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We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6Chigh) and granulocytic-like (CD11b+Gr1high) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1high cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ–dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.
In clinical transplantation, lifelong immunosuppression is often necessary to control graft rejection but could be associated with life-threatening complications, including opportunistic infections, malignancies, and cardiovascular diseases (1). Therefore, to establish and maintain donor-specific transplant tolerance is highly desirable and remains an unmet need. Previous work in our laboratory has demonstrated that infusions of donor splenocytes (SPs) treated with the chemical cross-linker 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (ECDI) induce long-term donor-specific tolerance to islet allografts (2–4), and, when combined with a short course of rapamycin or anti-CD20, also to heart allografts and islet xenografts (5), respectively. A first in-human clinical trial based on the same principle using peptide-coupled autologous cells in patients with multiple sclerosis was recently published and established the clinical feasibility, tolerability, and safety of this novel tolerance strategy (6). Ongoing preclinical studies in nonhuman primate models of allogeneic and xenogeneic transplantation are attempting to establish the efficacy of this tolerance strategy for clinical transplantation. Mechanistically, treatment of splenocytes with ECDI induces their apoptosis and phagocytosis by recipient splenic phagocytes (3, 7), and the subsequent graft protection appears to be dependent on the presence of Gr1+ cells and the enzyme IDO (8). However, the direct effect of donor ECDI-SPs on the Gr1+ cell populations and the source of IDO in this model remain unclear.
Myeloid-derived suppressor cells (MDSCs) bearing the cell surface markers CD11b and Gr1 have been described in cancer and inflammation (9). Although a barrier to promoting antitumor responses, they have been shown to play an important role in establishing allograft tolerance (10, 11). Murine CD11b+Gr1+ cells can be subdivided into monocytic or granulocytic MDSCs according to their surface expression of the components of the Gr1 Ag, Ly6C and Ly6G, and are defined as CD11b+Ly6ChighLy6G− and CD11b+Ly6CINTLy6Ghigh cells, respectively (12). A number of conditions, including inflammation, infection, and tumors, can induce MDSCs [reviewed in (13)]. They suppress T cell proliferation in both Ag-dependent and independent manners through a variety of effector mechanisms, including NO, arginase, and reactive oxygen species (12, 14, 15); and they promote regulatory T cell (Treg) induction through production of IL-10, TGF-β, and IDO (16, 17).
There is also evidence that most MDSC subsets require IFN-γ, both for their induction and effector function, through IFN-γ–inducible genes such as inducible NO synthase (iNOS) (18). Studies in transplantation have demonstrated that IFN-γ can be protective via a variety of mechanisms, including mediating the suppressive function of MDSCs (10, 19–21). The potential sources of such IFN-γ are thought to be macrophages, T, NK, and/or NKT cells and can also result from phagocytosis of apoptotic cells in the spleen (22). More recent studies have suggested that immature myeloid cells expressing Gr1 can themselves produce IFN-γ after pathogen infection for host protection (23).
In this article, we show that infusions of apoptotic donor ECDI-SPs induce an increase in two cell populations that resemble monocytic and granulocytic MDSCs. These cells are distinct in their phenotype and function and protect cardiac allografts in part through IFN-γ production themselves and subsequent IFN-γ–mediated pathways of T cell suppression. Our study thus describes a novel therapeutic approach for the manipulation of these suppressor cell populations to facilitate the development of transplant tolerance.
Materials and Methods
Eight- to 10-wk-old male BALB/c (H-2d), C57BL/6 (H-2b), and SJL/J (H-2s) mice were purchased from The Jackson Laboratory and Harlan. All mice were housed under specific pathogen-free conditions at Northwestern University, and protocols were approved by Northwestern University Institutional Animal Care and Use Committee.
Preparation and injection of ECDI-SPs
Donor (BALB/c) splenocytes were treated with ECDI, as previously described (2). Briefly, spleens were processed into single-cell suspensions, and erythrocytes were lysed. Splenocytes were incubated with ECDI (Calbiochem; 150 mg/ml per 3.2 × 108 cells) on ice for 1 h, followed by washing. A total of 108 cells was injected i.v. into recipient C57BL/6 (B6) mice on days −7 and +1 with reference to transplantation on day 0.
Heterotopic cardiac transplantation
Abdominal heart transplantation was performed, as previously described (24). BALB/c (donor, H-2d) to C57BL/6 (recipient, H-2b) combination was used. In some cases, SJL/J mice (H-2s) were used as donor for third-party heart allografts. Briefly, the donor heart was excised en bloc via median sternotomy. The ascending aorta and pulmonary artery of the donor were anastomosed end to side to the recipient abdominal aorta and inferior vena cava, respectively. Direct abdominal palpation of the heart beating was used to assess graft survival. Rejection is defined by loss of palpable cardiac impulses.
Ab treatment
In experiments in which cardiac recipient B6 mice were treated with the anti-Gr1 Ab, the Ab (clone RB6-8C5; BioXCell) was injected i.p. at a dose of 200 μg on day −8, followed by 100 μg on days −7, −3, −1, and +1 with reference to transplantation on day 0 (10, 25–27).
Cell isolation and culture
For isolating cells from the spleens and the cardiac allografts, animals were sacrificed, and spleens and cardiac grafts were excised and digested at 37°C with collagenase type IV (Worthington) for 25 min, followed by lysis of erythrocytes. For graft cells, leukocytes were further enriched by density centrifugation (Lympholyte; Cedarlane) to remove the myocytes. For functional assays, Gr1+ cells were enriched by negative selection using the following biotinylated Abs (clones): B220 (RA3-6B2), CD4 (GK1.5), CD8α (53-6.7), Ter119 (erythroid cells), CD49b (DX5), and CD25 (7D4), and streptavidin-conjugated magnetic beads, according to manufacturer's instructions (Miltenyi Biotec). Cell purity was determined by flow cytometry and ranged from 70 to 90%. For indicated assays, Gr1high and Ly6Chigh cells were further purified by FACS to a purity of >98%. All cell cultures were performed in RPMI 1640 (Life Technologies) supplemented with 10% FCS (Life Technologies), 1% PenStrep, 1% L-glutamine (Life Technologies), 1% HEPES (Lonza), 0.5% gentamicin (Life Technologies), and 0.1% 2-ME (Life Technologies). Splenic CD4+ or CD8+ T cells were isolated by negative selection first using the following biotinylated Abs (clones): CD11b (M1/70), Gr1 (RB6-8C5), Ter119, CD49b (DX5), and B220 (RA3-8B2), followed by positive selection for CD4 or CD8.
Proliferation assays
Splenic CD8+ T cells were purified, as described above, labeled with 0.5 mM CFSE (Life Technologies), and plated at 1 × 105 per well as responder cells for in vitro stimulation. T cells were activated by either anti-CD3/28 Dynabeads per manufacturer's instructions (Invitrogen), or 5 × 105 irradiated donor (BALB/c) APCs. FACS-sorted suppressor cells were added at a 1:1 ratio with the CFSE-labeled T responder cells and incubated at 37°C for 96 h. T cell proliferation was measured by CFSE dilution. For indicated studies, FACS-sorted suppressor cells were either pretreated at room temperature for 30 min with 10 μg/ml anti–IFN-γ (clone XMG1.2; BioXCell) prior to addition to the proliferation assays, or added to the proliferation assays in the presence of 5 mM L-NMMA or D-NMMA (Cayman Chemical,) or 2 mM 1-methyl-DL-tryptophan (1-MT; Sigma-Aldrich) or vehicle (2% carboxymethylcellulose). For suppression assays by graft Gr1high and Ly6Chigh cells, CFSE-labeled responder CD8+ T cells were plated at 1 × 104 per well and cocultured with 1 × 104 anti-CD3/28 Dynabeads or 5 × 104 BALB/c APCs and 1 × 104 FACS-sorted suppressor cells from the graft. T cell proliferation was determined by CFSE dilution after 96 h.
Flow cytometry
Protein measurement and cytokine detection
Tissue cytokines were analyzed by 32-Plex multiplex assays (Millipore). Tissues were homogenized to obtain cell lysates and centrifuged at 13,000 rpm for 2 min, and the soluble portion was collected and analyzed by the multiplex assays per manufacturer's instructions. Results were normalized to the amount of total protein as measured by the Bradford assay (Pierce Biotechnology).
Quantitative RT-PCR
Total RNA was extracted using the RNeasy kit (Qiagen), according to manufacturer's instructions. Total RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). RT-PCR amplifications were performed using TaqMan Universal Master Mix II and TaqMan gene expression assays (Applied Biosystems). The reactions were run at 50°C for 2 min, followed by 95°C for 10 min and 40 cycles of 95°C for 15 s, and 60°C for 1 min. Reactions were run on the 7500 Real Time PCR System, and data were analyzed using 7500 v2.0.1 software (Applied Biosystems). The Δ cycle threshold values for each duplicate sample were calculated with reference to 18S.
Graft histology and immunohistochemistry
Grafts were snap frozen in OCT compound with liquid nitrogen. All sections were 8 μm thick. Frozen sections were blocked with avidin/biotin blocking kit (Vector Laboratories), followed by staining with anti-mouse Foxp3 mAb (1:400, rat IgG2a, κ clone FJK-16s; eBioscience) or anti-mouse CD8 (1:250, rat IgG2a, κ clone 53-6.7; BD Biosciences). Samples were then stained with biotinylated goat anti-rat Ig for Foxp3 (1:200, goat Ig clone polyclonal; BD Biosciences) or biotin-SP-AffiniPure donkey anti-rat Ig for CD8 (1:250; Jackson ImmunoResearch Laboratories). Visualization of Foxp3 and CD8 was performed with Vectastain ABC kit (Vector Laboratories) and diaminobenzidine substrate kit (BD Biosciences).
Statistical analysis
Significance between groups was calculated by Student t tests, one-way ANOVA, or the Wilcoxon rank sum test, as appropriate. A p value <0.05 was considered significant. All statistical analysis was performed using Prism v5.0 (GraphPad).
Infusions of donor ECDI-SPs expand two cell populations in the spleen that resemble monocytic- and granulocytic-like MDSCs
To determine whether allogeneic ECDI-SP infusions could expand cells bearing features of MDSCs, naive B6 mice were injected with either one or two doses of BALB/c ECDI-SPs, and the spleens were harvested for phenotypic analysis for CD11b+Gr1+ cells on the indicated day (Fig. 1A). Clone RB6-8C5 was used to stain for Gr1. As cells expressing the Gr1 Ag can be subdivided according to their relative expression of Ly6C, a component of the Gr1 Ag (28), clone HK1.4, was used to further stain for Ly6C. As shown in Fig. 1B and 1C, one dose of BALB/c ECDI-SPs induced a significant increase in the percentage as well as the total numbers of the CD11b+Gr1INTLy6Chigh (hereafter referred to as Ly6Chigh) cells and the CD11b+Gr1highLy6CINT (hereafter referred to as Gr1high) cells. The total number of the Gr1high cells was further increased after an additional dose of BALB/c ECDI-SPs (Fig. 1C). In contrast, injection of syngeneic B6 ECDI-SPs did not significantly increase either population (Fig. 1B, 1C). Phenotypic analysis (Fig. 1D) revealed that the Ly6Chigh cells expressed CCR2 and F4/80, a marker profile consistent with that of monocytic-like MDSCs, whereas the Gr1high cells expressed Ly6G (stained with clone 1A8) and exhibited a high side scatter resembling granulocytic-like MDSCs (Fig. 1D). Other markers associated with the MDSC phenotype such as CD124 and CD115 were also measured, but were not expressed by either population (data not shown). Comparison of cells from naive untreated versus BALB/c ECDI-SP–treated animals revealed that markers of these two populations were not changed by a single injection of BALB/c ECDI-SPs (data not shown) or two injections of BALB/c ECDI-SPs (Fig. 1D). Collectively, these data suggest that treatment with allogeneic ECDI-SPs expands two cell populations in the spleen that resemble monocytic- and granulocytic-like MDSCs.
ECDI-SP–expanded Ly6Chigh and Gr1high cells suppress T cell proliferation
To further characterize ECDI-SP–expanded Ly6Chigh and Gr1high cells and to determine their similarities to MDSC populations, in vitro T cell suppression assays were performed. In this study, we primarily focused on CD8+ T cells, as they are the dominant effector cells in the graft rejection in our model (8). As shown in Fig. 2A, both subsets significantly inhibited CD8+ T cell proliferation in response to anti-CD3/28 stimulation, with the Gr1high cells suppressed more efficiently. The unexpanded Ly6Chigh and Gr1high cells from naive untreated mice were also tested and showed a similar pattern of suppression of CD8+ T cell proliferation (Fig. 2A, right bar graph), albeit slightly less potently than the expanded Ly6Chigh and Gr1high cells from ECDI-SP–treated mice. MDSCs are known to exert their effects in part through cytokine production (29). We next examined the cytokine profiles of the ECDI-SP–expanded Ly6Chigh and Gr1high cells after two infusions of ECDI-SPs. Neither IL-12 nor IL-10 was produced by either population (data not shown). Interestingly, IFN-γ was produced by the Gr1high cells (Fig. 2B), and they were the predominant source of IFN-γ within the CD11b+ fraction (Fig. 2C). Furthermore, this IFN-γ production was not detected in Gr1high cells from naive untreated mice (Fig. 2B, 2D), was expressed at a very low level after one dose of ECDI-SPs, but increased significantly after the second dose of ECDI-SP (Fig. 2D).
IFN-γ and IFN-γ–inducible genes are critical for the suppressive function of ECDI-SP–expanded Gr1high IFN-γ+ cells
We have previously demonstrated that pharmacological inhibition of IDO resulted in the loss of protection of cardiac allografts provided by infusions of donor ECDI-SPs, and that depletion of Gr1+ cells in the same model was concomitant with a reduction of IDO+ cells in the cardiac allografts (8). However, the exact source of IDO remained unclear. IDO is known to be an IFN-γ–inducible gene in other cell populations such as dendritic cells (30). Given that ECDI-SPs induce Gr1high cells to produce IFN-γ, we next sought to determine whether IDO could be produced by these Gr1high IFN-γ+ cells. CD11b+Gr1high cells from untreated mice or mice treated with ECDI-SPs were FACS sorted to a purity of >98%. Quantitative RT-PCR analysis of this cell population revealed that the Gr1high cells from untreated animals did not express ido, but they did modestly increase their ido expression after incubation with rIFN-γ (p = 0.008). However, after treatment with two doses of ECDI-SPs, not only was the baseline ido expression significantly higher than that of the control cells, there was a further significant increase after IFN-γ stimulation (p = 0.039) (Fig. 3A). iNOS is another IFN-γ–inducible gene and a known effector molecule of MDSCs. As shown in Fig. 3B, in response to IFN-γ stimulation, the Gr1high cells from ECDI-SP–treated mice showed a significant increase in the expression of nos2, the murine equivalent of inos (p = 0.033). This increase in response to IFN-γ was not observed in the Gr1high cells from untreated animals (Fig. 3B). Expression of arginase 1 and heme oxygenase-1, two additional molecules associated with MDSC function, was similarly examined but did not appear to be induced by treatment with either ECDI-SPs or IFN-γ stimulation (data not shown). Collectively, these data indicate that, compared with Gr1high cells from naive untreated mice, ECDI-SP–expanded Gr1high cells are induced to produce IFN-γ themselves and have a further enhanced capacity to upregulate the expression of IFN-γ–inducible genes. These characteristics may therefore form a positive feedback loop to enhance their suppressive function in vivo in addition to their increase in numbers by the ECDI-SP treatment.
To determine whether IDO, iNOS, or IFN-γ played a role in the suppression mediated by the Gr1high expanded by ECDI-SP treatment, we performed similar suppression assays, as described in Fig. 2. Addition of pharmacological inhibitors to IDO (1-MT) or iNOS (L-NMMA) to the suppression assays partially reversed Gr1high cell–mediated suppression (Fig. 3C). Inhibition of arginase 1 did not affect T cell suppression (data not shown), consistent with the lack of its expression, as determined by qRT-PCR. Most strikingly, pretreatment of Gr1high cells with an anti–IFN-γ–neutralizing Ab completely abrogated their suppressive function (p < 0.0001) (Fig. 3D). Collectively, these data indicate that ECDI-SP–expanded Gr1high cells critically depend on the presence of IFN-γ and its downstream effector molecules to exert their suppressive function.
Cardiac allografts from recipients treated with ECDI-SPs show enhanced accumulation of Ly6Chigh and Gr1high IFN-γ+ cells
To determine how the Ly6Chigh and Gr1high cells behave after cardiac transplantation in recipients with or without ECDI-SP infusions, allogeneic heterotopic cardiac transplants were performed. Spleens and grafts from untreated control or ECDI-SP–treated animals were harvested 7 and 21 d after transplantation. In the spleen (Fig. 4A), Ly6Chigh cells significantly increased in percentages and total numbers at day 7 in transplant recipients compared with untransplanted recipients shown in Fig. 1B and 1C. This increase was similar in both untreated control (white bars) and ECDI-SP–treated recipients (black bars) and sustained at day 21. Similarly, Gr1high cells also significantly increased in percentages and numbers at day 7 compared with untransplanted recipients shown in Fig. 1B and 1C, but this increase appeared to be more profound in untreated recipients (white bars) compared with ECDI-SP–treated recipients (black bars) at day 7 (p = 0.059) and subsided in both to baseline at day 21 (Fig. 4A). Interestingly, the splenic Gr1high cells from ECDI-SP–treated recipients were no longer positive for IFN-γ expression (Fig. 4A, right histograms).
In the cardiac allografts, however, a very different picture emerged (Fig. 4B), as follows: Ly6Chigh cells were found in considerably higher percentages and total numbers in the protected grafts from ECDI-SP–treated recipients compared with grafts from untreated recipients at day 7 (Fig. 4B). In contrast, the percentages and total numbers of Gr1high cells were significantly lower in the protected grafts in ECDI-SP–treated recipients compared with rejecting/rejected grafts from control untreated recipients at day 7. Importantly, the Gr1high cells from allografts of ECDI-SP–treated recipients continued to express IFN-γ at both day 7 and day 21 (Fig. 4B, right histograms), contrasting to the Gr1high cells from allografts of untreated recipients that were IFN-γ−. By day 21, the already rejected cardiac grafts from control untreated recipients were progressing to fibrosis, and considerably fewer graft-infiltrating cells, including Ly6Chigh cells and Gr1high cells, were recovered compared with the protected grafts from ECDI-SP–treated recipients.
We next measured cytokine and chemokine production of the graft tissue to determine whether there was a correlation between cell infiltration, cytokine/chemokine production, and graft outcome. Tissue lysates were prepared from day 7 and day 21 heart allografts. Multiplex analysis of heart graft tissue lysates revealed that IL-1β and M-CSF, factors important for MDSC induction, were significantly increased in the protected grafts compared with control grafts at day 21. IL-10, a factor known to be produced by MDSCs, was also significantly higher in protected grafts at day 21 compared with control grafts. CCL4, another soluble factor implicated in the recruitment of Tregs, was similarly increased in protected grafts at days 7 and 21 compared with control grafts. In contrast, factors associated with neutrophil accumulation, including G-CSF, CXCL1, and CXCL2, were significantly higher in control grafts at day 21 (Fig. 4C).
Collectively, these data indicate that the Gr1high cells induced by ECDI-SP infusions and those induced by allogeneic cardiac transplantation may be distinct from each other, and that cardiac allografts from recipients treated with ECDI-SPs show enhanced accumulation of Gr1highIFN-γ+ cells, as well as Ly6Chigh cells, and a graft environment consistent with their local induction and accumulation.
Cardiac allograft Ly6Chigh and Gr1high cells produce soluble mediators important for Treg induction and migration
To determine the functional significance of the Ly6Chigh and Gr1high cells from the protected cardiac allografts in recipients treated with donor ECDI-SPs, we first examined their ability to suppress T cell proliferation. We chose to examine this at day 21 posttransplant, a time point when the fate of the graft in ECDI-SP–treated recipients was readily distinguishable from that of the graft in control untreated recipients. As shown in Fig. 5A, left panel, neither population isolated from the cardiac allografts on day 21 suppressed in vitro CD8+ T cell proliferation stimulated by anti-CD3/28. MDSCs have been reported to also have the ability to exert Ag-specific suppression of T cell proliferation (12, 31). We next tested whether these cells suppressed CD8+ T cell proliferation stimulated by donor APCs. As shown in Fig. 5A, right panel, they also did not substantially suppress donor APC-stimulated CD8+ T cell proliferation. As a control, in vitro suppression assays using splenic Ly6Chigh and Gr1high cells from the protected recipients on day 21 were also set up and showed similar inhibition of proliferation, as observed in Fig. 2A (Supplemental Fig. 1). Interestingly, when we examined cytokine production in the supernatants from the suppression cultures by multiplex analysis, we found that coculture with allograft, but not splenic, Ly6Chigh and Gr1high cells led to a significantly higher production of IL-10 and CCL4, two soluble mediators implicated in the induction and homing of Tregs (Fig. 5B). Supporting this possibility, cardiac allografts retrieved from donor ECDI-SP–treated recipients showed a progressive increase of the number of Foxp3+ cells compared with those from control untreated recipients (Fig. 5C). This characteristic was unique to graft Ly6Chigh and Gr1high cells, as spleen Ly6Chigh and Gr1high cells from the same recipient mice in the same cocultures did not lead to an increased production of IL-10 or CCL4 (Fig. 5B). Collectively, these data indicate that posttransplant the graft, but not the splenic, Ly6Chigh and Gr1high cells may function by establishing an environment conducive to local Treg induction and homing, pointing to the functional difference between the graft and the splenic Ly6Chigh and Gr1high cells.
Anti-Gr1 Ab depletes both Ly6Chigh and Gr1high cells, concomitant with a significant increase in graft-infiltrating CD8+ T cells and graft loss
To test the in vivo protective role of the Ly6Chigh and Gr1high cells, we treated mice with the Gr1-depleting Ab (clone RB6-8C5). As shown in Fig. 6A, this Ab depleted both Ly6Chigh cells and Gr1high cells in vivo. In this study, the Gr1high cells were shown as SCChighLy6CINT cells (as shown in Fig. 1B, 1D) to avoid the use of the same clone (clone RB6-8C5) for cell surface staining. Given that these cells suppress CD8+ T cells ex vivo (Fig. 2A), we sought to confirm these findings in vivo. As schematically outlined in Fig. 6B, B6 mice were treated with BALB/c ECDI-SPs and anti-Gr1 Ab, and BALB/c cardiac transplants were performed. Consistent with our previous findings (8), in sharp contrast to recipients treated with ECDI-SPs alone, all recipients additionally treated with the anti-Gr1 Ab promptly rejected the cardiac allograft by day 10 (Fig. 6B). Of note, injection of BALB/c ECDI-SPs did not protect third-party SJL cardiac allografts (Fig. 6B), consistent with our previous demonstrations of the Ag specificity of this regimen (2, 5, 8). At day 10, grafts were harvested and graft-infiltrating cells were analyzed by histology and by flow cytometry. It was observed that allografts from recipients treated with ECDI-SP infusions only had preserved graft architecture, overall few graft-infiltrating cells (Fig. 6C, top image), and specifically few CD8+ T cells (Fig. 6D, left image), consistent with graft protection. However, with concomitant anti-Gr1 Ab treatment, there was a discernable loss of graft integrity with a significant increase in overall graft-infiltrating cells (Fig. 6C, bottom image) and specifically graft-infiltrating CD8+ T cells (p = 0.043) (Fig. 6D, right image). These data suggest that the presence of Ly6Chigh and Gr1high cells is critical for the protection of the cardiac allograft, possibly through their control of graft-infitrating CD8+ T cells.
In the current study, we demonstrate that infusions of apoptotic donor cells in the form of ECDI-SPs expand two myeloid suppressor populations (Ly6Chigh and Gr1high) that function to inhibit T cell proliferation. Moreover, treatment with ECDI-SPs induces the Gr1high population to produce a high level of IFN-γ and enhances the responsiveness of this population to IFN-γ to produce downstream effector molecules, including IDO and iNOS, which mediate their ability to suppress T cell proliferation. Finally, these cells traffic to and protect cardiac allografts through inhibiting graft CD8 T cell accumulation, and potentially also through inducing a Treg-rich environment.
Phenotypic analysis of Ly6Chigh cells expanded in our model revealed similarities with monocytic MDSCs (M-MDSCs); however, this population was distinct in its absence of expression of the IL-4R (CD124) and the M-CSFR (CD115) (data not shown). Cell subset heterogeneity is a hallmark of MDSCs, with cell surface phenotype dependent on the model, stimulus, and anatomic location. M-MDSCs can be further classified as M1 or M2, dependent on the polarizing conditions. M1 M-MDSCs are defined by their production of iNOS and TNF-α and expression of IFNγR, whereas M2 M-MDSCs produce arginase and IL-10 and express CCR2 (32). As shown in Fig. 1D, ECDI-SP–expanded M-MDSCs appear to exhibit a M2-like phenocyte by their expressing CCR2. The function of these Ly6Chigh cells also appears to correspond to that of M2 M-MDSCs in their ability to suppress CD8 effector T cells and produce factors for Treg induction (Figs. 2A, 5B) (32).
ECDI-SP–expanded Gr1high cells are Ly6Ghigh, Ly6CINT, SSChigh, and CCR2− (Fig. 1B, 1D). This phenotype is indicative of neutrophil-like cells or granulocytic MDSCs (G-MDSCs). Several recent studies have attempted to phenotypically and functionally differentiate between neutrophils, G-MDSCs, and a further population of neutrophil-like cells termed tumor-associated neutrophils (TAN) (33, 34). G-MDSCs can be subcategorized according to their phenotype, with G2 G-MDSC expressing arginase, IL-10, and CCL2 (32). Graft-infiltrating Gr1high cells in the ECDI-SP–treated cardiac transplant recipients appeared to have an enhanced ability to induce IL-10 production in comparison with their splenic counterpart (Fig. 5B), indicating that the graft environment may skew the Gr1high phenotype to resemble that of G2 G-MDSCs, which mirrors that of TAN from tumor-bearing mice (35). Interestingly, the TAN phenotype has been shown to be dependent on tumor TGF-β, which may also perpetuate Treg development in conjunction with IL-10; and depletion of TAN resulted in a significant increase in local CD8+ T cell proliferation (35). In contrast, bone marrow neutrophils did not exhibit these characteristics (34, 35). Collectively, our data indicate that ECDI-SP–expanded Gr1high cells resemble G-MDSC or TAN rather than bone marrow neutrophils, a phenotype that may be further perpetuated by the graft microenvironment.
Flow cytometric analysis of Gr1high cells revealed that these cells produced a high level of IFN-γ after repeated infusions of ECDI-SPs (Fig. 2C, 2D). Although not yet reported in transplant models, CD11b+Gr1+ cells producing IFN-γ have been previously demonstrated in models of infection. In 2002, two groups demonstrated that the immune response to Nocardia asteroides and Salmonella infections induced CD11b+Gr1+ cells to produce IFN-γ (36, 37). This was corroborated recently in a Streptococcus infection model, in which phenotypic analysis demonstrated that these cells were CD11b+Gr1+, and Ly6G+, Ly6Clow, CCR2−, and CX3CR1+ (23). Interestingly, this expression profile closely resembles that of ECDI-SP–expanded Gr1high cells. In each of these studies, the IFN-γ–producing cells were defined either as neutrophils (36, 37) or as immature myeloid cells based on their induced differentiation with addition of growth factors (23). However, only the immature myeloid cells could suppress T cell responses (23). Therefore, although more than one subtype of CD11b+Gr1+ cells may be capable of producing IFN-γ it is clear that not all may be suppressive.
It is interesting to note that our previous attempts to induce transplant tolerance to islet allografts using donor ECDI-SPs were unsuccessful in IFN-γ–deficient mice (3), indicating an obligatory role of IFN-γ in mediating tolerance induced by the ECDI-SP–based regimen. We have previously shown that IFN-γ contributes to the contraction and deletion of allospecific T cells (3). Our data in this study provide an additional role of IFN-γ in graft protection by promoting and perpetuating the function of MDSCs, in part via IDO and iNOS-mediated suppression (12, 38). These findings indicate the complex role that IFN-γ may play in transplantation tolerance, consistent with studies by others demonstrating a protective role of IFN-γ in transplant models via a variety of mechanisms. For example, exogenous IFN-γ prevented graft-versus-host disease in a murine bone marrow transplantation model through posttransplant inhibition of T cell proliferation (19, 20). IFN-γ stimulation of dendritic cells resulted in an upregulation of IDO production, which mediated allograft protection in a rat liver transplantation model (21). Most relevant to our findings, a recent study by Garcia et al. (10) in a tolerant heart transplant model demonstrated that a monocytic suppressive cell population bearing the phenotype CD11b+Gr1+CD115+ mediated their suppression via signaling responses to IFN-γ, as Ifngr−/− Gr1+ monocytes could not provide graft protection as wild-type Gr1+ cells did. Interestingly, the source of the IFN-γ was not the Gr1+ monocyte themselves, as Ifng−/− Gr1+ monocytes were equally efficacious in graft protection as wild-type Gr1+ cells (10). Therefore, to the best of our knowledge, ours is the first report of IFN-γ production by MDSCs themselves potentially mediating the induction of transplant tolerance.
It is unclear at the moment what signals are transmitted to these cells by the injection of ECDI-SPs to allow the induction of IFN-γ in the Gr1high cells, as these cells themselves do not directly interact and phagocyse the injected ECDI-SPs (J. Bryant and X. Luo, unpublished observations). On a broader base, determining the exact mechanism through which ECDI-SPs induce the expansion of both the Ly6Chigh and Gr1high cells is of clinical relevance, as it will potentially allow for a more direct and effective manipulation of this response therapeutically. The pathways through which MDSCs are differentiated and expanded have been extensively studied in other model systems. Inflammatory mediators arising from infections and tumors have been implicated by many studies, including IL-1β, PGE2, cyclooxygenase-2, GM-CSF, IL-6, vascular endothelial growth factor, S100A8/9, and IFN-γ (13). Interestingly, the process of apoptotic cell uptake (efferocytosis) also induces PGE2 production (39). Apoptotic ECDI-SPs are phagocytosed in the recipient spleens by subsets of dendritic cells (3), which may generate subsequent mediators associated with MDSC differentiation or expansion. This may provide a link between efferocytosis and the expansion of Ly6Chigh and Gr1high cells in our model. Further studies are ongoing to delineate this relationship.
Previous work in our laboratory demonstrated that IDO was critical for cardiac allograft tolerance after ECDI-SP infusions (8). In the current study, we demonstrate that this molecule can be produced by the Gr1high cells and it mediates the T cell–suppressive function of the Gr1high cells. Although the role of IDO in positive allograft outcome has been extensively studied, its production by MDSCs has not been fully investigated. Two recent studies in human tumor MDSCs showed that IDO was directly produced by these MDSCs, and that it played a role in T cell suppression and Treg induction by this cell population (40, 41). Additionally, in allogeneic stem cell transplant recipients, monocytic MDSCs were also shown to produce IDO, playing a role in T cell suppression (42). Our studies demonstrate that infusions of ECDI-SPs induce a MDSC population with a heightened ability for IDO production in response to IFN-γ, resulting in a positive graft outcome via both mediators through CD8+ T cell suppression and potentially Treg induction. Collectively, ours and published data, demonstrating IDO production by MDSCs provides a link between T cell suppression and Treg induction by the same cell population, therefore point to an attractive target for therapeutic exploitation in transplantation.
Characterizing the ECDI-SP–expanded MDSCs before (Fig. 1C) and after (Fig. 4A, 4B) cardiac transplantation suggests that, prior to transplantation, these cells are present in the spleen and function to suppress proliferation of T cells via IFN-γ–dependent mechanisms, whereas, after transplantation, they migrate to the cardiac allograft and protect the graft via possible induction and homing of Tregs. Migration of MDSCs has been best examined in tumors and is thought to be mediated by multiple tumor-secreted factors, including IL-1β (43) and M-CSF (17), two factors that we have also observed in our protected cardiac allografts (Fig. 4C). Furthermore, it has been well recognized that functional differences exist between the MDSCs isolated from the spleen and the MDSCs isolated from the site of tumors (44–46). Similarly, migration of MDSCs to transplanted allografts has also been described (10), although there the expression of the ligands for P- and E-selectins on the MDSCs was found to be crucial for the homing. Thus, findings of the spleen versus the graft MDSCs in our model are consistent with these notions. However, the exact signals necessary for their trafficking to the allograft and the basis for their functional evolution warrant further investigation.
In summary, we demonstrate that ECDI-SPs expand MDSCs that function to protect transplant allografts paritally through their own production of IFN-γ and its downstream effector molecules. These studies thus expand the recent advances in the understanding of regulatory myeloid cells in transplantation (47) and provide the basis for future therapeutic manipulations of the population size and functionality of MDSCs for the purpose of transplant tolerance induction.
The authors have no financial conflicts of interest.
We acknowledge the Northwestern University Interdepartmental ImmunoBiology Flow Cytometry Core Facility for support of this work.
This work was supported by National Institutes of Health Training Grant T32 DK077662 (to N.M.L.) and National Institutes of Health Directors New Innovator Award DP2 DK083099 (to X.L.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide
- granulocytic myeloid-derived suppressor cell
- iNOS
- inducible NO synthase
- myeloid-derived suppressor cell
- 1-methyl-DL-tryptophan
- splenocyte
- tumor-associated neutrophil
- Treg
- T regulatory cell.
- Received October 15, 2013.
- Accepted April 10, 2014.
- Copyright © 2014 by The American Association of Immunologists, Inc.


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Cognitive psychology and neuropsychology bring together a range of different theoretical frameworks. This MSc programme provides an overview and critical evaluation of the major issues, investigative strategies, and empirical findings of recent attempts to integrate these different approaches to 'brain cognition'.
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subset: commoncrawl


text:
A Wave of Bankruptcies is Coming in Europe
Steffen Müller
in: The Economist, May 16, 2020
The epidemic is obstructing the economic recovery in Germany. Foreign demand is falling, private households forgo domestic consumption if it comes with infection risk, and investments are postponed. Assuming that the spread of the disease can be contained in short time, GDP growth in 2020 is expected to be 0.6% according to IWH spring economic forecast. Growth in East Germany is expected to be 0.9% and thus higher than in West Germany. If the number of new infections cannot be decreased in short time, we expect a recession in Germany.
Trade disputes are causing international trade in goods to decline this year. The manufacturing industry in Germany is particularly affected by this. However, a robust labour market is supporting the economy. According to IWH autumn economic forecast, German gross domestic product (GDP) will increase by 0.5% in 2019. At 1%, output growth in East Germany is likely to be significantly higher than in West Germany.
Members & Research Doctoral Students Annika Backes ; Doctoral thesis project: tba Dmitri Bershadskyy ; Doctoral thesis project: "Experimental Analysis...
Four Research Clusters ...


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Details Sold Per MeterMELCOâ"¢ tape is used to seal neoprene seams and stitches on a variety of equipment and garments. Use MELCO-TQ5000 on neoprene .
Amazon.com: Seam Sealing Tape Melco T-5000 – Hot Melt Wetsuit/Scuba. Waterproof Iron-on Seam Sealing Repair Tape for Gore-tex Wetsuit Drysuit Fabrics.
Looked good on the roll and melted fine on the drysuit,but when flexed the tape let loose and did not adhere to the tri laminate material it looked good so long as
A 7/8" heat applied seam tape for sealing seams on neoprene, outdoor garments and. your iron and fabric while sealing seams with Melco Iron On Seam Tape.
Melco iron on seam tape is used for sealing seams on neoprene and outdoor wear. Purchase this easy to apply 7/8 inch Melco seam tape from Seattle Fabrics!
Melco Iron On Seam Tape 12" Sold by the inch. A 12" heat applied seam tape for sealing seams on neoprene, outdoor garments and outdoor gear. Can be .
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24 Downloads
Updated 19 Sep 2012
The X-Rite Color Checker Chart is a very common standard used for standardizing different images and imaging systems. (This chart has been known in the past as the Gretag Macbeth ColorChecker Chart,
and the Munsell ColorChecker Chart.) You can take a photo of one chart and a different photo at a different time or on a different system, and then develop a transformation to match the two images based on the colors of the chart's chips.
This script makes (synthesizes) a perfect X-Rite Color Checker Chart from the X-Rite supplied sRGB values. (Note: This script just creates a nice perfect image (which you can save) so it can't be used for standardizing two different images or imaging systems. It's just for looks, for example to put in a publication.) You have options on the size of the image, whether you want informational text (chip name & color) in the overlay, and whether you want to save the image.
Image Analyst (2020). Color Checker Chart (https://www.mathworks.com/matlabcentral/fileexchange/38236-color-checker-chart), MATLAB Central File Exchange. Retrieved .
Dear Image Analyst
Great job, thanks for sharing, this is very useful for color analysis.
Actually, I have two more questions.
Firstly, when evaluating the color accuracy of cameras, a colorchecker chart will be captured, my question is how to get these pixel value with gui-programming in matlab?
Another question is about color correction, say that most popular ISP gives a 3x3 RGB to R'G'B' transformation, I've tried polyfit, regress to derive such matrix, neither of them showed confident results, the average color difference is too high, about 8 deltaE, do you have any suggestion to optimize the fitting process?
Thanks a lot!
thank you very much
Found this, and thought "Oh, this is great! Gives me ideal RGB values - now I just need to modify the script to remove the text, and then figure out how to save it off as an image". Amazingly, the first time I ran, I found these features to be already implemented! Prompted me first to generate it with or without text, then asked me if I'd like to save it. Perfect! Thank you!
Hi Image Analyst. I'm a student currently working on a project that involves detecting the presence of certain colors within images all taken under the same lighting conditions. I've been learning a lot from your Simple Hue Detection code, but am finding that I'm having trouble getting constant values for each color in different pictures (specifically dark brown, light brown, red, blue-grey, black and white). I've decided to use a color checker chart, but don't know how to fully use it to modify my images. Currently I'm using a modified method of the masks that you used for your yellow color detection that can successfully detect each color, but only for a narrow range of images. If you could, can you explain how I could use the color checker chart (a physical one) to modify the color in an image?
Thanks for this helpful submission! Just what I needed.
Tim: I'm quite aware of color science. I teach color courses around the world, take color courses, and attend professional society symposia on color where I talk with top experts in the world. I'm one of the top color scientists in one of the largest corporations in the world. I develop calibrated color imaging systems full time, all the time. I think you misunderstood the intent of this submission, which was to simply create an image using the X-rite supplied values of their Color Checker chart. Nothing more. No one who images a real chart should expect to get the sRGB values. No one would be able to use this image for any calibration or anything at all other than creating a synthetic image, which is all it's meant for. I'm sorry you expected more, despite my disclaimer in the second paragraph of the description, and gave it the lowest possible rating.
No, no, no, NO. You cannot state R,G,B values for such targets, "Image Analyst". The RGB colorspace is HIGHLY non-perceptually-uniform. Conversion to LAB would make it as perceptually uniform and standardized as you can get these days. And yes, you state that you use the sRGB values, but which sRGB whitepoint do you use? What observer degree? And so on. Convert your image to the appropriate LAB values and take the color space into consideration. Then compare LAB values.
I'm really impressed with your contribution . gr8. Thanks for sharing.
Inspired by: export_fig
Inspired: Convert between RGB and Color Names
Create scripts with code, output, and formatted text in a single executable document.


text:
Building Maintenance Proposal
Hickory Grove Baptist Church currently has over 600,000 square feet of buildings located on almost 175 acres. As our buildings and facilities age, they face critical maintenance and improvements that must be addressed.
Based on the recommendation of the Finance and Long Range Planning Committees and affirmation of the deacons, this proposal is asking the church to extend our current debt by $2,000,000 to address these issues.
In 2005, following completion of Education Building 3 at Harris Campus and the Worship Center at Mallard Creek Campus, the church owed a total of $13,000,000 in debt. Because of the faithful and sacrificial giving of our members, that debt has been reduced to $1,019,949 as of November 1, 2019. At our most recent interest rate of 3.31%, the additional loan will add approximately four years to our debt payoff without changing the monthly payment.
- Roof Replacement (Mallard Creek Campus) - $600,000
- Climate Control Systems Replacement - $300,000
- Flooring Replacement (Harris Campus) - $400,000
- Parking Lot Maintenance (Harris Campus) - $100,000
- Chapel (Harris Campus) - $150,000
- Worship Center Bathrooms (Harris Campus) - $125,000
- Ministry Offices (Mallard Creek Campus) - $125,000
- Early Education Center Enhancements (Mallard Creek) - $50,000
- Exterior & Interior Signage - $50,000
- New Shuttle Buses - $100,000
In order to allow adequate time for our members to process this request, we will not vote on this proposal until the Sunday morning worship services on December 15. Until then, please address any questions to Steve Adams or any member of the Finance or Long Range Planning Committees.
Elmer Dryden, Chairman
Todd Bennett
Marson Berry
Mark Foster
Rob Manning
Rich Pacetti
Joe Swann
Joe Swarengin
Rene Toadvine
Jay Manning, Treasurer
Craig Duet, Assistant Treasurer
Don Ceccarelli, Chairman
Marson Berry
Dave Cornett
Kevin Day
Craig Duet
Tim Feeney
Mike Fogle
Danny King
Garrett Olin
Randy Scribner
Mike Worley


text:
MILWAUKEE, Wis. (April 15, 2020) — A. O. Smith Corporation (NYSE: AOS)
announced today that executive chairman Ajita Rajendra will be retiring
effective May 1, 2020.
Company's Board of Directors has elected Kevin J. Wheeler as chairman of the
Milwaukee-based water technology company effective upon Rajendra's retirement.
Wheeler will continue to serve as president and chief executive officer of the
company. Rajendra will remain a member of the company's Board of Directors.
has been an inspiring and visionary leader since the moment he joined the
company in 2005 to run A. O. Smith's water products division, as well as during
his time at the helm of A. O. Smith as chief executive officer and president
from 2013 to 2018," said William Greubel, lead director. "He built a strong
team and developed the strategy that has resulted in the Company's current
success in water technology."
O. Smith has flourished under Ajita's guidance and tenure as chairman and CEO,"
Greubel noted. "A. O. Smith went from being a $1.9 billion company when he
began as chief executive officer and president to a $3 billion organization,
and our earnings per share had a compound annual growth rate of 22.3%. Under
his watch, A. O. Smith acquired two water treatment companies in North America
and built a stronger presence in China, India, and Vietnam, further building
and cementing A. O. Smith's position as a global leader in water technology.
Board has the utmost confidence about the Company's future with Kevin as our
tenth chairman and chief executive officer," said Greubel. "He and Ajita have
worked together 15 years in shaping the company's strategy. Like Ajita, Kevin
has an obsessive focus on caring for our customers and rewarding our
shareholders. He is an outstanding businessperson with strong values, strategic
vision and excellent leadership skills."
joined A. O. Smith in January 2005 as president of its Water Products Company,
a manufacturer of residential and commercial water heaters and boilers, and he was
named an executive vice president of the corporation in 2006. He was promoted
to president and chief operating officer in August 2011 and was elected to the
company's Board of Directors in December of that year. Rajendra was named
executive chairman of the corporation in September 2018 as he continued to lead
the company's Board of Directors.
In addition to the A. O. Smith Board, Rajendra is a member of the Board of Directors of Donaldson Company and the Timken Company. He is currently a member of the Advisory Board for the University of Wisconsin - Milwaukee Lubar School of Business, the Board of Trustees of Alverno College in Milwaukee and a member of the Board of Directors of the Metropolitan Milwaukee Association of Commerce.
joined A. O. Smith in 1994 as a regional sales manager for the former Water
Products Company. Prior to being named president and chief executive officer,
he held numerous senior leadership positions during his two and a half decades
with the company, including vice president-international with responsibility
for all European and Asian water heating operations, senior vice president and
general manager of the U. S. retail water heater business, and senior vice
president and general manager of the North America, India, and Europe water
heating business.
Wheeler is a member of the Business Roundtable, Manufacturers Association of Productivity Improvement (MAPI) division leadership council and the President's Roundtable. He has served as an at-large director of the Air-conditioning, Heating, and Refrigeration Institute (AHRI).
statementsThis release contains statements that the company believes
are "forward-looking statements" within the meaning of the Private Securities
Litigation Reform Act of 1995. Forward-looking statements generally can be
identified by the use of words such as "may," "will," "expect," "intend,"
"estimate," "anticipate," "believe," "forecast," "continue," "guidance" or
words of similar meaning. All forward-looking statements are subject to
risks and uncertainties that could cause actual results to differ materially from
those anticipated as of the date of this release. Important factors that could
cause actual results to differ materially from these expectations include,
among other things, the following: a further weakening of the Chinese economy
and/or a further decline in the growth rate of consumer spending or housing
sales in China; negative impact to the company's businesses from international
tariffs and trade disputes; potential weakening in the high efficiency boiler
segment in the U.S.; significant volatility in raw material prices; inability
of the company to implement or maintain pricing actions; potential weakening in
U.S. residential or commercial construction or instability in the company's
replacement markets; foreign currency fluctuations; the company's inability to
successfully integrate or achieve its strategic objectives resulting from
acquisitions; competitive pressures on the company's businesses; the impact of
potential information technology or data security breaches; changes in
government regulations or regulatory requirements; and adverse developments in
general economic, political and business conditions in key regions of the
world. Forward-looking statements included in this press release are made only
as of the date of this release, and the company is under no obligation to
update these statements to reflect subsequent events or circumstances. All
subsequent written and oral forward-looking statements attributed to the
company, or persons acting on its behalf, are qualified entirely by these cautionary
About A. O. SmithA.
O. Smith Corporation, with headquarters in Milwaukee, Wis., is a global leader
applying innovative technology and energy-efficient solutions to products
manufactured and marketed worldwide. Listed on the New York Stock Exchange
(NYSE), the company is one of the world's leading manufacturers of residential
and commercial water heating equipment and boilers, as well as a manufacturer
of water treatment and air purification products. For more information, visit www.aosmith.com.
SOURCE: A. O. Smith Corporation
# # #
A. O. Smith ProLine water heaters are available through local plumbing contractors across the country.For more information about ProLine water heaters and installation options, visit www.hotwater.com.View ProLine
A. O. Smith Signature Series water heaters are exclusively available through Lowe's.For more information about Signature Series water heaters and installation options, visit www.aosmithatlowes.com.View Signature Series


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M5A1 Detailed Turret Roof and Hatches (for AFV Club)
M5A1 Corrected Turret Roof & Hatches (for AFV Club)
Detailed and corrected turret roof with separate hatches for the AFV Club M5A1 Stuart kit. This set corrects the mistakes in the AFV Club kit.
Master by: Karl van Sweden
Scale: 1/35th
Old Item#: 35-74454
Additional information
|1.5 oz


text:
Thales Academy, a college preparatory network of Pre-K-12 independent schools based in North Carolina, is expanding to include its first Tennessee campus, with a new campus location in Franklin opening for the 2020-2021 school year.
The Franklin campus will open this July, initially offering grades K-3 for the 2020-2021 school year, with more grades to come in future years.
"We look forward to offering our Thales Academy model of the highest quality education at the most affordable tuition price to Nashville area families," says Bob Luddy, founder of Thales Academy. "We are excited for the opportunity to grow and serve students outside of North Carolina as we expand to Tennessee and Virginia this summer."
A parent interest meeting is scheduled for the following dates- March 10th, April 14th, May 12th, and June 9th at 6:30 p.m. at The Gate Community Church, 3835 Carothers Parkway, Franklin, TN 37067. Mr. Luddy and two campus administrators will present about Thales Academy and will be available to answer questions. Children are welcome to attend. For more information and to register for this event, visit www.thalesacademy.org/Franklin.
Thales Academy currently operates eight campuses in the Raleigh and Charlotte, North Carolina areas, instructing over 3,000 students, and continues to add campuses as demand for its successful school model grows. Student formation is designed to develop the entire person, which is achieved through a research-based teaching method, best-in-class instructors, and a culture that embodies traditional values. Hallmarks of Thales Academy include: rigorous, college-prep academics that emphasize learning to mastery; character formation; real world skills development; personalized attention and affordable tuition with no extra fees or fundraising. Tuition is $6,000 per year, with payment plans, discounts, and scholarships available. Thales Academy does not accept vouchers.
About Thales Academy
Thales Academy is a Pre-K-12 college preparatory network of schools that assists parents in cultivating virtuous, critical thinking, compassionate individuals. Thales Academy fosters high standards of accountability, academic achievement, and civic responsibility through the use of a rigorous curriculum, character development, and technical and non-cognitive skills development. Thales Academy seeks to deliver the highest quality education at the lowest cost to parents and the community. Students are well-prepared for success in college and beyond, consistently outranking nationwide peers on objective, standardized tests and mastering skills that last a lifetime. For more information and to apply for admission online, visit www.thalesacademy.org.


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For 15 Invitations, Hong Kong-based curator Inti Guerrero presents his research on the work of Filipino experimental musician and composer José Maceda (1917–2004). The exhibition includes archival and audiovisual materials on two of Maceda's groundbreaking projects: Ugnayan (1974) and Udlot-udlot (1975), which involved thousands of participants seizing public space and mass media during the years of martial law in the Philippines. Maceda's work is placed in dialogue with video pieces, short films, and artefacts by Pio Abad, Anand Patwardhan, Jan Švankmajer, and Koki Tanaka to expand on collective creativity and social oppression found in the contradictory context of the arts during the Marcos regime.
Documentation on the 1972 large-scale sound performance Udlot-udlot can be found in AAA's The Chabet Archive from which Guerrero sourced a selection of material for the exhibition. Notes and reflections on his research are recorded in AAA's Field Notes, providing unique insight into his curatorial process.


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Iman Shumpert will miss 3 months after wrist surgery, as Cavs take another hit
In his opening press conference of the 2015-16 NBA season during Monday's Media Day session, LeBron James said that for the Cleveland Cavaliers this year, it all comes down to health. Just 24 hours later, the club was forced to deal with a significant challenge on that score:
[Follow Dunks Don't Lie on Tumblr: The best slams from all of basketball]
If, like me, you're not so up on your extensors, it looks like Iman Shumpert tore the covering of the tendon that runs from the forearm down the wrist to the fifth metacarpal, the bone connecting to your pinky finger. Cavaliers general manager David Griffin said Shumpert sustained the injury "on the rim during a workout last week," according to USA TODAY's Jeff Zillgitt. A magnetic resonance imaging exam taken after Shumpert reported the incident to the Cavs' medical staff upon reporting to training camp revealed the injury; he'll have surgery at Cleveland Clinic on Wednesday.
"In a situation like this, when the tendon's out of the sheath, it's not optional to just wait," Griffin told reporters at training camp Tuesday.
being traded to Cleveland with J.R. Smith midway through last season.A three-month layoff would leave Shumpert on the shelf for about 25 games. That, obviously, is disappointing for the former New York Knicks first-round draft pick, who became a valuable contributor and postseason starter after
"Tough day but I will be back better than ever," Shumpert wrote in the caption to an Instagram post Tuesday afternoon.
[Yahoo Fantasy Basketball: Sign up for a league today]
The 6-foot-5 Georgia Tech product showed flashes of being an explosive backcourt contributor and perimeter stopper in three-plus seasons in Manhattan, but who typically alternated stretches of strong play with periods of ineffectiveness born out of a struggle to finish around the basket, a flagging touch from long distance, wavering on-court confidence and myriad injuries. Knee ligament tears ended his rookie season and limited him to just 45 games as a sophomore; a dislocated left shoulder sidelined him out for 20 games last season, and cropped up again during the playoffs. (He also battled a groin strain in the postseason.)
After getting healthy in Ohio, Shumpert settled into a spot on the second unit, helping check opponents' top perimeter threats while chipping in 7.2 points, 3.8 rebounds, 1.5 assists and 1.3 steals in 24.2 minutes per game in 38 appearances. He was forced into a larger role in the second round of the playoffs, when head coach David Blatt had to juggle his rotation to make up for losing starting power forward Kevin Love to a season-ending shoulder injury and starting off-guard Smith to a two-game suspension for cracking Boston Celtics wing Jae Crowder in the face.
Shumpert performed admirably against the Chicago Bulls and Atlanta Hawks in the Cavs' run to the Eastern Conference crown, averaging 11.4 points and 5.3 rebounds in 36.4 minutes per game. He shot 38.3 percent from 3-point land; more important, he helped make life difficult for the likes of Derrick Rose and Kyle Korver.
"Look at those All‑Defensive teams, and — obviously he has a small window with us — but he will be on the All‑Defensive team in this league very soon," James said of Shumpert after Cleveland's Game 2 win over Atlanta in the Eastern Conference finals.
Shumpert, like the rest of the non-LeBron Cavs, struggled mightily in shorthanded Cleveland's NBA Finals loss to the Golden State Warriors without stars Love and Kyrie Irving. But despite that rough dismount, Griffin and owner Dan Gilbert re-upped the 25-year-old wing on a four-year, $40 million contract in restricted free agency this summer, part of a cash-splashing display that keeps Cleveland in place as the team to beat in the East but will have major luxury-tax (and possibly roster-management) implications down the line.
All those expenses — max deals for LeBron and Love, new deals for J.R. and Matthew Dellavedova, agreements with veterans Mo Williams, Richard Jefferson and Russian center Sasha Kaun — have Cleveland well over the salary cap, and that's before you consider whatever deal the club might come to with lingering restricted free agent power forward Tristan Thompson. Trading away veterans Brendan Haywood and Mike Miller created a combined $13 million in trade exceptions they can use to absorb more salary in deals should the Cavs feel they need more help, but it sounds like Griffin's content to leave things alone for now and see what develops.
"I don't think, in the immediate future, it does [affect the roster]," Griffin said Tuesday. "We look at this as a very similar situation to what we went through in the playoffs last year. Next man up. We have a roster we feel is deep enough to withstand one injury like this, and so we're going to give people a chance to kind of absorb it from within. But obviously, we'll be paying a lot of attention to opportunities that we may be able to improve the group. We'll just play it by ear."
You can understand why Griffin doesn't necessarily feel compelled to start burning up the phone lines. While Shumpert's a better and more versatile defender than Smith, Dellavedova, Williams, Jefferson and second-year man Joe Harris, his minutes are not exactly irreplaceable. And though it's certainly not ideal to lose another member of the backcourt rotation while All-Star Irving's still working his way back from knee surgery, with no evident superpower competition looming in the conference, the Cavs seem like they can afford to scuffle a bit in the early going before turning it on and proving themselves the class of the conference … just like last year.
Love, who's also coming back from surgery, figures to take on a larger role in the Cleveland attack to help make up for the backcourt injuries. Smith will likely return to his starting spot, with Williams and Dellavedova sharing duties and time at both backcourt spots; Harris, who saw a surprising amount of minutes early last season after the Cavs tabbed him in the second round of the 2014 draft before the trade for Smith and Shumpert send him to the end of the bench, could get a longer look. The Cavs will figure it out, as James said Tuesday, according to Tom Withers of The Associated Press:
"Obviously it's a big blow. Injuries will probably be the only thing than can stop us long-term," LeBron James said following the team's first training camp practice. "This is a short-term thing. He will be there when we most need him." […]
"If there's one place we have depth it's at the guard spot," he said. "With this injury, our guys should be able to step in."
They probably will. This'll probably be fine. If Cleveland struggles early, though — without Irving's playmaking, without Shumpert's defense, with Love and Anderson Varejao knocking the rust off, etc. — you wonder if LeBron will wind up seeing a more significant season-opening workload than he, Blatt or Griffin would prefer, and whether some of these "no big deal" early nicks and bruises could wind up having a greater negative impact down the line, when Cleveland needs all their big guns as fresh as possible to reach the heights they were too banged up to achieve last postseason.
– – – – – – –
Dan Devine is an editor for Ball Don't Lie on Yahoo Sports. Have a tip? Email him at [email protected] or follow him on Twitter!
Stay connected with Ball Don't Lie on Twitter @YahooBDL, "Like" BDL on Facebook and follow Dunks Don't Lie on Tumblr for year-round NBA talk, jokes and more.


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Sunday, 3 November 2019
World War IX - Fired for Partying
As you may have noticed, not being a proper writer I don't really do reviews. These few paragraphs are more introductions to the day's song than a review but nevertheless there's still a little bit of added pressure on me today to write something decent as the last time I featured Brooklyn punks World War IX on here they used part of my write up to promote their new ep. I called them "an adrenaline rush, a band with fire in their veins. A throwback to the 80's when the term faster and louder reigned supreme" which isn't exactly exactly going to have proper reviewers worrying about their jobs being under threat but wasn't bad for me. This time round I'm going to have to reinforce that 80's hardcore punk vibe I get from these guys and tell you that the 5 track Phoning It In ep is akin to getting into Dr Emmett L. Brown's retrofitted DMC Delorean and heading back to an era when throughout a Reagan led country, sweaty teens were going wild down their local flea pits to some of that period's most beloved bands. Those teens might be a little older and greyer now, and their t shirt sizes a little larger, but they'll still be working up a sweat as they mix with an appreciative younger generation as World War IX give it their best shot at carrying on the legacy of their heroes. In Johnny C they've a frontman with a perfect voice for this kind of thing. He definitely brings to mind the likes of Jello Biafra or Henry Rollins (I doubt very much it's coincidental that the song you'll find at the bottom of this update reminds you so much of Black Flag's TV Party). He's ably backed by a band that's both talented and energetic; Justin (guitar), Mike Moosehead (guitar) Brian 'Chinatown' Jackson (bass) and Jon (drums).
Don't listen to the reviewer though, no matter how much he's flailing around trying to paint you an accurate picture, just click on the link to the music and make your own mind up : https://worldwarix.bandcamp.com/
So, here's the raison d' etre for this blog, the song. They've just released a video to accompany it and if any of you guys are a member of the Just Some Punk Songs group on Facebook the graphics may look a little familiar (https://www.facebook.com/justsomepunksongs/). The band's guitarist Justin Melkmann is a talented artist who not only produces his own comic books but who kindly contributed the artwork for the JSPS page. Now his work graces the new video. If you've read Justin's illustrated history of the band in the comic books you'll know it's quite possible this song is autobiographical. It's called Fired For Partying...
Posted by mickfletcher at 02:03
Labels: 2019, punk, World War IX
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The head coaching job of the West Linn football team has been on-again, off-again since the 2019 season ended.
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Welcome to my blog,
I answer to all my guests on all the websites and i did say the changes will come,
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