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TRANSFUSION MEDICINE| May 26, 2017
Human and murine splenic neutrophils are potent phagocytes of IgG-opsonized red blood cells
Sanne M. Meinderts,
Sanne M. Meinderts
1Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
2Department of Molecular Cell Biology, VU Medical Center, Amsterdam, The Netherlands;
Per-Arne Oldenborg,
Per-Arne Oldenborg
3Department of Integrative Medical Biology, Umeå University, Umeå, Sweden;
Boukje M. Beuger,
Boukje M. Beuger
Thomas R. L. Klei,
Thomas R. L. Klei
Johanna Johansson,
Johanna Johansson
Taco W. Kuijpers,
Taco W. Kuijpers
4Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Takashi Matozaki,
Takashi Matozaki
5Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan;
Elise J. Huisman,
Elise J. Huisman
6Sofia Children's Hospital, Erasmus Medical Center, University of Rotterdam, Rotterdam, The Netherlands; and
Masja de Haas,
Masja de Haas
7Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, The Netherlands
Timo K. van den Berg,
Timo K. van den Berg *
Robin van Bruggen
Robin van Bruggen *
Blood Adv (2017) 1 (14): 875–886.
https://doi.org/10.1182/bloodadvances.2017004671
Sanne M. Meinderts, Per-Arne Oldenborg, Boukje M. Beuger, Thomas R. L. Klei, Johanna Johansson, Taco W. Kuijpers, Takashi Matozaki, Elise J. Huisman, Masja de Haas, Timo K. van den Berg, Robin van Bruggen; Human and murine splenic neutrophils are potent phagocytes of IgG-opsonized red blood cells. Blood Adv 2017; 1 (14): 875–886. doi: https://doi.org/10.1182/bloodadvances.2017004671
In steady state, where no IgGs against RBCs are present, macrophages are the primary phagocytes of RBCs.
In conditions where RBCs are IgG-opsonized, neutrophils can have a major effect on RBC clearance.
Red Cells, Iron, and Erythropoiesis, Transfusion Medicine
immunoglobulin g, mice, neutrophils, phagocytosis, retail clinics, spleen, phagocytes, cd47 antigen, antibodies, erythrocytes
Visual Abstract
Red blood cell (RBC) clearance is known to occur primarily in the spleen, and is presumed to be executed by red pulp macrophages. Erythrophagocytosis in the spleen takes place as part of the homeostatic turnover of RBCs to remove old RBCs. It can be strongly promoted by immunoglobulin G (IgG) opsonization of RBCs, a condition that can occur as a consequence of autoantibody or alloantibody formation. The purpose of our study was to investigate which phagocytes are involved in IgG-mediated RBC clearance in the human spleen. We developed a highly specific in vitro assay to monitor RBC phagocytosis in total human splenocytes. Surprisingly, we have found that whereas homeostatic clearance of RBCs is primarily a task for splenic macrophages, neutrophils and, to a lesser extent, also monocytes can be a major factor in clearance of IgG-opsonized RBCs. Erythrophagocytosis by neutrophils is strongly dependent on the degree of opsonization of the RBCs. Additionally, the process is enhanced after blocking the "do not eat me" signal CD47 on the opsonized RBCs, which binds signal regulatory protein α, a myeloid inhibitory receptor that restricts phagocytosis. Moreover, RBCs isolated from autoimmune hemolytic anemia patients, opsonized by auto-IgGs, were shown to be readily phagocytosed by neutrophils. Finally, priming of neutrophils by inflammatory mediators such as tumor necrosis factor α and lipopolysaccharide further increases the magnitude of erythrophagocytosis. Collectively, our data suggest that neutrophils contribute significantly to the phagocytosis of antibody-opsonized RBCs, especially under inflammatory conditions. This indicates a hereto unanticipated contribution of neutrophils in RBC phagocytosis, especially under pathological conditions such as alloimmunization or autoimmunization.
The spleen is the biggest lymphoid organ of the body and acts primarily as a blood filter. It is the main site of red blood cell (RBC) clearance.1 Erythrophagocytosis is important for RBC turnover and is believed to be executed by the red pulp macrophages or Kupffer cells in the liver.1,2 The mechanism of RBC clearance under homeostatic conditions is not fully understood and there is no consensus about the removal signals on the RBCs that trigger phagocytosis by macrophages.2 In general, it is accepted that an accumulation of "eat me" signals triggers phagocytosis of the RBC at the end of its lifespan.2-4 Additionally, CD47 is an important regulator of RBC homeostasis by functioning as a "do not eat me" signal.3-9 CD47 on red cells inhibits phagocytosis of erythrocytes by binding the inhibitory myeloid receptor signal regulatory protein α (SIRPα). CD47-SIRPα interactions negatively control effector functions such as phagocytosis.3,5-9 CD47 acts as a molecular switch for erythrophagocytosis. Through a conformational modification, the protein can change from a "do not eat me" signal to an "eat me" signal.3 In a general sense, the balance between "eat me" and "do not eat me" signals seem to determine whether a RBC is cleared from the bloodstream.
Autoantibody formation against RBCs can occur in conditions such as autoimmune hemolytic anemia (AIHA) or upon alloantibody formation as a result of blood transfusion. Immunoglobulin G (IgG) opsonization of RBCs promotes phagocytosis of the RBCs through Fc-receptor activation on phagocytes.
Studies investigating RBC clearance have mainly focused on the role of macrophages. In this study, we have investigated the role of different populations of human spleen leukocytes in phagocytosis of antibody-opsonized RBCs. Surprisingly, we found that, depending on the conditions, neutrophils have a prominent role in phagocytosis of opsonized RBCs. So far, there have been no studies on erythrophagocytosis by neutrophils in humans. However, there are several case reports of patients with hemolytic conditions such as AIHA in which circulating neutrophils are identified that carry internalized RBCs.10-17 Nevertheless, this phenomenon has remained unexplained, as it seems highly unlikely that neutrophils capture RBCs in the bloodstream where the shear force is markedly high. Furthermore, it is not known whether neutrophils, under these and other conditions, have a substantial contribution to erythrocyte clearance in humans. If so, this would certainly contrast with the current dogma that neutrophils do not play a major role in the clearance of IgG-opsonized RBCs. Here, we demonstrate in vitro and in vivo that neutrophils from the spleen can make a significant contribution to phagocytosis of IgG-opsonized RBCs.
Information on antibodies used for phagocytosis assays can be found in supplemental Figure 2. The following antibodies were used for opsonization of human RBCs: anti-RhD (human polyclonal antibody, RheDQuin; Sanquin), anti-glycophorin A (anti-GPA) (CD235a, mouse monoclonal IgG1; Sanquin Reagents), isotype control monoclonal mouse IgG1 (Ref: MG100; ThermoFisher), anti-CD47 (mouse monoclonal IgG1, clone B6H12; generously provided by E. Brown, University of California, San Francisco, San Francisco, CA),18 anti-CR1 (rabbit polyclonal antibody; a kind gift from M. R. Daha, Leiden University Medical Center, Leiden, The Netherlands), anti-human CD147 (mouse monoclonal IgG2a, conjugate phycoerythrin, clone 8D12; eBioscience), anti-CD55 (mouse monoclonal IgG1, conjugate fluorescein isothiocyanate [FITC]; Pelicluster), anti-CD59 (mouse monoclonal IgG2a, conjugate FITC; Pelicluster). B6H12 F(ab′)2 fragments were generated by pepsin digestion. Antibody opsonization was measured using goat-anti-human (conjugate Alexa 488; Invitrogen), goat-anti-mouse (conjugate Alexa 488; Invitrogen), or goat-anti-rabbit (conjugate Alexa 488; Invitrogen). Complement deposition was measured using iC3b antibody (Quidel) or C3 antibody (clone anti-C3-19; a kind gift from Diana Wouters, Department of Immunopathology, Sanquin, Amsterdam, The Netherlands). Monocytes or macrophages were identified by anti-CD14 (conjugate FITC; Santa Cruz Biotechnology) and anti-CD163 (conjugate phycoerythrin; Trillium Diagnostics) staining.
Patient material
Blood samples of 6 AIHA patients were provided by the treating physician or by the diagnostic department of Sanquin Amsterdam (excess of blood for diagnostic procedures was used in an anonymous fashion for research in the present study, in accordance with the Dutch law regarding the use of rest material for research purposes). Patients were selected based on the clinical diagnosis and a positive Coombs test for IgG at time of sample collection. Patients were tested negative for IgM and IgA. RBCs of the patients were used in the phagocytosis assay with neutrophils isolated from 2 healthy donors.
Human spleen tissue
Splenocytes were isolated as previously described.19 Splenic material was obtained from organ transplant donors without clinical signs of infection or inflammation. Written informed consent for organ donation was obtained according to national regulations regarding organ donation. Splenic tissue of the donors was collected during transplantation surgery as part of the standard diagnostic procedure for HLA typing. Excess splenic material for diagnostic procedures was used in an anonymous fashion and used for research purposes.
Isolation of RBCs
RBCs were isolated as previously described3 from blood from healthy RhD+ donors, after obtaining informed consent. Studies on human blood samples were approved by the Sanquin Research institutional medical ethical committee, in accordance with the standards laid down in the 1964 Declaration of Helsinki.
Isolation of human neutrophils and monocytes
Neutrophils (polymorphonuclear neutrophils) or monocytes were isolated as previously described.20,21 All blood was obtained after informed consent and according to the 1964 Declaration of Helsinki.
Human spleen phagocytosis assay
RBCs were biotinylated by incubating cells in 6 μg/mL biotin (Thermo Scientific) at 37°C for 30 minutes and then washed and incubated with streptavidin MicroBeads (Miltenyi Biotec) for 30 minutes at 4°C. RBCs were washed and stained with DiD lipophilic membrane dye (1:100 for 108 RBCs, 20 minutes, 37°C; Life Technologies). The RBCs were incubated for 1 hour at 37°C with cells isolated from the spleen (1:2 ratio, 80 × 106 RBCs + 40 × 106 splenocytes) under various conditions. After incubation of the RBCs with the splenocytes for 1 hour at 37°C in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) medium,20 remaining RBCs were lysed using an isotonic ammonium chloride buffer for 10 minutes at 4°C followed by a second lysis step of 5 minutes at 4°C. After washing, the phagocytotic cells were selected using a LS magnetic-activated cell sorting (MACS) column (Miltenyi Biotec) analyzed by cytospin followed by May-Grünwald Giemsa staining and flow cytometry using Canto II or LSRII + HTS (BD Biosciences) equipped with FACSDiva software (BD Biosciences) (for gating strategy, see supplemental Figure 1).
Human blood neutrophil phagocytosis assay
RBCs were stained with DiD lipophilic membrane dye (Life Technologies). Subsequently, RBCs were incubated with neutrophils isolated from blood for 1 hour at 37°C (10:1 ratio, 5 × 106 RBCs + 0.5 × 106 neutrophils). Next, RBCs were lysed using an isotonic ammonium chloride buffer for 10 minutes at 4°C followed by a second lysis step of 5 minutes at 4°C. Cells were washed and resuspended in stop buffer containing 0.5% (wt/vol) paraformaldehyde, 1% (wt/vol) bovine serum albumin, and 20 mM NaF in phosphate-buffered saline. Phagocytosis was measured using flow cytometry.
Male and female CD47-deficient (CD47KO) mice on the Balb/c background,22 SIRPα-mutant mice (lacking most of the SIRPα cytoplasmic domain) on the C57BL/6 background,23,24 and their respective homozygous wild-type (Wt) littermates were used. Mice were kept in accordance with local guidelines, maintained in a specific pathogen-free barrier facility, and experiments were performed in compliance with relevant Swedish and institutional laws and guidelines and approved by the Umeå research animal ethics committee (A14-12).
Phagocytosis experiments in mice in vivo
RBCs were isolated from the blood of Wt or CD47KO mice, labeled with PKH26, and IgG-opsonized with the anti-murine RBC monoclonal antibody (mAb) 34-3C (mouse IgG2a; kindly provided by Shozo Izui, University of Geneva, Geneva, Switzerland) at 1 µg/1 × 108 RBCs, as previously described.8 After washing, 150 µL of RBCs at 10% hematocrit was injected IV into a lateral tail vein of anesthetized mice (isofluorane). At 90 minutes, mice were euthanized, blood was harvested by heart puncture into heparin (5000 IU/mL), and spleens were removed and kept on ice. Whole blood was incubated with Fc block (mAb 2.4G2), followed by addition of allophycocyanin-conjugated anti-mouse Gr-1 and FITC-conjugated anti-mouse CD11b at room temperature for 15 minutes. After washing and RBC lysis, samples were analyzed by flow cytometry (FACSCalibur; BD Biosciences) to quantify the fraction of PKH26+Gr-1hiCD11b+ neutrophils. Single-cell suspensions were prepared from spleens by mechanical disruption, as previously described.25 Following incubation in Fc block, splenocytes were labeled with mAbs against Gr-1 and CD11b followed by fluorescence-activated cell sorter (FACS) analysis of the Gr-1hiCD11b+ neutrophil population, as described for neutrophils from whole blood.
Phagocytosis experiments in mice in vitro
Bone marrow neutrophils were isolated from femurs of 2- to 4-month-old mice, using a Percoll gradient as previously described,26 and were resuspended at 5 × 106/mL in cold Hank balanced salt solution (HBSS) containing Ca2+ and Mg2+ (HBSS++). RBCs were isolated from the blood of Wt or CD47KO mice and opsonized with 5 µg of mAb 34-3C/1 × 108 RBC as described in the previous section. Neutrophils were incubated with IgG-opsonized RBCs at a 1:20 ratio for 60 minutes at 37°C. Following lysis of noningested RBCs using ice-cold sterile H2O, cells were resuspended in phosphate-buffered saline/1% human serum albumin (Octapharma) and cytospun onto glass slides at 200g for 5 minutes. The slides were stained with May-Grünwald Giemsa and mounted using DPX (BDH Prolabo). A phagocytosis index was calculated by counting ingested RBCs in 200 to 300 neutrophils per slide, and expressed as number of RBCs per 100 neutrophils.
Statistical analysis was performed using GraphPad Prism 6.02. For comparison of phagocytosis levels, the 1-way analysis of variance test was used followed by the Sidak post hoc test for correction of multiple comparison (****P < .0001; ***P < .001; **P < .01; *P < .05).
Unopsonized RBCs are primarily phagocytosed by macrophages
We wanted to identify, in an unbiased fashion, the relevant phagocytic cells in the human spleen that engulf IgG-opsonized RBCs. To examine this, we developed an in vitro assay to monitor RBC phagocytosis in total human splenocytes (Figure 1). Instead of isolating specific subsets of phagocytic cells, such as the red pulp macrophages, we used all nucleated cells from the spleen. As a result, the different subsets of splenocytes are present in their different natural ratios in our phagocytosis assay. Approximately 2% of the cells isolated from human spleen (n = 9) are macrophages (high autofluorescence), 7% are monocytes (side scatter low [SSClo]–forward scatter high [FSChi]), 19% are neutrophils (SSChi-FSCint), and 51% are lymphocytes (SSClo-FSClo) (supplemental Figure 1; Figure 2A).
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Magnetic sorting of RBC-phagocytosing cells from the human spleen. The following assay was used to investigate erythrophagocytosis by cells of the human spleen in vitro. RBCs were biotinylated and coupled to magnetic beads. RBCs were opsonized and stained with the membrane dye DiD. Cells of the spleen were isolated and incubated with the RBCs for 1 hour. RBCs were lysed and the remaining spleen cells were put over a MACS column. Cells elutriated from the column were analyzed by flow cytometry and cytospin (original magnification ×50; May-Grünwald Giemsa stain).
IgG opsonization of RBCs induces phagocytosis by neutrophils and monocytes. (A) Percentages of cell types in the spleen (n = 9). (B) RBCs were unopsonized (unops), anti-RhD-opsonized, or anti-GPA-opsonized. Splenocytes and RBCs were incubated for an hour and the phagocytic fraction was obtained using MACS. The absolute numbers of phagocytes are shown. Anti-GPA opsonization causes a drastic increase of phagocytosis. (C) The absolute number of splenocytes that take up RBCs per cell type. Neutrophils are the primary phagocytes in case of anti-GPA opsonization (n = 5-12). ns, nonsignificant difference.
In our phagocytosis assay, we evaluated the heterogeneous splenocyte population for their phagocytic capacity with magnetic RBCs. After incubation, RBCs were lysed and the remaining cells were selected by magnetic sorting and analyzed (Figure 1). This allowed us to extract the phagocytes that had taken up a magnetic RBC. First, we determined phagocytosis of unopsonized RBCs representative for homeostatic erythrophagocytosis (Figure 2B-C; supplemental Figure 3). Using unopsonized RBCs as targets, analysis by flow cytometry showed that the phagocytes isolated in this manner contained 81.6% macrophages, 14.3% neutrophils, and 4% monocytes, consistent with the idea that splenic macrophages represent the primary phagocytes for homeostatic turnover of RBCs in vivo.1,2
RBC opsonization leads to phagocytosis by spleen neutrophils and monocytes
We next determined the effect of IgG opsonization of RBCs on phagocytosis. Using anti-RhD, which causes a low level of opsonization (Figure 3A), there was a slight but significant increase of phagocytosis by monocytes (Figure 2B-C; supplemental Figure 3). Compared with the unopsonized condition there was no substantial increase in phagocytosis in the total number of phagocytes. Of note, there was a shift toward the monocytes within the phagocytic fraction, now representing 34% of the phagocytic fraction, which occurred at the expense of the phagocytosis by macrophages, suggesting that anti-RhD opsonization selectively promoted monocyte phagocytosis (Figure 2B). However, using anti-GPA, which leads to a very high level of opsonization (Figure 3A), we observed a significant increase in the total number of RBCs that were phagocytosed. In absolute terms, the phagocytosis by neutrophils was most notably enhanced (up to 65.7% of the phagocytic cells) and monocyte phagocytosis was too, albeit to a lesser extent, by opsonization with the anti-GPA antibody. Thus, opsonization of RBCs leads to a gradual recruitment of first monocytes (at low opsonizing levels) and later neutrophils (at higher levels) to the pool of spleen erythrophagocytic cells (supplemental Figure 4A). This indicates that there are different thresholds for erythrophagocytosis among the different phagocytes, and that antibody opsonization is a particularly powerful trigger for lowering the threshold in neutrophils.
Erythrophagocytosis by neutrophils is strongly dependent on the degree of opsonization of the RBCs. (A) Anti-GPA shows a much higher mean fluorescence intensity (MFI) compared with anti-D after secondary antibody staining (n = 3). (B) Phagocytosis by neutrophils depends on the degree of opsonization; extent of phagocytosis explained as MFI of anti-GPA-opsonized RBCs (left) or the percentage of phagocytic neutrophils (right) (n = 10). Error bars denote the standard error of the mean. (C-D) Cytospins of the phagocytic fraction using (C) anti-RhD-opsonized RBCs (original magnification ×50; May-Grünwald Giemsa stain) and (D) anti-GPA-opsonized RBCs (original magnification ×50; May-Grünwald Giemsa stain). These images were blindly chosen and are representative for 5 different experiments.
Phagocytosis of IgG-opsonized RBCs by neutrophils is dependent on the degree of opsonization
Because neutrophils are the most abundant phagocyte in the spleen, their contribution to RBC phagocytosis can be quite substantial once they participate in the process. Therefore, we subsequently explored factors that are of potential influence on RBC phagocytosis by neutrophils. First, we investigated how the degree of opsonization influences RBC phagocytosis by neutrophils. Therefore, we opsonized RBCs with increasing amounts of anti-GPA and determined the effect on phagocytosis by neutrophils from blood (Figure 3B-C). The results indicate that with increasing amounts of antibody deposited on the RBC, phagocytosis by neutrophils increases as well. An isotype control did not induce phagocytosis (supplemental Figure 4B). The effect of antibody opsonization was not restricted to anti-GPA antibodies. Using antibodies directed against various other RBC antigens, phagocytosis by neutrophils was found to correlate with the degree of antibody binding (supplemental Figure 5A-B). We also determined the effect of different IgG subclasses on RBC phagocytosis by neutrophils. Using monoclonal IgG1, IgG2, or IgG3 anti-RhD for RBC opsonization, we found that the IgG subclass of anti-RhD does not influence RBC phagocytosis (supplemental Figure 5C). Our anti-GPA is from murine origin so we next investigated whether other mouse monoclonal IgG1 antibodies could be as potent in inducing phagocytosis of RBCs by neutrophils. Phagocytosis of RBCs opsonized with anti-CD44 or anti-CD55 showed a far lower level of phagocytosis compared with RBCs opsonized with anti-GPA (supplemental Figure 5D). This coincided with the lower level of opsonization of these 2 antibodies (supplemental Figure 5E) which suggests that not antibody origin but rather the level of opsonization is the determining factor for RBC phagocytosis by neutrophils.
Blocking of CD47 increases erythrophagocytosis by neutrophils and monocytes
Antibody-dependent phagocytosis of RBCs is mediated through Fc-receptor signaling. To counterbalance prophagocytic signals, RBCs display the "do not eat me" signal CD47 on their cell membrane. Interaction of CD47 with the inhibitory myeloid receptor SIRPα inhibits phagocytosis.5,6,27 Using F(ab′)2 blocking antibodies against CD47, we determined the role of CD47 in IgG-mediated phagocytosis of RBCs in the spleen. For these studies, we used low opsonizing anti-RhD that by itself does not induce erythrophagocytosis. The data show that merely blocking CD47 is not enough to induce RBC phagocytosis by neutrophils. However, the combination of anti-RhD opsonization and blocking of CD47 enhances phagocytosis by neutrophils (Figure 4A,C). With a conventional phagocytosis assay using stained RBCs instead of magnetic RBCs the same results were obtained (supplemental Figure 6A). Moreover, CD47 blocking was able to further augment phagocytosis of anti-GPA-opsonized RBCs (supplemental Figure 6B). In conclusion, CD47 blocking is able to lower the threshold for neutrophil phagocytosis, which enables them to phagocytose RBCs opsonized with low levels of IgG. Thus, IgG opsonization and CD47 are major counterbalancing determinants for phagocytosis and, by lowering the threshold for phagocytosis through CD47 blocking, neutrophils can also be engaged in erythrophagocytosis.
Erythrophagocytosis is greatly enhanced after blocking the "do not eat me" signal CD47 on the opsonized RBCs in vitro. (A) CD47-SIRPα interaction was blocked on unopsonized and IgG-coated RBCs with F(ab′)2 CD47. Splenocytes and RBCs were incubated for an hour and the phagocytic fraction was obtained using MACS. Absolute amounts of phagocytic cells are shown (n = 7-10). (B) Cytospins of the phagocytic fraction using anti-D-opsonized RBCs with CD47 blocking. These images where blindly chosen and are representative for 5 different experiments (original magnification ×50; May-Grünwald Giemsa stain). (C) F(ab′)2 fragments of anti-CD47 do not significantly increase RBC phagocytosis by neutrophils. However, F(ab′)2 CD47 in combination with anti-RhD opsonization does significantly increase RBC phagocytosis (n = 7-10). Error bars denote the standard error of the mean. Asterisks represent highly significant differences (*P < .05, ****P < .0001).
Neutrophils of the blood and spleen phagocytose IgG-opsonized Wt or CD47-deficient RBCs in vivo
Our in vitro experiments indicate an important role for human neutrophils in IgG-mediated RBC clearance in the spleen. To establish the potential of neutrophils to phagocytose IgG-opsonized RBCs in vivo, we used a murine model. For this, labeled IgG-opsonized RBCs were injected IV into Wt Balb/c mice and neutrophils in blood and spleens were analyzed for RBC uptake at 90 minutes after injection, using flow cytometry (Figure 5A-B; supplemental Figure 7). Because murine neutrophils were found to phagocytose IgG2a-opsonized, but not IgG1-opsonized, RBCs in vitro (data not shown), an IgG2a antibody was used to opsonize RBCs in the present study. Our results showed that IgG-opsonized RBCs were taken up by neutrophils in the spleen (supplemental Figure 7B) and were also found, albeit to a much lesser extent, in neutrophils in the blood. Additionally, erythrophagocytosis was substantially enhanced when RBCs lacked CD47 (Figure 5A-B). Collectively, these data show that neutrophils can significantly contribute to erythrophagocytosis, also in vivo, and thus support our in vitro findings with human splenocytes. To further pinpoint whether the ability of CD47 to regulate neutrophil phagocytosis required SIRPα signaling in neutrophils, we investigated in vitro phagocytosis of IgG-opsonized RBCs by neutrophils from Wt or SIRPα-mutant mice. In SIRPα-mutant mice, the extracellular SIRPα-domain is expressed whereas the cytoplasmic signaling domain is deleted.23,24 Although phagocytosis of CD47KO RBCs by Wt neutrophils was about 4 times more efficient than that for equally opsonized Wt RBCs, CD47KO and Wt RBCs were phagocytosed equally by SIRPα-mutant neutrophils (Figure 5C). These data therefore show that the phagocytosis-inhibitory function of CD47 on RBCs requires SIRPα signaling in neutrophils.
The CD47-SIRPα interaction regulates neutrophil phagocytosis of syngeneic RBCs in vivo. (A-B) Neutrophil uptake of IgG-opsonized Wt or CD47-deficient RBCs in vivo. PKH26-labeled IgG-opsonized RBCs were injected IV into Wt recipient mice and neutrophils in blood or spleen were analyzed for RBC uptake at 90 minutes after injection using flow cytometry (n = 4-5). (C) Phagocytosis of equally IgG-opsonized CD47 Wt or CD47-deficient (CD47KO) RBCs by Wt or SIRPα mutant bone marrow neutrophils in vitro in the presence of 1 µM fMLF. Error bars denote the standard error of the mean. Asterisks represent highly significant differences (****P < .0001).
RBCs of AIHA patients are more readily phagocytized by neutrophils compared with RBCs of healthy individuals
To further address whether neutrophils may play a role in erythrophagocytosis in AIHA, we tested whether RBCs of AIHA patients (n = 6) could be phagocytosed by neutrophils (Figure 6). To compare different patients tested on different occasions, we used RBCs of healthy controls opsonized with anti-RhD or anti-GPA as a reference for IgG-induced phagocytosis, and normalized the data of each patient to the phagocytic response seen with anti-GPA on the same day. All patients showed a positive Coombs test (direct antiglobulin test) for IgG, but with varying levels of total IgG observed by flow cytometry (supplemental Figure 8A). Furthermore, the IgGs were shown to be of different specificities (supplemental Figure 8D). Nevertheless, in 4 of 6 patients, the overall opsonization appeared sufficient to induce neutrophil erythrophagocytosis, and in 3 of 6 patients the level of phagocytosis was close to that seen with anti-GPA-opsonized healthy control RBCs (Figure 6). This was striking because the level of opsonization with anti-GPA appeared substantially higher (Figure 3A) than that with the endogenous autoantibodies of AIHA patients. As expected, based on the data in the previous 2 sections, blocking of CD47 could further enhance neutrophil phagocytosis of AIHA RBCs, but not of RBCs from a healthy control in 2 of 3 cases (supplemental Figure 8E). Taken together, these data show that the prophagocytic signals are certainly sufficient, in the majority of AIHA patients tested here, to induce neutrophils to phagocytose RBCs from AIHA patients.
RBCs of AIHA patients show increased phagocytosis compared with RBCs from healthy individuals. (A) RBCs of an AIHA patient with auto-IgG antibodies and RBCs of a healthy control were labeled with DiD and incubated with neutrophils of 2 to 3 healthy donors. Erythrophagocytosis of patient I, III, and VI RBCs was comparable to erythrophagocytosis of anti-GPA-opsonized RBCs from a healthy control. Phagocytosis of RBCs of patient II and V by neutrophils is not significantly increased compared with RBCs of a healthy control. Phagocytosis of RBCs of patient IV by neutrophils is increased compared with RBCs of a healthy control. Error bars denote the standard error of the mean. Asterisks represent highly significant differences (*P < .05, ***P < .001, ****P < .0001). (B) Results of the Coombs test (direct antiglobulin test). All patients were tested positive for IgG and negative for IgM and IgA in the Coombs test performed by diagnostics. Patient II and IV showed low positivity for complement and remaining patients were tested negative for complement (+, positive; −, negative; (+), low positivity). Error bars denote the standard error of the mean.
Neutrophil priming enhances erythrophagocytosis
In 20% to 80% of the cases, AIHA is secondary to an underlying illness such as an infection, a lymphoproliferative disorder, or an autoimmune disease.28,29 In idiopathic as well as in secondary AIHA, the production of proinflammatory cytokines has been reported to be increased.30-33 We hypothesized that the immunological status of the patient can be of influence on the degree of erythrophagocytosis by neutrophils. For this reason, we investigated the effect of neutrophil priming on erythrophagocytosis. Neutrophil priming prior to incubation with unopsonized RBCs or anti-RhD-opsonized RBCs did not have any effect on phagocytosis (Figure 7A-B). Yet, depending on the stimulus, phagocytosis of anti-GPA-opsonized RBCs was significantly increased after stimulating neutrophils (Figure 7C). FSL-1 (ligand for Toll-like receptor 2 [TLR2] and TLR6), tumor necrosis factor α (TNFα; TNFR1 agonist), and lipopolysaccharide (LPS; TLR4 agonist) all induce phagocytosis of IgG-opsonized RBCs by neutrophils (Figure 7A). Priming with C5a (C5aR agonist), interferon γ, or interleukin-8 (IL-8) did not increase phagocytosis. Neutrophil priming using LPS in combination with CD47 blocking resulted in augmented phagocytosis of IgG-opsonized RBCs by neutrophils (Figure 7D). Although Bian et al showed this same effect in vivo in mice for multiple stimuli,34 in our assay other stimuli did not have this effect. Overall, our data demonstrate that various inflammatory mediators can lower the threshold for phagocytosis of IgG-opsonized RBCs by neutrophils.
Neutrophil priming enhances erythrophagocytosis. (A-C) Stimulation of human neutrophils isolated from the blood. Stimuli can enhance phagocytosis of anti-GPA-opsonized RBCs but not of unopsonized RBCs or anti-RhD-opsonized RBCs. (D) CD47 blocking combined with neutrophil priming using LPS enhances phagocytosis by neutrophils. Other stimuli do not have this effect. Error bars denote the standard error of the mean. Asterisks represent highly significant differences (****P < .0001). INF, interferon.
In this study, we took an unbiased approach to investigate phagocytosis of IgG-opsonized RBCs in the human spleen. The aim of the study was to examine the role of different phagocytes in the process of RBC clearance in the presence of antibodies. The spleen is the primary site for IgG-mediated RBC clearance whereas IgM-associated extravascular hemolysis occurs predominantly in the liver.33 For our study, the spleen is therefore the organ of interest.
Using magnetic RBCs, we have developed a unique assay to investigate the role of different cell types in RBC phagocytosis in the human spleen (Figure 1). Previous methods for studying erythrophagocytosis have difficulties differentiating between RBC uptake, RBC adherence, and trogocytosis. By lysing remaining RBCs after incubating RBCs with splenocytes and subsequently selecting for magnetic splenocytes, we efficiently obtain the cells that have taken up RBCs. In line with the current literature, we found that under steady-state conditions (in the absence of anti-RBC antibodies) macrophages are responsible for RBC clearance.2,35 For IgG-opsonized RBCs, as in AIHA, the mechanism of RBC clearance appears different. Namely, neutrophils, and to a lesser extent monocytes, can contribute significantly to IgG-opsonized RBC clearance not only in our in vitro assay, but also in a murine model of AIHA. These data fit with the current opinion that tissue-resident macrophages, such as the red pulp macrophages, have a homeostatic function in clearance whereas infiltrating myeloid cells take over this function under pathological conditions.36-38
The threshold for neutrophils to phagocytose RBCs seems to be much higher than that of macrophages (whereas monocytes have an intermediate one). Different factors were shown to determine whether the threshold to phagocytose IgG-opsonized RBCs by neutrophils is reached. First, erythrophagocytosis by neutrophils was found to be strongly dependent on the degree of opsonization of the RBCs. Second, activation of the neutrophils, which can be increased by different stimuli, such as ligands for TLRs (FSL-1 and LPS) and TNFα, increases phagocytosis of IgG-opsonized RBCs. Lastly, RBC phagocytosis by neutrophils is under negative control by the inhibitory CD47-SIRPα pathway.
Our data indicate that once the threshold for RBC phagocytosis by neutrophils has been reached, neutrophils may become an important factor in the process of RBC clearance. To investigate the possibility that neutrophils participate in the clearance of RBCs in AIHA, we studied phagocytosis by neutrophils using RBCs from AIHA patients. RBCs from AIHA patients were more readily phagocytosed than RBCs of healthy controls. This is despite the fact that in vivo IgG opsonization of patient RBCs is always lower than that of in vitro–opsonized RBCs. An explanation for this may be that RBCs of AIHA can have additional "eat me" signals besides the IgG opsonization that enhances phagocytosis by the neutrophils, such as complement. Even though the Coombs tests for complement deposition were negative or low in all cases, in some patients, C3 fragments were detectable by flow cytometry (supplemental Figure 8B). It could also be that the specificity of the antibody has an effect on the type of immune activation. Anti-GPA is highly potent in inducing phagocytosis because GPA is the most abundant protein of the cell surface of the RBC membrane. Besides the abundance of the epitope, the density may also affect phagocytosis. Additionally, the glycosylation status of the antibody may also be of influence. Low IgG-Fc fucosylation has been shown to enhance platelet phagocytosis by neutrophils and monocytes via increased binding to FcγRIIIa/b.39 Moreover, low IgG fucosylation has been found to correlate with hemoglobin levels and disease severity in hemolytic disease of the fetus and newborn.40,41
This project has focused on FcγR-mediated phagocytosis, yet in vivo antibody-mediated RBC clearance can also be promoted by complement, which induces phagocytosis via complement receptors.42 Both Fcγ- and complement receptor–mediated phagocytosis are regulated by CD47-SIRPα interactions.6 It would be interesting to investigate the role of neutrophils in complement receptor–mediated RBC clearance.
In vitro, RBC phagocytosis by neutrophils can further be potentiated by inflammatory mediators, indicating that in AIHA the inflammatory status of the patient may also contribute to the severity of RBC clearance and hence anemia. Indeed, there is a large body of evidence that AIHA can be induced by an underlying disease in the patient which may lower the threshold of the neutrophils to phagocytose IgG-opsonized RBCs, and thus exacerbate the hemolytic anemia. It is important to keep in mind that during these pathological conditions the cellular composition of the spleen can change43,44 which subsequently may influence phagocytosis. Additionally, IV immunoglobulin administration, a common therapy in AIHA can change cellular distribution in the spleen which may affect erythrophagocytosis.45
Overall, our data show that neutrophils residing in the spleen can contribute significantly to the destruction of IgG-opsonized RBCs. We have identified IgG levels on the RBC as well as the presence of inflammatory mediators to be important factors to drive phagocytosis of IgG-opsonized RBC by neutrophils. Therefore, in a clinical setting where antibody opsonization of RBCs and inflammatory mediators are present, neutrophils may strongly contribute to RBC clearance. Due to their abundance, the impact of erythrophagocytosis by neutrophils may be of great significance in conditions such as AIHA or blood transfusion reactions.
The full-text version of this article contains a data supplement.
The authors thank Swi Fan Cheung (Sanquin, Amsterdam, The Netherlands) for providing tissue samples, Conny Brouwer (Sanquin, Amsterdam, The Netherlands) for collecting patient material, and Peter Ligthart (Sanquin, Amsterdam, The Netherlands) for providing human monoclonal anti-RhD antibodies.
S.M.M. was supported by a grant from the Dutch Ministry of Health awarded to T.K.v.d.B. P.-A.O. was supported by the Swedish Research Council (2012-2702) and the Faculty of Medicine Foundations at Umeå University.
Contribution: S.M.M. performed the experiments, analyzed the data, and wrote the manuscript; B.M.B. and T.R.L.K. performed additional experiments; P.-A.O. and J.J. performed the animal experiments, analyzed the data, and wrote the manuscript; T.M. provided the SIRPα mutant mice; T.W.K., E.J.H., and M.d.H. enrolled patients; T.K.v.d.B., R.v.B., and S.M.M. designed the study; T.K.v.d.B. and R.v.B. supervised the study, edited the manuscript, and served as principal investigators; and all authors revised and approved the final manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Robin van Bruggen, Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands; e-mail: r.vanbruggen@sanquin.nl.
Mebius
Kraal
Structure and function of the spleen
Nat Rev Immunol
de Back
Kostova
van Kraaij
van den Berg
van Bruggen
Of macrophages and red blood cells; a complex love story
Front Physiol
Hilarius-Stokman
de Korte
CD47 functions as a molecular switch for erythrocyte phagocytosis
Sosale
Spinler
Discher
Macrophage engulfment of a cell or nanoparticle is regulated by unavoidable opsonization, a species-specific 'Marker of Self' CD47, and target physical properties
Curr Opin Immunol
Oldenborg
Zheleznyak
Lagenaur
Role of CD47 as a marker of self on red blood cells
CD47-signal regulatory protein alpha (SIRPalpha) regulates Fcgamma and complement receptor-mediated phagocytosis
J Exp Med
Dose-dependent inhibitory effect of CD47 in macrophage uptake of IgG-opsonized murine erythrocytes
Biochem Biophys Res Commun
CD47 on experimentally senescent murine RBCs inhibits phagocytosis following Fcgamma receptor-mediated but not scavenger receptor-mediated recognition by macrophages
Marker-of-self becomes marker-of-senescence
Erythrophagocytosis by neutrophils
Chandrashekar
Soni
Florid erythrophagocytosis on the peripheral smear
J Lab Physicians
Depcik-Smith
Brecher
Transfusion medicine illustrated. RBC rosetting and erythrophagocytosis in adult paroxysmal cold hemoglobinuria
Homenda
Mital
Complak
Erythrophagocytosis by neutrophils--a rare morphological phenomenon resulting in acquired haemolytic anaemia?
Int J Lab Hematol
Lobreglio
Valacca
Images in clinical medicine. Erythrophagocytosis
N Engl J Med
Transfusion medicine illustrated. Erythrophagocytosis in Epstein-Barr virus IgM-mediated hemolytic anemia
Mukhopadhyay
Souid
Erythrophagocytosis in paroxysmal cold hemoglobinuria
Am J Hematol
Barbot
Freitas
Erythrophagocytosis by neutrophils in paroxysmal cold haemoglobinuria
Eur J Haematol
Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation
J Immunol
Nagelkerke
aan de Kerk
Kuijpers
Failure to detect functional neutrophil B helper cells in the human spleen
van der Schoot
Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation
Dekkers
Kustiawan
Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages
Beaudet
Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice
Inagaki
Yamao
SHPS-1 regulates integrin-mediated cytoskeletal reorganization and cell motility
EMBO J
Okazawa
Motegi
Ohyama
Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system
Koskinen Holm
Non-hematopoietic and hematopoietic SIRPα signaling differently regulates murine B cell maturation in bone marrow and spleen
Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions
J Cell Biol
The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target
Annu Rev Immunol
Zwicker
Further studies on the relationship of anti-Ena and anti-Wrb in warm autoimmune hemolytic anemia
Autoimmune haemolysis: an 18-year study of 865 cases referred to a regional transfusion centre
Br Med J (Clin Res Ed)
Barcellini
Clerici
In vitro quantification of anti-red blood cell antibody production in idiopathic autoimmune haemolytic anaemia: effect of mitogen and cytokine stimulation
Br J Haematol
Vigevani
Pozzetto
High cytokine serum levels in patients with autoimmune hemolytic anemia (AIHA)
Immunol Invest
Abenante
Lymphocyte activation and cytokine production in autoimmune hemolytic anaemia (AIHA)
Gehrs
Autoimmune hemolytic anemia
Bian
Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells
Proc Natl Acad Sci USA
Macrophages and systemic iron homeostasis
J Innate Immun
, et al. Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities. Nat Commun.
;7:ncomms11852.
Haldar
Kohyama
Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages
Kurotaki
Uede
Functions and development of red pulp macrophages
Microbiol Immunol
Kapur
Vestrheim
A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy
Della Valle
Sonneveld
Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn
Koelewijn
Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn
Engelfriet
The immune destruction of red cells
Transfus Med
van de Laar
Saelens
De Prijck
Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages
Aslam
The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia
Exp Hematol
Thymic-derived tolerizing dendritic cells are upregulated in the spleen upon treatment with intravenous immunoglobulin in a murine model of immune thrombocytopenia [published online ahead of print 25 November 2016]
doi:10.1080/09537104.2016.1246718
T.K.v.d.B. and R.v.B. contributed equally to this study.
Document 1. Supplemental Data (PDF, 1.38 MB)- pdf file
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text:
Fracking industry advances with phase one exploratory applications in South Africa
Jasper Finkeldey
Traditional leaders fear that grazing land and agriculture will suffer if fracking goes ahead in South Africa
Hydraulic fracturing is still a 'known unknown' in South Africa's ongoing energy debate. And whilst two weeks ago communities in the KwaZulu-Natal province made it clear they don't want fracking, President Jacob Zuma does. Jasper Finkeldey reports
Granting a prospecting licence would open up a veritable Pandora's box - Francois du Toit, CEO of African Conservation Trust
Proposals to get fracking started in South Africa first surfaced in 2008 when three multinational oil and gas companies asked for permission to prospect in the Karoo. Those applications are still pending and so whilst to date, fracking has not commenced either in the Karoo or elsewhere in the country those companies anxious to get started have not gone away and if current applications are successful, an initial fracking exploratory phase could start in a matter of weeks.
While oil and gas companies insist on the prospect of job creation, economic growth and energy sovereignty, concerned communities including farmers, conservationists, and environmental activists warn that arable land and water resources would be rapidly depleted if fracking were to go ahead.
Several transnational as well as local companies have registered their interest in prospecting for unconventional gas resources in South Africa but whilst the application to frack the Karoo came to a halt due to a government-initiated strategic assessment that was released earlier this month, there are still sustained efforts to advance with the applications to prospect in large parts of KwaZulu-Natal province, as well as in the Free State and in the Eastern Cape.
A joint report by the Academy of Science in collaboration with the Department of Technology concluded there has not been enough research to date to take an informed decision and in addition, scientists have expressed concern that regulatory oversight for potential fracking might also be insufficient.
Under existing laws, oil and gas companies have to go through three to four separate environmental impact assessments before they can actually start extracting. However, the initial scoping rights have been granted by South African Petroleum Agency (PASA), which considers fracking 'a viable option' to obtain gas in principle. President Jacob Zuma is also willing to give fracking a chance stating: "We will find practical opportunities to enhance the economic opportunities the shale gas sector has to present."
But even the promised prospect of significant job creation is in doubt following a debate in the recently published Shale Gas report which pointed out the specialist skills set needed for shale gas exploration would be virtually non-existent in the country.
The most advanced exploration application proposing to explore a vast stretch in the Eastern Cape from South of Lesotho to Mount Fletcher is currently under review with PASA. Texas-based Rhino Oil & Gas has submitted an environmental impact assessment that might trigger the first exploration phase in a matter of weeks in and around the Matatiele municipality.
Local residents are concerned not only that the exploratory boreholes might cause harm, but that future fracking operations would wreak havoc to the agriculture and water supply. Matatiele local municipality has identified "Nature, Agriculture and Tourism" as its "investments of choice". And councillors have argued that fracking would be incompatible with the municipality's agenda, an anti-fracking view that is shared by traditional leaders who fear that grazing land and agriculture would also suffer if fracking were to go ahead.
In the pubic consultation process Rhino Oil & Gas - one of the four big players - has admitted the company is not even sure about the potential profitability of going ahead (subject to permission) since there has been no prior exploration of the area. Rhino's report, submitted to PASA, states: "Our application only includes work aimed at determining the presence of a petroleum resource. Approval is not being sought for any work to determine the commercial viability of any such resource.
A maximum of "10 core boreholes" are being proposed as part of this initial exploratory phase whilst additional applications by Rhino in other catchment areas only propose to use equipped airplanes to start the exploration phase. These measures are deemed "non-invasive" however anti-fracking lobbyists debate that it is unfair to discuss the prospecting application on the basis of its limited scope only. As Francois du Toit, CEO of African Conservation Trust, warns the prospecting licence would open up a veritable "Pandora's box"
At five Rhino's public meetings across KwaZulu-Natal a fortnight ago, residents voiced strong opposition towards unconventional gas exploration. Matthew Hemming, an environmental consultant who manages the environmental assessment process, said that those parties attending these meetings to object to fracking are taking the view they would rather "close the door on exploration before it opens."
Notwithstanding declining numbers at last week's meetings (the result of consultation fatigue) the opposition to fracking appears undeterred. A gathering of environmental and community organizations earlier this month in Matatiele, Eastern Cape assembled NGOs, potentially affected communities, and faith-based organizations from around the country. In total, 16 different organizations decided to send delegates to the small town at the foothills of the western formation of the Drakensburg.
Chilling temperatures did not keep delegates from engaging in heated debates over what they consider 'an assault' on their livelihoods. And in a manifesto that came out of those deliberations at Matatiele, delegates noted "the critical absence of relevant government departments and elected political leadership at public meetings where the exploration proposals are being pushed," concluding that this could amount to a silent endorsement of fracking from the Government's side.
Jasper Finkeldey is a PhD Researcher at the University of Essex and a Visiting Scholar at University of KwaZulu-Natal
Anti Fracking

redpajama_set_name: RedPajamaCommonCrawl


text:
package com.github.uchan_nos.c_helper.suggest;

import java.util.ArrayList;
import java.util.Collection;

import org.eclipse.cdt.core.dom.ast.*;
import org.eclipse.jface.text.BadLocationException;

import com.github.uchan_nos.c_helper.resource.StringResource;

import com.github.uchan_nos.c_helper.util.ASTFilter;
import com.github.uchan_nos.c_helper.util.PrintfFormatAnalyzer;
import com.github.uchan_nos.c_helper.util.TypeUtil;
import com.github.uchan_nos.c_helper.util.Util;

public class CastSuppressingErrorSuggester extends Suggester {

    @Override
    public Collection<Suggestion> suggest(final SuggesterInput input,
            AssumptionManager assumptionManager) {
        ArrayList<Suggestion> suggestions = new ArrayList<Suggestion>();

        Collection<IASTNode> castExpressions =
            new ASTFilter(input.getAst()).filter(
                new ASTFilter.Predicate() {
                    @Override
                    public boolean pass(IASTNode node) {
                        return node instanceof IASTCastExpression;
                    }
                });

        for (IASTNode node : castExpressions) {
            Collection<Suggestion> s = suggest((IASTCastExpression) node, input);
            if (s != null) {
                suggestions.addAll(s);
            }
        }
        return suggestions;
    }

    private Collection<Suggestion> suggest(
            IASTCastExpression castExpression, final SuggesterInput input) {
        ArrayList<Suggestion> suggestions = new ArrayList<Suggestion>();

        IType castedType = castExpression.getExpressionType();
        IType operandType = castExpression.getOperand().getExpressionType();

        if (castedType instanceof IPointerType && operandType instanceof IBasicType) {
            String message = "";
            String suggestion = "";

            IASTFunctionCallExpression fce =
                    getParentFunctionCallExpression(castExpression);
            if (fce != null) {
                IASTName name = Util.getName(fce.getFunctionNameExpression());
                IASTExpression[] args = Util.getArguments(fce);
                if (name != null && Util.equals(name.getSimpleID(), "printf")) {
                    String formatString = null;

                    // 書式指定文字列を取得する
                    IASTLiteralExpression le;
                    if ((le = TypeUtil.asIASTLiteralExpression(args[0],
                                IASTLiteralExpression.lk_string_literal)) != null) {
                        formatString = String.valueOf(le.getValue());
                    }

                    if (formatString != null) {
                        // castExpression が何番目の引数か検索
                        int indexOfCastExpression = -1;
                        for (int i = 1; i < args.length; ++i) {
                            if (args[i].contains(castExpression)) {
                                indexOfCastExpression = i;
                                break;
                            }
                        }

                        PrintfFormatAnalyzer.FormatSpecifier[] formatSpecifiers =
                                PrintfFormatAnalyzer.removePercentSpecifier(
                                        new PrintfFormatAnalyzer().analyze(formatString));

                        if (indexOfCastExpression >= 1 && (indexOfCastExpression - 1) < formatSpecifiers.length) {
                            PrintfFormatAnalyzer.FormatSpecifier spec =
                                    formatSpecifiers[indexOfCastExpression - 1];
                            if (spec.type == 's') {
                                suggestion = StringResource.get(
                                        "式%sが1文字を表しているなら%%cで表示可能。",
                                        castExpression.getOperand().getRawSignature());
                            }
                        }
                    }
                }
            }

            if (suggestion.length() == 0 && message.length() == 0) {
                IBasicType opt = (IBasicType) operandType;
                if (opt.getKind() == IBasicType.Kind.eChar
                        || opt.getKind() == IBasicType.Kind.eInt) {
                    if (castExpression.getOperand().getRawSignature().equals("0")
                            || castExpression.getRawSignature().equals("NULL")) {
                        // 整数リテラル0からポインタへのキャスト、NULLマクロの使用は例外として認める。
                    } else {
                        suggestion = StringResource.get(
                                "整数からポインタ型へのキャストは危険。");
                    }
                }
            }

            if (suggestion.length() > 0) {
                try {
                    suggestions.add(new Suggestion(
                            input.getSource(), castExpression,
                            suggestion, message));
                } catch (BadLocationException e) {
                    // TODO Auto-generated catch block
                    e.printStackTrace();
                }
            }
        }

        return suggestions;
    }

    private IASTFunctionCallExpression getParentFunctionCallExpression(
            IASTExpression expression) {
        IASTNode parent = expression;

        while (parent != null && !(parent instanceof IASTFunctionCallExpression)) {
            parent = parent.getParent();
        }

        return (IASTFunctionCallExpression) parent;
    }

}

redpajama_set_name: RedPajamaGithub


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text:
Skift Global Forum: Hilton's Tech Innovations Drive Brand Growth
Activism / Arts and Culture / Camping / Climbing / Hiking / History / Learning / News / North America / United States
National Park Service prepares for its second century
by Kraig Becker on Sep 1, 2011
Last week the National Park Service celebrated its 95th anniversary. Over the past nine-and-a-half decades, the NPS has been protecting and managing some of America's most amazing wild places, with the charge of ensuring that those places are "unimpaired for the enjoyment of future generations." That hasn't always been an easy task however, and now more than ever, the parks face incredible challenges. Yesterday, the Park Service released a comprehensive plan that will chart its course over the next five years, leading up to its centennial in 2016, and beyond.
In that plan, which is entitled A Call to Action: Preparing for a Second Century of Stewardship and Engagement, the Park Service challenges its employees and partners to work together towards a common goal, namely preparing the NPS for a second 100 years of service. The plan outlines 36 different areas of opportunity for the Park Service in which they can more fully engage visitors in the parks, continue to preserve America's favorite places, improve educational outreach, and become more efficient as an organization.
A Call To Action identifies a variety of goals for the Park Service, including creating a "State of the Park" report for each of the 50 States, studying the economic impact that parks have on surrounding communities, and finding ways to enhance cultural diversity across the entire system. The report also acknowledges the importance of addressing the impact of climate change and establishing a national parks endowment, amongst other things. In short, it is an ambitious, important outline of where the Park Service wants to go by the end of its first century, so it can be better prepared to serve in its second.
The National Parks Conservation Association, was quick to praise the Park Service plan as well. The organization's President, Tom Kiernan, called A Call To Action "a very strong and important step towards ensuring our spectacular national park landscapes, wildlife, and American history and heritage are better protected for future generations." But the NPCA says that for this plan to succeed, it needs the support of Congress and the President. With a $600 million annual budget shortfall, the Park Service obviously faces serious difficulties in completing its mission on a yearly basis.
U.S. national parks continue to be very popular travel destinations for both Americans and foreign visitors alike. With this plan, the Park Service has set its sights on the future, while identifying its biggest challenges and ways to overcome them. Those challenges, at times, can seem very daunting, but the Park Service, the NPCA, and their partners are prepared to take them on.
Tags: adventure travel, national park service, national parks, national parks conservation association, NationalParks, NationalParksConservationAssociation, NationalParkService, npca
Interview: Abercrombie & Kent CEO on the Roots of Experiential Travel
Animal Planet Builds Travel Series Around Adventure GoPro Shots
Before You Book: Eco-Friendly Hotel Or Just Greenwashing?
Ziplines and Farms are Fertile Ground for Adventure Travel
Lindblad Expeditions CEO Reflects on Travel Entrepreneurship


Vladimir Kara-Murza, a leader in the Russian opposition who believes he was the target of a Russian security service poisoning, in Arlington, Va., Aug. 19, 2016. "In the space of about 20 minutes, I went from feeling completely normal to having a rapid heart rate, really high blood pressure, to sweating and vomiting all over the place, and then I lost consciousness," Kara-Murza said. Mysterious deaths and close calls have some opposition figures worried that political murder is resurgent in Russian foreign policy.
MOSCOW – From a certain perspective, certainly the Kremlin's, Vladimir Kara-Murza's behavior in Washington could be seen as treasonous, a brazen betrayal of his homeland.
In a series of public meetings on Capitol Hill, Kara-Murza, a leader in the Russian opposition, urged U.S. lawmakers to expand economic sanctions against the Russian government under a law known as the Magnitsky Act. That would hasten political change in Russia, he argued.
Back in Moscow a month later, in May 2015, the changes Kara-Murza detected were going on in his own body.
Midway through a meeting with fellow dissidents, beads of sweat inexplicably dotted his forehead. His stomach churned.
Though Kara-Murza survived, few others in his position have proved as lucky. He said he was certain he had been the target of a security service poisoning.
Used extensively in the Soviet era, political murders again are playing a prominent role in the Kremlin's foreign policy, the most brutal instrument in an expanding repertoire of intimidation tactics intended to silence or otherwise intimidate critics at home and abroad.
Though typically not traceable to any individuals and plausibly denied by government officials, poisonings leave little doubt of the state's involvement – which may be the point.
No other major power employs murder as systematically and ruthlessly as Russia does against those seen as betraying its interests abroad.


text:
SBIJI Innovation
D-Fend Solutions expands US headquarters!
Nov. 12th 2020
D-Fend Solutions has opened its first North American office in McLean, Virginia, to expand its operations and meet the increasing demand for counter-drone products and solutions.
Sanofi, Israel's Biond Biologics to collaborate on new cancer drug
JERUSALEM (Reuters) - Israel's Biond Biologics Ltd said on Tuesday it had forged an exclusive global licence agreement, potentially worth more than $1 billion, with French drugmaker Sanofi for the dev
Alstom Invests In Cybersecurity Specialist Cylus And Signs Strategic Cooperation Agreement
9 December 2020 – Alstom has finalised a deal to invest US$7 million in Cylus, an Israel-based cybersecurity specialist, thus acquiring a minority stake in the company. The investment will give Alstom
Trigo Raises $60M to Scale Its Frictionless Checkout Grocery Platform
Trigo, a global leader in frictionless checkout technology, has raised a $60 million Series B funding round led by 83North. The latest round brings Trigo's total funding to $94 million. The new funds
A Member firm of SBI Group, Japan
© 2020 SBI JI Innovation Advisory Ltd. All rights reserved


text:
Results tagged "IDENT1" from Spy Blog - SpyBlog.org.uk
National Policing Improvement Agency annual report and accounts 2008-09 - mobile handheld computers, ANPR database, DNA database, PNC, IMPACT, Aircell for the Tube, Prüm
on July 15, 2009 6:28 AM | Permalink
Reading the
National Policing Improvement Agency annual report and accounts 2008-09
we were reminded of our previous Spy Blog article:
Police Receive £50 Million For 10,000 Hand Held Computers i.e. £5,000 each - are they gold plated ?
<40 MANAGEMENT COMMENTARY
During 2008, the Home Office allocated £80 million of funding over the next three years to support the delivery of handheld computers to frontline police officers. The funding was provided to increase the number of devices used by officers to 30,000 by March 2010.
The roll-out, managed by the NPIA has already exceeded its first milestone to have 10,000 devices in force by September 2008, and the second phase of roll-out is well
underway. All forces in England, Scotland and Wales have now received a portion of the funding.
So £50 million has now magically become £80 million !
MANAGEMENT COMMENTARY page 71
Table 2: Summary of other protected personal data related incidents
in 2008/09
Incidents deemed by the Data Controller not to fall within the criteria for report to the Information Commissioner's Office but recorded centrally within the Department are set out in the table below. Small, localised incidents are not recorded centrally and are not cited in these figures.
Category Nature of incident Total
I Loss of inadequately protected electronic equipment, devices or paper documents from secured Government premises - 0 (nil)
II Loss of inadequately protected electronic equipment, devices or paper documents from outside secured Government premises - 4 (four)
III Insecure disposal of inadequately protected electronic equipment, devices or paper documents - 0 (nil)
IV Unauthorised disclosure - 1 (one)
V Other - 0 (nil)
N.B. the carefully worded "inadequately protected devices or paper documents"
How many of the 10,000 portable devices which NPIA have issued have been lost or stolen ?
It is hard to believe that none of them at all have gone missing.
These may or may not prove to be "adequately protected"
Some other points of interest in the report:
Airwave,
ANPR,
IDENT1,
NPIA,
Police Computer,
Continue reading National Policing Improvement Agency annual report and accounts 2008-09 - mobile handheld computers, ANPR database, DNA database, PNC, IMPACT, Aircell for the Tube, Prüm.
Home Office spinning about 6 and 12 years data retention for innocent people's DNA profiles and Fingerprints
on May 7, 2009 4:46 PM | Permalink
The smug and complacent, yet incompetent and untrustworthy, Labour Home Secretary Jacqui Smith and her side kick Vernon Coaker, have been spinning their grudging response to the European Court of Human Rights Judgment against them in the Marper case, regarding their illegal and immoral retention of innocent people's DNA human tissue samples and analysed DNA profiles on the Police National DNA Database. This also applies to the 10 finger and palm print Fingerprints of innocents on the IDENT1 database.
Read their latest fake "public consultation" (as if anyone believes now that they will somehow "listen" to the public who bother to submit formal responses):
Keeping the right people on the DNA database (1Mb .pdf)
Keeping the right people on the DNA database - Annexes (3Mb .pdf)
Annex B contains a list of the the names of the data fields which currently make up a DNA Database profile record.
Annex C has some academic research commissioned by the Home Office from the Jill Dando Institute to explain the Home Office's dubious choice of 6 and 12 year retention periods. This paper by the criminologist Professor Ken Pease OBE, is based on a "sample" of Metropolitan Police Service cases, in the "No Further Action" (NFA) category, not from the entire National DNA Database.
There is a hint that the Home Office were lobbying for 15 years data retention in the case of Fingerprints. Note that Professor Pease mentions the evils of Eugenics, something which the NuLabour police / nanny / database / surveillance state politicians, apparatchiks and spin doctors did not dare to mention in their press releases and interviews.
Annex D has a "monetised impact assessment" full of obscure figures e.g. they assume it costs 90p per year to refrigerate each DNA human tissue sample.
All of these figures look to be guesses, or evidence of bloated costs e.g. £15,000 to write and test a computer program to automatically delete records off the databases after a set period of time, something that should only take a couple of lines of computer code to program (such standard routines almost certainly already exist in the data backup software)
These new style "impact assessments" are starting to appear across Government, but they still do not provide enough detail of the basic assumptions and omissions, to make much sense.
There is no good reason why the other "samples" which the Police are legally empowered to take from you (by force, without your consent, if necessary) should not also be destroyed once they have been properly forensically analysed and processed:
i.e. oral swabs (skin cells from the inside of the mouth, used for DNA analysis) , saliva, blood, urine, semen, pubic hair, other human tissue.
That should also apply to the Non-Biological Samples i.e. photographs ("mug shots"), footwear impressions and dental impressions (classified as "intimate" samples), and to Samples taken for non-DNA analysis purposes e.g. alcohol or drugs related analyses.
N.B. Obviously none of this applies to Crime Scene samples.
Read the GeneWatch UK Press Release, who, like all the other numerate and principled people who oppose this potentially genocidal policy, are unlikely to be mollified by these "fake consultation" documents.
Home Office drags its feet on DNA database removals
Data Retention,
National DNA Database
Continue reading Home Office spinning about 6 and 12 years data retention for innocent people's DNA profiles and Fingerprints.


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President Cyril Ramaphosa chief guest at India Republic Day celebrations
South Africa's President Cyril Ramaphosa heads to Delhi as the chief guest for the India's Republic Day.
The time will be spent in efforts to improve existing ties between the two countries including social and defense sectors.
Moving beyond the shade of the Jacob Zuma era
It is understood that President Ramaphosa is working towards improving relations with India and move past the era of the erstwhile President Jacob Zuma, who resigned from his post in February last year after a nine-year reign tainted by corruption scandals including those involving the Indian-born Gupta brothers, said people aware of the matter.
Ramaphosa, like Prime Minister Narendra Modi, has listed skill development, health services and digitization of governance and services as his priorities.
The upcoming visit on January 25-26 will enable Ramaphosa to seek alignment in policies in areas of common priorities.
Arms deals on the agenda
South Africa has Africa's most thriving defense industry and the two sides are keen to explore partnership in joint production.
South African defence firm Denel, which had been blacklisted prior to September 2018, will explore joint ventures with Indian firms under the Make in India programme.
The ban was lifted following a May 2018 judgement of the Supreme Court, which dropped all corruption charges against the defence company. It came after a personal request from Ramaphosa to Modi during a July meeting in Johannesburg on the sidelines of 2018 BRICS Summit.
Denel was blacklisted in 2005 by the then UPA government over allegations that the company paid kickbacks to secure a deal for the Indian Army's global tender to purchase of 1,000 NTW-20 anti-materiel rifles along with 398,000 rounds of ammunition.
Ramaphosa to turn on the charm to Indian business community
During his stay, Ramaphosa will also seek to engage the Indian business community and invite it to take advantage of South Africa's pre-eminent position in conducting trade with several other African countries.
South Africa is the biggest African economy and there is substantial potential for trade growth between the two countries, according to experts.
South Africa has a 1.5 million strong Indian-origin community, which accounts for about 3% of the country's population.
Ramaphosa's visit comes at a time when, saddled with Chinese loans, many countries from western, eastern and southern Africa are looking at India owing to its goodwill and non-interfering approach to untangle themselves from the debt trap.
Africa has been a foreign policy priority for the Modi government and there have been several high-level engagements with countries in the continent since May 2014.
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Bano Qudsia was a writer, intellectual, playwright and spiritualist from Pakistan who is regarded among the best Urdu novelist and short story writers of modern times. She is best known for her novel Raja Gidh. She wrote for television and stage in both Urdu and Punjabi languages. She is the wife of famous novelist Ashfaq Ahmed. Bano Qudsia is recognized as a trendsetter in the realm of television plays.


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Have a look at all our latest publications.
All our protocols and reviews are published online in the Cochrane Library.
A list of our completed reviews and protocols and registered titles is shown below, with links to abstracts where available.
The Cochrane Consumer Network has a web site which includes summaries of Cochrane Reviews of evidence and more than half of these have additional consumer summaries.


Thanks to ABDULLAH AL MAMUN for this awesome Mockup!
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What was the inspiration behind putting a team together for this Relay for Life event? Here at Wairau Pharmacy we have a great team spirit both at work... Here at Wairau Pharmacy we have a great team spirit both at work...... Note: If you want to delete your account, you can do so by providing proof of authority over the account. What constitutes "proof of authority" will vary depending on the circumstances, but generally will require sufficient identifying information so that we can be confident you are the account owner.
Emily Clark One Step Closer to a Cure: Relay For Life How it All Began 1985, Tacoma, Washington, Dr. Gordon Klatt walked around a track for 24 hours Signified a day of your life for someone who may not have one Raised $27,000 for the American Cancer Society Relay For Life was born Relay Today http... Reading Scottish will be playing at the start of Ascot's Relay for life on Saturday 22nd July just before the start of the relay at 11am. It is held in the Silver Ring, Ascot Racecourse.
Relay For Life is a fantastic community event, where we get the chance to celebrate our cancer survivors and carers, while remembering those who we have lost in their cancer journey.
Relay for life helps us as a community be more aware on how to show compassion, love, comfort, and lots of supports to families due to losing beloved ones.
Relay For Life. For 20 years, Relay For Life has been the Canadian Cancer Society's signature fundraising event. Relay is an event like no other, where you join your family, friends, co-workers and neighbours to celebrate life and give hope to those living with cancer.
Thank you for your interest in the Nyngan Relay for Life Facebook page. We'd love for you to share your thoughts, comments, opinions and your personal experience on topics we talk about on this page and your passion for your local Relay for Life.


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Director Terry Prince
Services/Benefits
IDVA Benefits Guide
State Employment and Licenses
Statewide Veteran Organizations
Veterans' Legal Aid
Fishing and Hunting Permits
Credit for Military Learning
Veterans' Cash
Serving Those Who Served
Homeless Program
Illinois Joining Forces
Veteran Service Officers
IDVA Jobs
Committees/Task Forces
IDVA Procurement
Veterans' Accountability Unit
Notice to Officials
VAU Staff
Terry Prince - Director - Illinois Department of Veterans' Affairs
Terry Prince was appointed Acting Director of the Illinois Department of Veterans' Affairs by Governor JB Pritzker on April 1st, 2021 and confirmed by the Senate Executive Appointment Committee on March 28, 2022.
As Director, he leads a team of 1,200 professionals, in partnership with numerous local and state organizations, to provide beneficial services for more than 600,000 veterans and their families across the State of Illinois.
Upon joining IDVA, Director Prince developed a Six-Point Plan for the Future of IDVA, which he and his team have diligently worked to execute change and maximize opportunities. Over the course of the year, IDVA's leadership team has been reimagined. It has recruited and hired staff for key roles across IDVA. Communication and culture have been enhanced. Infection control systems have been elevated and many policies, procedures, and technology have been updated and implemented. The five Illinois Veterans' Homes across the state continue to provide excellent care delivered by trained medical professionals and staff. The Veteran Service Officers interface with veterans and their families to help them identify and obtain earned benefits and services.
Prior to this appointment, Director Prince served as the Superintendent of the Ohio Veterans Homes. In this capacity, he led 850 employees providing skilled nursing and domiciliary care for 801 veterans at three separate facilities, including the 5th largest Veterans home in the country. At the conclusion of this assignment, he was awarded the department's inaugural Major General Manning Force Award, an honor that recognized his distinguished service in a position of significant responsibility.
Director Prince began his military career in 1986 and went on to serve at 14 different duty stations around the world. His most recent tours include: Command Master Chief at Naval Hospital Camp Lejeune, NC and the Walter Reed National Military Medical Center in Bethesda, MD. Later, he was handpicked to be the 1st Senior Enlisted Advisor at the Defense Health Agency in Falls Church, VA. He closed out his illustrious 31-year career in 2017 as the 13th Director of the U.S. Navy Hospital Corps and Force Master Chief of Navy Medicine.
Force Master Chief Prince's military awards include the Legion of Merit, Defense Superior Service Medal (two awards), Meritorious Service Medal (two awards), Navy and Marine Corps Commendation Medal (six awards), Navy and Marine Corps Achievement Medal (three awards) and the U.S. Army Order of Military Medical Merit. He is also triple warfare qualified as a Surface, Aviation and Fleet Marine Force Warfare Specialist.
Director Prince earned a bachelor's degree in organizational leadership from Chapman University and is a graduate of the National Defense University Keystone Leadership Fellowship, Navy Senior Enlisted Academy and the Navy Command Master Chief course. He is a proud Eagle Scout and finisher of the Ironman Hawaii Triathlon World Championship.
There is a strong history of military service in Director Prince's family, with his grandfather, three uncles and two brothers all having worn the cloth of our nation. He and his wife, Dr. Jennifer Prince, a 12-year Navy veteran, reside in Chicago.
CommunicationsCurrently selected
Illinois Department on Aging
State of Illinois G.I. Bill & Education Programs
State of Illinois Benefits for Veterans
State of Illinois Programs for Veterans
State of Illinois Department of Employment Security
Healthcare Transformation Collaboratives
U.S. Department of Veteran Affairs Center for Women Veterans
U.S. Department of Veteran Affairs Welcome Kit
U.S. Department of Veteran Affairs Newsletter Links
Illinois Association of County Veterans Assistance Commissions


"There are two things, which I have always looked upon as difficult. The one is, to make the wicked sad; the other is, to make the godly joyful. Dejection in the godly arises from a double spring: either because their inward comforts are darkened, or their outward comforts are disturbed. To cure both these troubles, I have put forth this ensuing piece, hoping, by the blessing of God, it will buoy up their desponding hearts, and make them look with a more pleasant aspect.
I would prescribe them to take, now and then, a little of this Cordial: all things work together for good to them that love God. To know that nothing hurts the godly, is a matter of comfort; but to be assured that all things which fall out shall co operate for their good, that their crosses shall be turned into blessings, that showers of affliction water the withering root of their grace and make it flourish more; this may fill their hearts with joy till they run over." - Thomas Watson (Preface).
Click the button below to add the A Divine Cordial, or, The transcendent priviledge of those that love God and are savingly called (1663) by Thomas Watson to your wish list.


A huge thank you to the 146 Dale fans who made the trip to Plymouth Argyle on Saturday.
A gallery of photos, courtesy of DySporting, from Dale's League One fixture v Plymouth Argyle at Home Park.
A selection of images, courtesy of DySporting, from Dale's League One fixture v AFC Wimbledon at the Crown Oil Arena.


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Diamesa koshshimai är en tvåvingeart som beskrevs av Ole Anton Saether och Willassen 1987. Diamesa koshshimai ingår i släktet Diamesa och familjen fjädermyggor.

Artens utbredningsområde är Nepal. Inga underarter finns listade i Catalogue of Life.

Källor

Fjädermyggor
koshshimai

redpajama_set_name: RedPajamaWikipedia


Gamuda Land is set to unveil an impressive collection of artwork for the soon to be completed 661 Chapel St development in South Yarra.
Andy Dinan, Director of Mars Gallery which is renowned for exhibiting and promoting contemporary Australian artists, has been invited to specially curate the art collection throughout the development; included will be some of Australia's most collected artists.
Dinan's curation for the project adopted a slightly different approach, treating the shared spaces of 661 as part of a 'home' environment rather than as a 'corporate curation' with the each of the chosen pieces reflecting the spaces which they occupy, as well as the relationship between the artworks themselves.
Andy Dinan recently took time out of her schedule to chat to Urban Melbourne about Mars Gallery's curation of 661 Chapel St's shared public and resident spaces, and the growing demand for artist involvement in property development.
According to Dinan a number of developers are realising the potential shared spaces have to add value to their projects, and this has in turn created a growing number of opportunities for artists to be brought on for commissioned work in a curatorial, or consulting role. This growing demand for property development collaborations represents a key new pillar for Mars Gallery.
In commissioning artwork as part of their developments, the more culturally atuned and younger developers are providing a point of difference to their respective development in an increasingly competitive market, creating what Dinan terms as 'destination developments'.
Dinan says that developers and developments are moving away from just street art to other media such as video and audio art to enhance and compliment their spaces. Marina Tower's artwork which comprises optic fibre light to create a giant glow worm is set to be unveiled in February, with an art installation as part of Golden Age's Opera development on St Kilda Road to be launched mid-year.
Previously Mars Gallery has been involved in creating a sound sculpture as part of an Elenberg Fraser development on Albert Road South Melbourne, which featured sounds that the artist recorded in Japan that was activated when the visitor sat down.
Another project previously covered by Urban Melbourne and curated by Mars Gallery is Melbourne Central's Melbourne Art Loop on the lower ground level.
In explaining about how Mars Gallery won the curatorial commission for 661 Chapel St, Dinan says she was invited to submit via a tender process with a brief that called for a more 'homely' experience rather than something corporate.
This is not a corporate curation, nothing is "plonked" into place. This project is highly considered.
We have curated this like a personal home using all Australian artists to evoke a sense of continued discovery.
The process to select the artists and curate the works was slow but considered says Dinan, largely in response to the brief, with each piece chosen for its suitability for a particular space with a lot of negotiating and consultation as part of an open dialogue between architects Bird de la Coeur, Gamuda Land and the builder, Crema Constructions.
This has resulted in a carefully curated experience for both visitors to the building, and its soon to be residents. A stainless steel sculpture in the form of the building address by sculptor Matthew Harding greets residents upon arrival at the porte-cochere, and visually introduces the art project to visitors of 661 Chapel St.
Once inside visitors will find themselves within the high vaulted ceiling Grand Library - a space designed to capture light and create a sense of calmness. 'The Relic' - a cardboard sculpture with an industrial aesthetic by acclaimed artist Daniel Agdag who works entirely in cardboard and is known for his attention to detail, will feature in this space.
According to Dinan it was critical that the artworks selected were 'an appropriate fit for the spaces.' In the context of the Grand Library this extended beyond The Relic to the adjoining wall, which will feature a print of a photo by Agdag while a catalogue of his work will also sit within the space.
Fabrice Bigot, a French-born photographic artist, will have a series of pieces centred around botanical studies located above the concierge desk, and throughout the building's public spaces and private dining rooms.
Simon Finn has several works curated throughout the building, including a statement piece in the Grand Library, and three framed works entitled Liquid Tower in the meeting room which draw on his practices exploration of the synthesis between artist, environment and technology.
Woven polypropylene sculptures by Emma Davies are featured in the common spaces alongside Jenny Crompton's acrylic ink on paper frames in the newspaper room.
The artworks will be officially unveiled in February as part of the official building opening.