name implies. She wasn't sure if she did that for the fact that people seemed to carry it in different places more and more or if that's just where she liked it most herself, perhaps it was even a bit a both.
With herself set, she left a note on the table and headed out towards the gate. With a few minutes to spare the gate had come into full view. In front of it stood a rather small figure that appeared to be somewhat out of place. It wasn't just their body language that was showing that they weren't full comfortable that made them standout, the clothes they were wearing were also quite present. While she personally didn't care so much on this type of mission, but if they were on a different type of mission it might have been a lot more impactful. Fuyuko shrugged her shoulders, she was informed that they were still quite new so she didn't have any expectations to begin with, everyone has to start from somewhere after all.
Chiaki looked up from the paper in a half panicked and half embarrassed look. Sighing in relief she nodded to the girl in greeting before saying, "Hello!" The girl then asked if she was doing the same mission as her and she nodded once more in acceptance, "Yes, I suppose that I am. Do you know if there are any others that are going to be doing this mission with us or is it just you and me?" She looked around, trying to pin the situation down.
It was one of her first missions, if not for Takao she would say it was her first, but she was still a bit lost in the whole world of shinobi missions and didn't know what to do. She supposed that they should designate a leader. This girl seemed like she knew a thing or two, and so Chiaki stated, "I think perhaps you should take the lead on this mission, it's only my second one and I am more of a medical kunoichi than anything. Oh uhm… I am Guanyin Chiaki. Pleased to meet you." She held out a hand and smiled to Fuyuko. Perhaps this awkward greeting wouldn't put off the other girl.
Being up close to the girl made Fuyuko realize how small they really were. She was able to tell from a distance that they were going to be shorter than that they were themselves. However she didn't expect them to be about an entire head shorter. It was somewhat cute and their reaction just added on top of it. "As far as I'm aware it should just be you and me doing this together. I wasn't notified of there being anyone else." Fuyuko replied to their question.
The girl continued talking after hearing her reply, she stated something that was kind of obvious to herself at least, but it was probably more of reassurance thing for them. It was only a C-rank mission they were going to do, there was no reason for there to be two people on this mission if the other one wasn't capable of handling it alone. But before replying to that bit, she took the girls hand and shook her hand and smiled back. "Miki, Fuyuko, but you can just call me Fuyuko. And sure that's no problem." Her assumption from before was correct, but that didn't really matter, everyone had to start somewhere. "So seeing as your still fairly fresh to it all, if there is anything you want to know, just ask, I won't bite. But lets do it on the move, the location of the mission is quite a bit away." If there wasn't some sort of pressing matter that would make her stop and address it first, she would start the journey towards the village.
She nodded as the girl introduced herself as Fuyuko, and she was, perhaps, a bit younger than Chiaki herself. "Fuyuko-chan." She bowed her head a bit in deference, as this girl was clearly more experienced in missions than her. She nodded to the asking on the move, smiling at Fuyuko as she started to head out of the giant gates that guarded Konoha and out towards the village that was under attack. It would be faster if they didn't talk, but she had one pressing question on her mind as she took to the trees, using their great branches as a push off, leaping through them as every academy student was taught.
"What happens if someone dies on this mission?" She asked, "I mean, what happens if I accidentally…" She made a slitted motion across her throat and her eyes went wide, not particularly looking at anything, but really looking inward. "I mean, that's- that's what Ninjas do right? They kinda are the military class of Konoha, and I am not sure if I am ready for that sort of responsibility. But the bills-" She cut herself off and sighed, knowing that it was an over share.
"Sorry, sorry. You don't have to answer that. I mean, I am ready for whatever this mission brings." She gave a brave face as she kicked off from another tree, the village, luckily wasn't too far away, and the village also was going to put up as much of a fight as they could, seeing as they didn't want their things stolen as much as Chiaki didn't want them stolen. She cleared her throat and checked her hair as she jumped through the forest. Wouldn't do to get caught on a branch and fall out of a tree, especially on just her second mission.
Without there being something that prevented the two of them of at least heading towards their destination they took off. Jumping through the forests, leaves brushing against her face from time to time. Their travel had gone mostly quiet, the two of them just moving to their destination as fast as they could get their together. However at some point Chiaki broke the silence and posed her question.
It was a question that took her a couple of years back by now. It was something that had definitely crossed her mind more than once. It didn't matter how much time had passed, but she could still hear that agonizing scream from that day. From the way Chiaki asked it, not finishing her sentences and instead using motions it became clear she too struggled with it.
If no other question came Fuyuko would kick off a branch or two just a tad harder so she had pulled ahead just a smidge, just enough to make sure that Chiaki couldn't see her face. There was no need for a stranger to see the tears that started to take shape. On top of that she had no clue what kind of effect it would have on them.
Eventually the pair would reach the village and as soon as they did they could hear a commotion going on in town. "So what do you want to do? Or I mean, how do you want to approach this?" Fuyuko asked with a soft voice to ensure no one but Chiaki would be able to hear her. Seeing as the mission was relatively easy, it was a good opportunity to let her learn a bit about how to approach things.
Chiaki nodded, and waited for the eventuality of needing to kill people, she knew that she would have to do it at some point, and that she would be asked to and she hated that eventuality. But what she feared never came to pass, instead she was told that she doesn't need to kill. "I ah…" she nodded. "I believe the same thing. I actually developed a technique for just subduing people, believing that it is better to do that than to kill someone. I am going to make it stronger as I gain power too." She said, as Fuyuko kicked off to, well, probably scout ahead.
They came to the village, a small little thing on the edge of Hi No Kuni, barely in the jurisdiction of the hidden leaf. The village was bustling, and there seemed to be people running in different directions with weapons, both makeshift and no.
Fuyuko asked what Chiaki wanted to do, and the former gulped, thinking of the possibilities. It seemed like the village was built on a north to south axis, the fairway of the village was where most of the commotion was coming from, and probably where most of the revenue of the village was. She nodded to herself.
She lowered her voice to Fuyuko's level and smiled, "I have an idea. I will drive them to you using my jutsu, and we'll get them to run down the fairway to you. Do you know any subduing jutsu?" She asked, raising an eyebrow, "I think that it would be the best avenue, as most of them should run down the main street of the village. Maybe… maybe you could hide behind a cart or something and jump out and-" She cut herself off and made some nonsense hand seals and a small and faint explosion noise. She raised both of her eyebrows and grinned at Fuyuko. She hoped that would be enough of an explanation. She was nervous and wanted to not show it in front of her, and the nervousness turned into giddy energy.
Fuyuko listened patiently as Chiaki seemed to gather her thoughts, it would appear she had put her a bit on the spot, but that's not necessarily a bad thing. It would help her develop a mindset of always being ready, always thinking about how to approach things. It wouldn't take too long before the little girl, whom Fuyuko still assumed was younger than herself, had devised a plan. She listened with a lot of attention and nodded in agreement to her question if she knew any subduing techniques. The plan continued and sounded like a solid idea. Wouldn't be too hard to get a band of thugs to follow you blindly down a road. She couldn't help herself however when Chiaki stopped using words to explain the plan instead started using motions and sounds. Fuyuko tried her best to contain her laughter placing both of her hands on her lips. So instead of a laugh there was a lot of loud air blowing of her hands. When she had her giggle under control again she removed her hands and whispered back "sounds like a plan" with a big smile on her face.
She left the place they were at now, moving a couple of houses down the street and hiding in an alley way. Peeking around the corner capable of overlooking the entire main street. She made a single copy of herself with the shadow clone technique, just as an insurance policy. The clone would hide on top of the roof of the building that she was leaning against, ready in case things turned south.
Chiaki ran towards the other end of town, one, two... three bandits? They seemed to have a pattern to their looting, finding only the small things they could carry easily rather than the more expensive bigger things. She made her way to the main street and weaved a set of hand seals, lightning wound it's way through her arms and coalesced in her finger. She fired it and it zipped towards the nearest bandit, a pinpoint accuracy in her movement and hit the silver plate in his hand. He dropped it with a Yelp, his hand burned from conducting the blast.
With a crack of the air coming together again and the smell of ozone, all activity stopped. "I am Chiaki of the hidden leaf. Sent here to murder all of you bandits." She grinned as she had seen that serial killer in the hospital grin and as a show lightning crackled in her fingertips.
"Who is the first to die?"
With her head peeking around the corner Fuyuko watched intently as Chiaki made her way down the street towards the thugs. She noted that Chiaki was checking her sides, be that for getting a feel for what they were doing or making sure the houses appeared to be empty was a good thing. It could give her a feeling of security, which was very important, especially in the role of bait.
The palm of her hand slapped her forehead as she heard what Chiaki proclaimed and while the real her was to busy facepalming her clone moved up over the rooftops closer, just in case something went wrong the gap to help her would be smaller. "Why would she proclaim something like that, if she's not committed to those words?" was all she thought. Her palm slid down her face as she continued peeking to see what was going on.


text:
Four Weird Tales
0.0 0 5 Autor: Algernon Blackwood
This collection assembles four of Blackwood's greatest stories: "The Insanity of Jones," "The Man Who Found Out," "The Glamour of the Snow," and "Sand." Algernon Blackwood was truly one of the progenitors of the weird fiction/fantasy genre!
Język: Angielski Kategoria: Thriller Tłumaczenie:
Wydawca: Ozymandias Press
Chaos at Crescent City Medical Center
We'll Make You an Offer You Can't Refuse
Ice Trap: A Novel of Psychological Suspense
The Last Man
The Haunter of the Ring & Other Tales
The Man Who Was Thursday
Ghost Stories of Henry James
The Ripples of Ann Arbor
Varney, the Vampyre
Lawless: A Novel Based on a True Story
The Complete Fiction of H.P. Lovecraft
The Opposite of Art
Lethal Experiment
The Ballad of John MacLea
Mind Switch
Miracles, Inc.
The Last Night at Tremore Beach
Havana Bay
Edgar Allan Poe: Complete Tales and Poems: The Black Cat, The Fall of the House of Usher, The Raven, The Masque of the Red Death...

redpajama_set_name: RedPajamaCommonCrawl


text:
Hospitals And Gps
Primary Health Care faces major wages bill for Dorevitch Pathology unit
Carrie LaFrenzSenior Reporter
Updated Sep 14, 2018 — 3.36pm, first published at 8.53am
Primary Health Care has been forced to offer a pay rises of up to 20 per cent to nearly 1800 workers in its Victorian pathology division after the Fair Work Commission intervened in an 11-year workplace dispute.
The workers have also been awarded significant increases in allowances and backpay after a decision by the Commission's full bench.
The Fair Work terminated industrial action in August 2017 after the Victorian government raised concerns about that a strike by 600-odd pathology workers threatened community welfare.
Health Workers Union has long claimed that Dorevitch Pathology workers were some of the lowest-paid pathology workers in the country. Rohan Thomson
Health Workers Union secretary Diana Asmar said on Friday that the Fair Work decision will deliver workers a pay rise of up to 20 per cent and increase allowances by up to 30 per cent. Workers will also be in line for backpay to July 2017.
"This result is long overdue," she said. "The Dorevitch workplace agreement expired over 11 years [ago]. Our members have effectively been living with a 2007 level of income to pay 2018 costs. They've been left behind. This pay rise is a far cry from the zero per cent that was offered after a year of fruitless negotiations."
The decision applies to pathology collectors, laboratory assistants, couriers, clerical, stores and maintenance employees.
The union has long claimed that Dorevitch Pathology workers were some of the lowest-paid pathology workers in the country, making less than $21 an hour, despite playing an important role in maintaining the health of the community.
"Dorevitch and their parent company, Primary Health, have proven themselves to be an immoral employer," Ms Asmar said.
"Their entire business model is based on suppressing the wages and conditions of their employees."
'Primary takes these matters seriously'
Sources claimed the decision would cost Primary about $10 million per annum in higher wages, and suggested it may cause staff in other states to pursue similar pay deals.
In a statement to the market on Friday, Primary said that before any mitigation strategies the decision had the potential to drag down its underlying net profit in 2018-19 by $4.5 million. "Importantly, the company aims to fully offset the impact through a range of mitigation strategies," Primary said.
"In addition, the impact of the determination on underlying and reported earnings for full year 2018 is estimated to be approximately $4.8 million."
Dorevitch Pathology provides services to about 20 public hospitals and performs work for Justice Victoria and Corrections Victoria.
At the time of industrial action last year Dorevitch Pathology boss Neville Moller suddenly left Primary.
Primary on Friday acknowledged Fair Work's negative commentary about Dorevitch's historical approach to bargaining.
"Primary takes these matters seriously and had already made a number of changes at Dorevitch, including installing a new CEO in 2017," it said.
"Primary has also introduced a range of initiatives with the aim of promoting a better workplace culture throughout the group, including at Dorevitch. These include a new purpose mission and values launched group‐wide over July and August 2018."
Sources close to Primary said these are legacy issues, and under new Primary boss Malcolm Parmenter, along with head of human resources Yvette Cachia, programs were being implemented to make sure staff are rewarded equitably and sustainably, and therefore they are not expecting unrest in other parts of the business.
Primary's shares were flat at $2.90.
Carrie LaFrenz has more than 10 years experience as a business journalist having previously covered healthcare, retail/consumer goods, industrials and agribusiness. She is based in our Sydney newsroom. Connect with Carrie on Twitter. Email Carrie at carrie.lafrenz@afr.com.au


Pictures above: Insome opinion the saucer on the pictures a VRIL-Disc - other people say it it a early Haunebu-version.
Anyway - Jan van Helsing thinks this is a real picture of a german flying saucer.
Is there a real story behind the fake informations you will find anywhere in the Web?
And if YES - What pictures are REAL and what pics are FAKE?
please read the german books "NATIONALE SICHERHEIT"
and "GEBOREN IN DIE LÜGE" from Dan Davis.
and in books are fakes and illustrations.
There are very few creators who have the possibility of being genuine.
Many of them are also found on the Internet in very poor quality.
Some of these images can be found here.
Above: This image should show a german Flying Saucer from the Inside.
Like most pictures of HAUNEBU in the Web.
Anyway - important ist - Jan van Helsing and some other researchers thinks these are real pictures of a german flying saucer.
of the 130 photos who are - maybe - real.
Above: Later "HAUNEBU II"-Version in flight.
Above: An early predecessor of a HAUNEBU-II.
Above: An early version of the "HAUNEBU II" at dusk just a few feet above the ground.
Above: "HAUNEBU II" from below in flight.
IN A VIEW DAYS HERE!
presumably Faked or Illustrations, no originals.
who owns the originals, and ask him for an opinion.
BACK TO HAUNEBU-MAIN PAGE - CLICK HERE!


AL-HODAYDA(YEMEN), March 30, 2015 (Xinhua) -- Chinese citizens wait to get on the a navy frigate to leave Yemen, in the al-Hodayda port in western Yemen, on March 30, 2015. Four hundred and forty-nine Chinese nationals left the Yemeni coastal city of Al-Hodayda on Monday aboard a Chinese navy frigate. On Sunday, 122 other Chinese nationals were evacuated from the Yemeni city of Aden and have already arrived in Djibouti.


NEW YORK, Feb 13 — Global esports revenues will hit US$1.1 billion (RM4.4 billion) in 2019, up 27 per cent since last year amid ballooning revenues from advertising, sponsorship and media rights to competitive video gaming, a report said yesterday.
Brand investments through those three avenues will make up 82 per cent — or US$897 million — of total revenues. In all, brand support will have nearly tripled since 2015, according to data from Newzoo, a gaming industry analytics firm.
North America will generate US$409 million of esports revenues in 2019, the most of any region, Newzoo's report found. China will generate 19 per cent and South Korea 6 per cent, with the rest of the world comprising the remaining 38 per cent.
While advertising is the bulk of revenue, merchandise and ticket sales are also expected to grow 22 per cent to nearly US$104 million, though game publisher fees will likely drop 3 per cent to US$95 million, the report showed.
The total audience — comprised of both enthusiasts and occasional viewers — is expected to grow 15 per cent to 454 million.


i run windows 2000 and my desktop wallpaper comes up, but all my desktop icons are gone. I should tell you the taskbar won''''''''''''''''''''''''''''''''t even show. I use webroot antivirus. Can some one help me?
HiCustomer Thank you for asking your question on Just Answer. It sounds to me like explorer is crashing for some reason. Unfortunately, this can happen for reasons too numerous to list. If this is a new problem, I would start by removing any software you've recently added - especially drivers and programs that start with Windows.
A temporary fix would be to just restart explorer. You can do this through the Task Manager (Ctrl+Shift+Escape). In Task Manager, go to File>New Task (run), then type in "explorer". All of your icons and the taskbar should reappear within a few seconds.
Something else to try is Safe Mode. This is an easy way to tell whether we're dealing with software clashes, or something more serious, like data corruption. If everything works in Safe Mode, then it should be fixable by going through startup items. You can get into Safe Mode by repeatedly hitting F8 (don't hold it down) when your system boots. You should be presented with a boot menu. Select "Safe Mode", and hit enter.


With many notable brands succumbing to store closures and bankruptcies in recent years, retailers now face intense pressure to deliver a more engaging in-store experience. Challenges abound, but creating successful customer engagements remains within reach. Learn how you can enable your workforce to deliver a personalized and unified experience that delights your customers.
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An intensive long day hike to the source of the Nooksack River: Nooksack Cirque.
This long and strenuous hike in and out of the the Upper North Fork drainage is largely off trail on rocky terrain. Plan on a full day, hiking at a good pace, and wearing hiking boots. At the Cirque we will perform a ritual in the sacred space of many hanging glaciers and waterfalls.
A very early start is essential to have sufficient daylight for this long day hike. Meet at the Dharma Hall for carpooling at 5:00am or at Nugent's Corner at 5:30am. If you are unsure of your fitness for this hike please contact Bob before registering.


text:
Нордхалбен () град је у њемачкој савезној држави Баварска. Једно је од 18 општинских средишта округа Кронах. Према процјени из 2010. у граду је живјело 1.932 становника. Посједује регионалну шифру (-{AGS}-) 9476159.

Географски и демографски подаци

Нордхалбен се налази у савезној држави Баварска у округу Кронах. Град се налази на надморској висини од 586 метара. Површина општине износи 21,9 -{km²}-. У самом граду је, према процјени из 2010. године, живјело 1.932 становника. Просјечна густина становништва износи 88 становника/-{km²}-.

Референце

Спољашње везе

 Званични сајт општине
 Њем. Савезни завод за статистику
 Стална конференција градова и општина
 -{KommOn}- - Информациони систем градова, општина и округа.
 План насеља на мапи (-{Mapquest}-)

Нордхалбен
Википројект географија/Насеља у Немачкој

redpajama_set_name: RedPajamaWikipedia


text:
Ha due fratelli minori: Mikael, che ha anch'egli intrapreso la carriera tennistica, e Rafael, nato nel 2006, che ha dichiarato di volere seguire le loro orme.

Carriera

Junior
A livello giovanile raggiunge la finale dell'Orange Bowl 2012 e nel gennaio successivo ottiene la sua migliore classifica grazie al quinto posto.

Professionista
Nel 2014 vince cinque Futures e guadagna oltre cinquecento posizioni nella classifica ATP; inizia a giocare tornei del circuito principale nel 2015. La prima possibilità gli viene concessa a Chennai, grazie a una wild-card, dove elimina Igor Sijsling per poi arrendersi nell'incontro successivo a Gilles Müller. Fa il suo esordio negli Slam a Melbourne, dove riesce a superare le qualificazioni senza troppe difficoltà e una volta nel tabellone principale costringe al quinto set il più esperto Soeda prima di arrendersi. Durante il 2015 riesce a superare le qualificazioni per tutti e quattro gli Slam, tuttavia non ha mai vinto un incontro nel tabellone principale.

Il 23 ottobre 2016 vince il suo primo titolo in doppio in coppia con il fratello Mikael Ymer, al torneo di Stoccolma superando in finale Michael Venus e Mate Pavić con un doppio 6-1.

Statistiche

Doppio

Vittorie (1)

Tornei minori

Singolare

Vittorie (11)

Finali perse (3)

Doppio

Finali perse (1)

Note

Altri progetti

Collegamenti esterni


This curved wedding ring features 9 round, channel set stones with 1 carats in total. The ring is 2 millimeters wide and weighs approximately 5.23 grams. This contour band is made to fit a round or princess cut solitaire from 0.5 carats up to 1 carats, or a marquise solitaire from 0.25 carats up to 1 carat. Curved rings are perfect for most style engagement rings. If you are unsure what will fit with your engagement ring, please add Virtual Fit onto your order so we can email you images of our ring with yours for approval before shipping.
See below for which sizes fit this ring wrap if you do not select PerfectFit: Round Stone: 0.5 to 1 Carat, Marquise Stone:0.5 to 1 Carat Mounted in Traditional Solitaire.


text:
Another day, another candidate for President
Conservative Republican Mike Huckabee, seeking to repeat the success of another former governor from Hope, Ark., said Sunday he is taking the first step in what he acknowledged is an underdog bid for the White House in 2008. Huckabee, who left office Jan. 9 after serving 10 1/2 years as governor of a Democratic-leaning state, faces steep odds in a crowded GOP field that includes well-known and well-funded hopefuls such as Sen. John McCain of Arizona, former Massachusetts Gov. Mitt Romney and former New York City Mayor Rudy Giuliani.
Conservative Republican Mike Huckabee, seeking to repeat the success of another former governor from Hope, Ark., said Sunday he is taking the first step in what he acknowledged is an underdog bid for the White House in 2008.
Huckabee, who left office Jan. 9 after serving 10 1/2 years as governor of a Democratic-leaning state, faces steep odds in a crowded GOP field that includes well-known and well-funded hopefuls such as Sen. John McCain of Arizona, former Massachusetts Gov. Mitt Romney and former New York City Mayor Rudy Giuliani.
"One of the reasons that I'm running for president is because I think that America needs folks who understand what it is to start at the bottom of the ladder and climb their way to the top," Huckabee said. "We've got a lot of people who are born on third base and think they've hit a triple.
"America loves an underdog. America loves people who have had to struggle and for whom every rung of the ladder has been sometimes three rungs up and two back down, Thank God for the one you've gained, and keep climbing," Huckabee said.
He planned to travel to Iowa, an early nominating state, on Tuesday and Wednesday.
The 51-year-old Huckabee, who took over as governor at the height of Bill Clinton's Whitewater scandal, comes from Hope, Ark., as does the former Democratic president.
Huckabee is setting up an exploratory committee that will allow him to raise money and hire campaign staff in an effort to gauge his prospects.
Huckabee is a staunch opponent of abortion rights and gay marriage, but faces a tough fight from other conservatives in the field for support from the GOP's right flank. This is an important voting bloc in the nominating contests.
Kansas Sen. Sam Brownback, a favorite son of the religious right, is already in the race. Other conservatives, such as California Rep. Duncan Hunter and Colorado Rep. Tom Tancredo, will battle for their share of the vote.
While Arkansas is a predominantly Democratic state, Huckabee won two full terms in landslides. He championed tax increases for public schools, expanded state insurance programs for the children of the working poor and opposed banning state services for illegal immigrants.
Nationally, Huckabee is perhaps best known for his dramatic weight loss and his emphasis on healthy lifestyles. He shed 110 pounds after being diagnosed with diabetes. He also saw his political profile rise when he headed the National Governors Association for one term.
Since he left office, Huckabee has been on a nationwide tour to tout his book, "From Hope to Higher Ground: 12 Stops to Restoring America's Greatness." With chapters on taxes and foreign policy, Huckabee's book lays out his potential talking points for a presidential campaign.
Huckabee made his official announcement during an interview on "Meet the Press" on Sunday.
Copyright © 2007 The Associated Press


If you will like advertise in FUSE, just contact us on promo@fusemagazineonline.com.
We have dealt with some amazing companies, brands, photographers and models who advertise with us.
All of the three options above include promotion on our social networks for each issue.
If you are a clothing, lingerie or swimsuit and will like to sponsor one of our photo shoot. We also do accept stock of clothing and merchandise to be used in our shoots. Email us at promo@fusemagazineonline.com if you are interested (usually combined with Advertorial option).


Cloud computing allows access to your computer from anywhere over a secured data network. STC keeps your business data protected with our hybrid cloud product. Access critical business functions and share documents among groups from the cloud – virtually anywhere.
Security cannot be overlooked when setting up your systems for flexible easy access. Managing risk is always a concern. If proper attention is not given you could be leaving the door open for going out of business due to an unforeseen catastrophe. Network assessments and risk management survey and reporting gives you the details you need to prioritize your resources to limited risk in your organization and secure your sensitive systems and data. Many businesses are governed by HIPAA, PCI or other regulations. If you must meet industry, government or corporate standards a security assessment by STC can give you the network, security, risk and document insights that you need to mitigate risk.


text:
Международный аэропорт Вале́нсия () — второй по пассажиропотоку аэропорт в Валенсии, после аэропорта Аликанте и десятый по пассажиропотоку в Испании. Также известен как аэропорт «Манисес» (Manises Airport). Расположен в 8 километрах от города Валенсия, имеет воздушное сообщение с 20 странами, пассажиропоток 8,53 млн пассажиров за 2019 год.

История аэропорта
Идея создания аэропорта возникла в 1927 году из-за сильного увеличения населения и пассажиропотока в Валенсии. Местный морской вокзал был дооборудован для временного приема гидропланов. Для строительства аэропорта власти сначала рассматривали узкий перешеек, разделяющий Ла Альбуферу (озеро, которое называют «Внутреннее море Валенсии» и побережье Балеарского моря. Однако из-за прав на землю вынуждены были отказаться от этого варианта и строительство началось рядом с городом Манисес в 8 км от Валенсии.

Официально аэропорт был открыт в 1933 году, однако первый рейс состоялся 1 сентября 1934 года перелетом Мадрид — Валенсия.

В 1983 году был построен новый пассажирский терминал, заменивший старое здание, возведенное в середине 60-х. Новое здание частично использует солнечную энергию, получаемую от солнечных батарей.

В марте 2007-го появился новый терминал для региональных рейсов, начато строительство терминала для частных джетов, и терминала стоянки транспортных средств. Также была расширена взлетно-посадочная полоса, и появились дополнительные рулежные дорожки. В 2009-м году аэропорт принял первый трансатлантический рейс — Boeing 757 авиакомпании Delta Air Lines рейсом из Нью-Йорка.

В 2012-м году главный терминал был расширен еще двадцатью стойками регистрации и новым залом для получения багажа, а также новым зданием для парковки автомобилей.

Примечания

Аэропорты Испании
Валенсия


A simple method to use your laptop as a Raspberry Pi display if you don't have an HD monitor.
I came up with this tutorial one day when my friend Suvigya and I were struggling to work on the Raspberry Pi, because we did not have any HD displays. Hence, I came up with this tutorial so others in the same situation would be able to use their laptop display as a monitor for their Raspberry Pi.
As we know, Raspberry Pi is known as the "Pocket-Size PC", but for debugging and project purposes it's cumbersome to carry an additional display for it. Also, many of us don't have access to an HDMI display, so, we figured out a way to easily connect a Raspberry Pi to a laptop display. Sit back and enjoy this simple tutorial!
Open ".config" folder from the Pi's: user folder (it is a hidden folder).
Next time you reboot your Pi, vncserver will start automatically and will seamlessly connect your Raspberry Pi to a laptop display.
Whenever you want to do something with your Pi, just connect it to your laptop with an ethernet cable and power it. Then open VNCViewer, mention the IP address of your Pi, and you can use your laptop's display as the Raspberry Pi's monitor.


Morning and afternoon appointments available.
Maria Rapp, DC, practicing chiropractic for 25 years. Owner of Chiropractic Wellness Center in Salina, KS with her husband, Jeffrey Rapp, DC. Clinic offers chiropractic, acupuncture, massage therapy, thermography, cold laser therapy, detoxification foot baths, nutritional supplements, and Young Living essential oils.


Last year, we created our first annual report to give an overview of the work of the Board and staff. We received such great feedback that we'll continue to create an annual report each spring. The annual report is a way for us to let you know the big-picture things we are working on, be accountable with our finances, and give a snapshot of the work of UTO. You can download your copy of the annual report at www.episcopalchurch.org/uto.