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The tryptophan derivative, tranilast, and conditioned medium with indoleamine 2,3-dioxygenase-expressing cells inhibit the proliferation of lymphoid malignancies
Shihoko Suwa
Aya Kasubata
Miyu Kato
Megumi Iida
Osamu Miura
Tetsuya Fukuda
Affiliations: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan
Published online on: January 9, 2015 https://doi.org/10.3892/ijo.2015.2825
Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes tryptophan degradation and induces immunosuppression. Although IDO is an important factor that allows tumors to escape from immunological attack, its effect on lymphoid malignancies has not been fully revealed. We evaluated the expression of IDO in samples from patients with B-cell malignancies. The IDO expression in the tumor samples was comparable to those in peripheral blood mononuclear cells from healthy donors and had mainly originated from non-B cell populations. We introduced IDO gene into Chinese hamster ovary (CHO) cells. We then cultured various cell lines using CHO- or CHO-IDO-conditioned medium. Compared with the CHO medium (CHO-CM), the CHO-IDO medium (IDO-CM) decreased the viability of lymphoid cell lines but not those of the non-lymphoid lines. Next, we examined the effects of tryptophan metabolites on lymphoid tumors, and revealed that the drug N-[3',4'-dimethoxycinnamoyl] anthranilic acid (tranilast), a synthetic derivative of the tryptophan metabolite, was able to repress proliferation and dose-dependently induce cell death of lymphoid cell lines. Tranilast induced the activation of the c-Jun N-terminal kinase, which is activated by cellular stress, in lymphoid cells. The effect of tranilast on lymphoid cells was independent of the aryl hydrocarbon receptor (AhR) although tranilast has been reported to be an AhR agonist. Finally, the administration of tranilast decreased murine lymphoid tumor progression in vivo. These results indicated that IDO and tryptophan derivatives, particularly tranilast, can be tools for the therapy for lymphoid malignancies.
Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the essential amino acid tryptophan and leads to the starvation of tryptophan and the accumulation of tryptophan metabolites such as kynurenine. Therefore, it induces apoptosis or cell cycle arrest of activated T cells and the differentiation of new regulatory T cells, which results in immunologic suppression (1–3). This control may be essential for physiological acquired immunological tolerance. For example, IDO expressed by the placenta prevents a maternal allo-reaction against the fetus (4,5) and enables the maintenance of gestation as shown by the fact that the IDO inhibitor, 1-methyl-tryptophan (1-MT), induced the immune-mediated rejection of allogeneic concepti (6). IDO is also expressed in some tumor cells, and it plays a pivotal role in the escape from immunological attack by inducing immunological anergy in such tumors (7,8). 1-MT could potentiate antitumor immunity in these IDO-expressing tumors, and could induce tumor regression in animal models (9). Based on this result, several clinical trials evaluating the use of 1-MT against solid tumors are currently ongoing. However, the role of IDO in lymphoid tumors such as lymphoma or lymphocytic leukemia has not been fully revealed.
Experimental autoimmune encephalomyelitis (EAE) is a well-known animal model of multiple sclerosis and is mediated by autoimmune TH1 cells (10). Altered peptide ligands (APLs) that had modified the strength of T-cell receptor signaling had induced self-tolerance to autoantigens. Platten et al (10) showed that the IDO expression was induced by APLs and that the administration of N-[3′,4′-dimethoxycinnamoyl] anthranilic acid (tranilast), a synthetic derivative of the tryptophan metabolite anthranilic acid, could reverse paralysis in mice with EAE. IDO has also been reported to be associated with acute graft-versus-host disease (GVHD) in murine bone marrow transplantation. IDO expression is induced at the site of GVHD. Moreover, exogenous kynurenines can reduce GVHD lethality (11,12). These reports indicated that the tryptophan metabolites as well as the forced IDO expression may have some effect on the fate of activated lymphocytes.
It is possible that an analysis of the expression and effect of IDO in lymphoid malignancies will provide a clue regarding their treatment. In the present study, we found that the lymphoid malignant cells were susceptible to cytotoxicity by IDO and that tranilast also induced growth suppression of these cells.
After written informed consent was obtained in compliance with the Declaration of Helsinki, samples (peripheral blood, bone marrow, lymph node, ascites and pleural effusion) were collected from the patients. Approval was obtained from the ethics committee of the Tokyo Medical and Dental University. All animal studies were approved by the Animal Subjects Committee of the Tokyo Medical and Dental University and were performed in accordance with the institutional guidelines.
Cells and reagents
Splenocytes were obtained from 8-week-old female BALB/c mice using a RBC lysis buffer [0.155 M NH4Cl, 0.01 M NH4HCO3 and 0.1 mM EDTA (pH 7.5)]. Peripheral blood mononuclear cells (PBMCs) from patients were isolated through density-gradient centrifugation from freshly collected blood samples using SEPARATE-L (Muto Pure Chemicals, Tokyo, Japan). Cells were preserved with CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) at −80°C. Human and murine cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium or Dulbecco's modified Eagle's medium (DMEM) medium with 10% fetal calf serum (FCS). Tranilast was provided by Kissei Pharmaceutical (Nagano, Japan). IFNγ, concanavalin A (ConA), L-kynurenine, 3-HAA, Kynurenic acid, fludarabine, α-naphthoflavone, and 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-2 was purchased from R&D Systems (Minneapolis, MN, USA). CD19 Pan B Dynabeads and human T-activated CD3/CD28 Dynabeads were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). 4-Hydroperoxy Cyclophosphamide were purchased from Wako Pure Chemical Industries (Osaka, Japan). JNK inhibitor SP600125 was purchased from Enzo Life Sciences (Farmingdale, NY, USA).
Real time (RT)-PCR
Samples from patients with B-cell lymphoma were used. RNA was isolated using TRIzol (Gibco-BRL, Gaithersburg, MD, USA). Reverse transcription using an oligo dT primer was performed with a SuperScript II RT kit (Invitrogen-Life Technologies, Grand Island, NY, USA). The synthesized cDNA was amplified with primers specific for human IDO (forward, 5′-CCTGACTTATGAGAA CATGGACGT-3′ and reverse, 5′-ATACACCAGACCGTCTG ATAGCTG-3′); or mouse IDO (forward, 5′-TTCGAAAGGTG CTGCCCCGC-3′ and reverse, 5′-GCCCTTGTCGCAGTCCC CAC-3′); or human GAPDH (forward, 5′-CTGACTTCAAC AGCGACACC-3′ and reverse, 5′-TCCTCTTGTGCTCTTGC TGG-3′); or mouse GAPDH (forward, 5′-TGCGACTTCAAC AGCAACTC-3′ and reverse, 5′-CTTGCTCAGTGTCCTTGC TG-3′). Quantitative RT-PCR was performed with the LightCycler FastStart DNA Master Plus SYBR-Green I kit using LightCycler software version 3.5 (Roche Applied Science, Indianapolis, IN, USA).
Cloning of mouse IDO (mIDO)
The murine Ido-1 cDNA containing the full length open reading frame was amplified with the specific primers (forward, 5′-GGAGTAGA CAGCAATGGCAC-3′ and reverse, 5′-GAGCTTGCTA CACTAAGGCC-3′) using the RNA from splenocytes as a template. The purified product of 1250 bp was cloned into the pGEM-T Easy Vector system (Invitrogen-Life Technologies). After sequencing, it was subcloned into the expression vector, pcDNA3 (Invitrogen-Life Technologies) (pcDNA3-mIDO).
Chinese hamster ovary (CHO) cells were transfected with pcDNA3-mIDO using Lipofectamine LTX (Invitrogen-Life Technologies) and selected with 1 mg/ml geneticin (G418) (Roche Applied Science). The stable transfected CHO cells were subcloned, and the clones with high levels of mIDO protein expression were selected. Furthermore, pcDNA3-mIDO was transfected to the murine B lymphoma cell lines A20 and M12 by electroporation. RNA and protein were extracted from the transfected cells after selection with G418. A plasmid containing the dominant negative form of c-Jun N-terminal kinase (JNK) was obtained from the Addgene repository [13761; pcDNA3 Flag Jnk2a2 (apf)] (13). The coding region (DN-JNK2) was subcloned into retroviral vector pQCXIN (Clontech: Palo Alto, CA, USA). A20 cells were transduced with pQCXIN-DN-JNK2 and selected with G418.
Conditioned medium
We cultured CHO cells transfected with the mIDO gene or control vector in a minimum essential medium (αMEM) medium. On the following day, the αMEM in the dish was changed to RPMI-1640 medium and the CHO cells were cultured for 5 days at 37°C and in 5% CO2. The collected supernatant from the dish was adjusted to a pH of 7.0 with 1 N NaOH and then 2 mM L-glutamine (Invitrogen-Life Technologies) was added. This medium was sterilized by a filter.
Measurement of cell viability and viable cell number
In the present study, the determination of cell viability was based on an analysis of mitochondrial transmembrane potential using 3,3′-dehexyloxacarbocyamine iodine (DiOC6) (Molecular Probes, Eugene, OR, USA) and the cell membrane permeability to propidium iodide (PI) (Molecular Probes) by flow cytometry with a FACSCalibur™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described (14). The fluorescence-activated cell sorting (FACS) data were analyzed with FlowJo software version 6.3.3 (Tree Star, Inc., Ashland, OR, USA). The DiOC6-positive and PI-negative cells were considered to be viable cells. The viable cell number was calculated as the cell number that was acquired during 30 sec multiplied by the percentage of viable cells.
Immunoblot analysis
Cells were lysed in a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml each of aprotinin and leupeptin (Sigma-Aldrich, St. Louis, MO, USA). The cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted to polyvinylidene difluoride membranes. Membranes were probed with primary antibodies and probed with HRP that was conjugated anti-mouse or rabbit IgG (GE Healthcare Life Sciences, Piscataway, NJ, USA) as the second antibody. Antibodies against IDO (Oriental Yeast Co., Ltd., Tokyo, Japan), SAPK/JNK, phospho-Thr183/Tyr185-SAPK/JNK, AKT, phospho-S473-AKT, p38, phospho-Thr180/Tyr182-p38 (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Sigma-Aldrich) were used.
Eight-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan) and were housed under specific pathogen-free conditions. After intraperitoneal injection of anesthesia using 2,2,2-tribromoethanol (Avertin; Sigma-Aldrich), the bilateral hind flanks of each mouse were transplanted with 2×105 of A20, which was a B lymphoma cell line derived from the BALB/c mice. These cells generated palpable tumors at the site of injection in 100% of the injected mice. Seven days after the inoculation, tranilast that was suspended in 0.5% carboxymethyl cellulose (CMC) solution or CMC solution alone had been administered by oral gavages with a disposable flexible tube twice a day for 3 weeks. The tumor progression was monitored biweekly, and the tumor volumes were calculated with the following formula: Volume = (width)2 × length/2.
The significance of the differences between groups was determined by the Mann-Whitney U test, the Student's t-test, or the Student's paired t-test using GraphPad Prism software (La Jolla, CA, USA).
IDO expression in human B-lymphoid malignancies
First, we examined the IDO expression in 30 samples from the patients with B-cell malignancies by Rq-PCR using specific primers for IDO gene. We set HeLa cells that were treated with 10 ng/ml IFNγ and untreated HeLa cells as the positive and negative controls, respectively. The expression of IDO from 22 samples was detected as 10-fold higher than those of the negative controls although all samples were less than one tenth of the positive controls. The results were comparable to the expression of PBMCs from three healthy donors. The expression was heterogeneous in the samples and had no clear correlation with the malignancy types (Fig. 1A) except for Burkitt's lymphoma (BL), which had no detectable expression. We further evaluated the IDO expression in the samples that had a relatively high expression after the separation of the B cells and non-B cells. The IDO expression was mainly detected in non-B cell populations in five examined samples (Fig. 1B). We then examined IDO induction after IFNγ treatment (10 ng/ml) using the PBMCs from CLL patients because this cytokine strongly induced IDO in dendritic cells. IDO was clearly upregulated after the treatment in PBMCs from patients with CLL. However, the findings demonstrated that this induction was associated with the non-B cells, but not with the CLL-B cells after the separation of B cells and non-B cells (Fig. 1C).
Indoleamine 2,3-dioxygenase (IDO) expression in B-lymphoid malignancies. (A) IDO expression in 30 clinical samples from B-lymphoid malignancies [acute lymphocytic leukemia (ALL) n=5; chronic lymphocytic leukemia (CLL) n=9; diffuse large B-cell lymphoma (DLBCL) n=6; follicular lymphoma (FL) n=6; Burkitt's lymphoma (BL) n=2; splenic marginal zone lymphoma (SMZL) n=2], 3 samples from healthy donors, positive controls (HeLa cells treated with 10 ng/ml IFNγ), and negative controls (untreated HeLa cells) were analyzed using quantitative real-time PCR (Rq-PCR) and showed the ratio IDO to GAPDH (internal control). Each symbol represents an individual patient. (B) IDO expression was analyzed in the total cells, B cells and non-B cells from the clinical samples of the patients with FL and DLBCL after the separation with CD19 Pan B Dynabeads. (C) B and non-B cells were separated from peripheral blood mononuclear cells (PBMCs) from patients with B-CLL. IDO expression was analyzed after the addition of IFNγ for 1 day. Five samples of different patients were analyzed, and representative results are shown. (D) IDO mRNA and protein expression in Chinese hamster ovary (CHO) cells, and in murine B-cell lines A20 and M12 in which mouse IDO or control pcDNA3 vector was transfected.
Establishment of cell lines transfected with the murine IDO gene
Next, we cloned the murine IDO cDNA from the splenocytes of the BALB/c mice in the pcDNA3 vector containing the G418-resistant gene, and transfected it into CHO cells. Multiple clones were selected by G418 that had high cytoplasmic IDO protein as well as IDO mRNA expression confirmed by immunoblot and RT-PCR (Fig. 1D, CHO-IDO). We next introduced the IDO gene into the murine B-cell lines A20 and M12 to examine the effect of IDO on the development of lymphoid malignancies. Compared with the control vector without the insert, the IDO-containing vector produced an almost equal number of G418-resistant clones. Although comparable amounts of IDO mRNA to CHO-IDO could be detected in these cells by RT-PCR, IDO protein was not detected, which suggested post-transcriptional downregulation of IDO in these lymphoid cell lines (Fig. 1D). Although some proteins have been regulated by their stability post-translationally, the proteasome inhibitors could not induce the IDO expression in these transfectants (data not shown).
Conditioned medium with CHO-IDO is cytotoxic to lymphoid cell lines
This downmodulation allowed us to consider the possibility that IDO may be toxic to these lymphoid cell lines. To examine this possibility, we created the conditioned medium with CHO (CHO-CM) and CHO-IDO (IDO-CM), and cultured these murine lymphoid cell lines in this media. The viability of both A20 and M12 was markedly decreased in IDO-CM compared with those in CHO-CM (Fig. 2A).
Effect of conditioned medium with CHO-IDO (IDO-CM) on murine and human lymphoid cell lines. (A) Murine B-cell lines, human B-cell or T-cell lines, and (B) non-lymphoid cell lines were cultured in CHO-CM (white bar) or IDO-CM (black bar) for 2 days. The viabilities were determined by flow cytometry. Results are presented as means ± SD of triplicate cultures. The astersks indicate statistically significant differences determined by Student's t-test (*P<0.01, **P<0.001). (C) PBMCs from healthy donors were cultured with 30 U/ml IL-2. The viabilities of cells were analyzed after 2 days of culture in CHO-CM or IDO-CM added to 10 ng/ml of concavalin A (ConA) or human T-activated CD3/CD28 Dynabeads (*P<0.01, **P<0.001).
We then examined the viability of various cell lines cultured in CHO-CM or IDO-CM. All lymphoid cell lines examined, including both T-cell and B-cell lines, showed less viability in IDO-CM than in CHO-CM (Fig. 2A). In contrast, the viability of non-lymphoid cell lines in IDO-CM was not changed significantly in comparison with those in CHO-CM (Fig. 2B).
When T cells were cultured with only 30 U/ml IL-2, these cells showed similar viability in IDO-CM than in CHO-CM. However, when T cells were cultured with 10 ng/ml concanavalin A (ConA) or human T-activated CD3/CD28 Dynabeads, IDO-CM decreased their viability (Fig. 2C) indicating that signals from antigenic stimuli may induce susceptibility to IDO toxicity.
Collectively, the cytotoxic effect of IDO may be specific to activated lymphocytes and lymphoid lines.
Tryptophan metabolites inhibit the proliferation of lymphoid cell lines
Both the starvation of tryptophan and the production of tryptophan metabolites induced by IDO have been reported to be responsible for IDO immune suppression (2). Actually the concentration of trypatophan in IDO-CM was undetectable (data not shown). Therefore, we cultured IDO-sensitive lymphoid lines with added IDO-CM and an excess amount of tryptophan. The addition of tryptophan restored the viability in IDO-CM, though the restoration was not complete (Fig. 3A); this indicated that tryptophan metabolites as well as the starvation of tryptophan affected the lymphoid lines.
Effects of exogenous tryptophan or various tryptophan metabolites on viabilities of murine B cell line. (A) The viabilities of A20 were analyzed after culture with added IDO-CM and excess tryptophan (0, 0.1, 1 and 10 μM). The astersks indicate statistically significant differences (*P<0.01, **P<0.001). (B) The viabilities (right) and viable cell numbers (left) of A20 are shown after 2 days of culture with various tryptophan metabolites (L-Kynurenine, 3-HAA, Kynurenic acid, tranilast) at the indicated concentrations. The astersks indicate significant differences compared with untreated cells (*P<0.01, **P<0.001).
We then examined the effects of the various tryptophan metabolites on the viability of IDO-sensitive lymphoid line A20 (Fig. 3B). After treatment with L-Kynurenine or 3-HAA, the viable number of A20 cells was decreased although the viability had not changed. These results indicated that these molecules could suppress proliferation although they were not cytotoxic. Kynurenic acid could not change proliferation. Compared with these molecules, tranilast markedly decreased the viability of A20 cells. Tranilast not only induced cell death, but also attenuated the proliferation of A20 cells, as estimated by carboxyfluorescein succinimidyl ester (CFSE) dilution assay (data not shown). This effect appears to be independent of the cell cycle phase because the treated cells remained unchanged by the cell cycle analysis (data not shown). When tranilast was washed out from the medium after 1 day of treatment, A20 cells proliferated as much as the untreated cells, indicating that the effect of tranilast was reversible at this time-point. However, the A20 cells did not proliferate after 2 days of tranilast treatment (data not shown). We then examined the effect of tranilast in combination with chemotherapeutic reagents. Tranilast showed additive cytotoxicity with active metabolites of 4-hydroperoxycyclophosphamide and fludarabine (data not shown).
Tranilast induces cell death of lymphoid malignant cells
We examined the effect of tranilast treatment on various cell lines. Similar to IDO-CM, tranilast induced cell death in all lymphoid lines (Fig. 4A) but not in the myeloid or epithelial cell lines (Fig. 4B) examined; however, the effects of IDO-CM and tranilast were not totally equivalent. Tranilast induced more cell death of EW36 than IDO-CM, and conversely, IDO-CM was more toxic than tranilast for HS-Sultan. We also analyzed the effect of tranilast against the primary samples from six patients with CLL. Tranilast dose-dependently augmented the spontaneous apoptosis of CLL cells (Fig. 4C).
Effects of tranilast on the viability of cells. Lymphoid cell lines (A) or myeloid or epithelial cell lines (B) were cultured in a medium treated with or without 200 μM of tranilast for 48 h. Mean viability of the triplicate cultures is shown. The astersks indicate significant differences in compared with untreated cells (*P<0.01, **P<0.001). (C) PBMCs from 6 patients with CLL were treated with indicated concentrations of tranilast. The viabilities of cells were analyzed after 4 days of culture, and mean values from triplicates are shown. The fraction of CLL B cells determined with flow cytometry was >90% of every PBMC. The cell number (%) was expressed as a percentage relative to the untreated control cells. The astersks indicate significant differences in compared with untreated cells (*P<0.01, **P<0.001).
Tranilast induces the phosphorylation of JNK
To reveal the molecular mechanism of cytotoxicity induced by tranilast, we checked the activation of some kinases in A20, TMD2 (15), and Raji cells by immunoblot after tranilast treatment. The phosphorylation of 42 kD of extracellular signal-regulated kinase (Erk-2) was upregulated transiently after the addition of tranilast in these lines (Fig. 5A; and data not shown). In addition, JNK activation was induced in A20 cells 12 h after tranilast treatment (Fig. 5A). Although JNK activation was not obvious in the human B cell lines, TMD2 and Raji, after the addition of tranilast alone, tranilast clearly augmented the phosphorylation of JNK after B-cell receptor signaling using anti-IgM treatment (data not shown). Because the JNK activation could promote not only cell survival but also cell death depending on the cell type, we examined the effect of JNK inhibition on tranilast treatment. The addition of JNK inhibitor SP600125 augmented the cytotoxicity of tranilast only slightly (Fig. 5B). We also induced the dominant negative form of JNK2 into some A20 cells. Tranilast induced the cell death of these cells as compared to that of wild-type A20 cells, indicating that JNK was not required for the cytotoxicity of tranilast (Fig. 5C).
c-Jun N-terminal kinase (JNK) activation induced by tranilast. (A) A20 cells were treated with 200 μM of tranilast and harvested at the indicated time. The whole cell lysate was subjected to immunoblot using antibodies against the indicated anti-phosphorylated proteins (p-JNK, p-Akt, p-Erk and p-p38). The same membranes were reprobed with anti-total proteins (JNK, Akt, Erk and p38). The major band of p-JNK was the 46 kD form of JNK, and the major band of p-Erk was the 42 kD form of Erk (Erk-2) as determined by the comparison with bands of total JNK or Erk (arrows). (B) The human B cell lines, TMD2 and Raji, were treated with or without 200 μM of tranilast in addition to the indicated concentration of the JNK inhibitor, SP600125. After 2 days of culture, the viabilities were analyzed and shown as the means with SD of triplicates. (C) A20 was transfected with pQCXIN-dominant negative form of JNK2 and selected with G418. Enhanced 54 kD of JNK and diminished auto-phosphorylation of 46 kD of JNK were verified by immunoblot analysis (data not shown). The viabilities were analyzed after 2 days of culture with 200 μM of tranilast. The astersks indicate significant differences in compared with untreated cells (*P<0.01, **P<0.001).
The effect of tranilast is independent of aryl hydrocarbon receptor (AhR)
Kynurenines can act as endogenous AhR ligand (16,17). Furthermore, tranilast could act as a ligand for AhR (18). To examine if the cytotoxic effect of tranilast on lymphoid cells was mediated by AhR, the AhR antagonist, α-naphthoflavone (α-NF), was added to the culture with tranilast. The addition of α-NF did not change the effect of tranilast (Fig. 6A). In addition, the potent agonist of AhR, 2,3,7,8-tetrachlorodibenzodioxin (TCDD), was not as cytotoxic to A20 cells as tranilast (Fig. 6B). Collectively, the cytotoxic effect of tranilast appeared to be independent of AhR.
Cytotoxic effect of tranilast on lymphoid cells was independent of the aryl hydrocarbon receptor (AhR). (A) A20 cells were cultured with or without 200 μM tranilast in addition to α-naphthoflavone (α-NF), which is an AhR antagonist at the indicated concentration. The viability after 2 days of culture is indicated. The astersks indicate significant differences compared with untreated cells (**P<0.001). (B) The viability of A20 cells cultured in a medium treated with AhR agonist, 2,3,7,8-tertrachlorodibenzodioxin (TCDD), at the indicated concentration for 2 days are shown.
Tranilast attenuates the growth of lymphoid tumor in vivo
To examine the effect of tranilast in vivo, we inoculated A20 into BALB/c mice subcutaneously. After 7 days of inoculation, treatment with oral administration of 100 mg/kg of tranilast suspended in CMC solution or CMC solution alone was started. The treatment was applied twice daily for 3 weeks. The mice that underwent tranilast treatment had slower tumor development compared with the mice that were dosed with CMC solution alone. The tumor mass at 5 weeks after the challenge was significantly smaller in mice treated with tranilast compared with the control mice (P=0.02, Fig. 7).
In vivo efficacy of tranilast in a mouse lymphoma model. Six BALB/c mice were engrafted with A20 cells subcutaneously and treated with 100 mg/kg of tranilast suspended in 5% carboxymethyl cellulose (CMC) solution by oral administration. Mice dosed with CMC solution alone (n=7) were used as controls. Mean tumor volumes of mice are plotted. The difference at day 33 was significant (*P=0.02).
In the present study, we showed that conditioned medium with IDO-expressing cells and a tryptophan metabolite, tranilast, both decreased proliferation and induced cell death of activated lymphocytes and specifically lymphoid tumor cells. In agreement with our results, it has been reported that mesenchymal stem cells treated with IFNγ inhibited the proliferation of normal and follicular lymphoma B cells in an IDO-dependent manner (19). Because tryptophan is an essential amino acid, its starvation and the presence of metabolites could influence all tissues including the placenta (4) or dendritic cells (6), which contain a significant amount of IDO. The specificity of cytotoxicity induced by IDO against activated lymphoid cells may explain the machinery for the protection of IDO-expressing cells themselves. In agreement with this result, we could not get an increased exogenous expression of IDO protein in murine lymphoid cell lines; however, we detected IDO mRNA in these transfected cells. In the same way, it has been reported that IDO protein has weak or absent enzymatic activity in human B cells, although it can be induced by CD40L and IFNγ together or by a TLR agonist (20). We analyzed IDO expression in lymphoid tumor samples by RT-PCR (Fig. 1A). The expression in tumors was not increased compared with those in PBMCs from healthy donors. Conversely, the IDO expression in most tumor samples were weak and in some barely detectable, particularly in BL. Because the percentage of malignant B cells in BL were almost 100% (data not shown), the decrease of IDO in malignant tissues may represent the decrease of IDO-expressing non-B cells. In agreement of this, the IDO expression was mainly derived from non-malignant bystander cells from the samples examined. A report has described that the functional expression of IDO could be detected in a portion of diffuse large B-cell lymphoma (DLBCL) cases, and this expression was correlated with worse prognosis (21). Although we did not have any IDO-expressing DLBCL cases, these IDO-expressing lymphoma cells may have the same protective properties as non-lymphoid cells against IDO toxicity. It was also possible that this property could result in resistance to chemotherapeutic reagents and contribute to the poor prognosis and escape from immunological surveillance.
As tranilast was the most effective tryptophan metabolite for EAE as reported by Platten et al (10), it was also the most potent cytotoxic reagent for lymphoid tumor cells among the tryptophan metabolites examined. Tranilast has an inhibitory effect on various cancer cells including breast, prostate cancer and glioma (22). In our analysis, lymphoid cell lines were more susceptible to the cytotoxicity of tranilast than the non-lymphoid cells examined. As kynurenines can act as endogenous AhR agonists and suppress immune response, tranilast is also an AhR agonist. Its effect on breast cancer stem cells is AhR dependent (18). The effect of tranilast for lymphoid tumor was not inhibited by AhR antagonist α-NF indicating that the effect was independent of AhR in the present study. To date, the mechanisms of specific cytotoxicity to lymphoid tumor cells induced by tranilast have not been elucidated. Tranilast induced Erk-2 activation transiently and JNK activation after 1 day of treatment in A20 cells. In contrast, it was reported that tranilast suppressed LPS-induced Erk-2 activation in microglial cells (23) and IL-1β-induced JNK activation of mesangial cells (24). These opposite effects of tranilast on MAP kinase signaling may explain the specific suppression against lymphoid cells. JNK can mediate cell survival and cell death dependent on cell type (25,26). But the fact that tranilast could not be inhibited by the inhibitor or the dominant negative form of JNK indicated that JNK was not essential for the effect of tranilast. Because JNK was activated by cellular stress such as UV, tranilast may also induce similar stress in lymphoid malignant cells.
Tranilast has been widely used as oral anti-allergic reagent in Japan and South Korea, and its safety is well recognized. Because infectious complications have not been reported as the side-effect of tranilast, this reagent should not induce profound immunosuppression. Adjuvant use of tranilast that potentiates the effect of chemotherapy is warranted.
The authors would like to thank Ms. Kaori Okada, Ms. Sakie Endo, Ms. Ayumi Kiyokawa and Ms. Shihomi Endo for skillful technical assistance and Dr Toshie Suzuki and Dr Hideki Kudo for helpful discussions. The authors also thank Kissei Pharmaceutical Corporation for providing the tranilast, and Dr Roger Davis for providing the Jnk2a2 plasmid.
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March-2015
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1. (US/CA fares) Half of a round-trip fare. A one-way fare is never considered a half-round-trip fare. See also combinations. 2. (International fares) Half of a round-trip (Tag 2) fare or a one-way (Tag 1) fare doubled and halved. Half of a specified/published or constructed/unpublished round trip normal or special fare. Additionally, the one-way normal may be considered to be a half-round-trip normal fare. If a one-way special fare may be doubled to establish a round-trip special fare, the one-way special fare may be considered to be a half-round-trip special fare. A one-way Tag 1 fare doubled and halved may be considered to be a half-round trip-fare, but it is never considered to be a Tag 2 fare. A half-round-trip fare is never a one-way (Tag 3) fare that cannot be doubled. See also combinations.
The city that appears first in a market city pair. Also called origin city.
A city between the origin and destination of the through international fare component that has a higher fare.
1. O, Origin - Intermediate. The HIP application applies from the origin of the fare component to any ticketed point(s) within the fare component being assessed. 2. D, Intermediate - Destination. The HIP application applies from a ticketed point to the fare component destination of the fare component being assessed. 3. I, Intermediate - Intermediate. The HIP application applies between any two ticketed points within the fare component being assessed.


text:
La société impériale d'archéologie (appelée avant 1881 et après 1918 société moscovite d'archéologie) est une société fondée dans les années 1860 en Russie impériale à Moscou pour favoriser les travaux d'archéologie.

Histoire
Le premier mécène de cette société est le comte Stroganov (1792-1884), immensément riche, qui avec le comte Ouvarov (1825-1884) en est le fondateur en 1864. Elle s'occupe de numismatique, du recensement et de la restauration de sites antiques et publie le résultat de ses travaux. La société se réunit les premières années au N°1/7 de la rue Malaïa Dmitrovka. Elle reçoit son patronage impérial à partir de 1881. En 1868, l'empereur Alexandre II fait don à la société du petit palais Kirillov, hôtel particulier fin  début  sis au N°20 du quai Bersenevka (Bersenevskaïa), qu'elle se charge d'ailleurs de restaurer.

La restauration est une activité importante de la société, jusqu'en 1889, date à laquelle la commission archéologique, nouvellement créée, s'en charge. La société finance de 1869 à 1911 des expéditions et des campagnes archéologiques et découvre de nouveaux sites.

La société n'a jamais dépassé les cinq cents membres (423 en 1904, 362 en 1914). Elle commence à recevoir des subsides à partir de 1872, pour financer ses fouilles, mais ce n'est qu'en 1914, année du jubilé de son cinquantenaire, que l'État lui octroie des subsides de dix mille roubles.

La société supprime l'adjectif impérial en 1918 et cesse d'exister en 1923.

Présidents
 Comte Alexeï Ouvarov, 1864-1885
 Comtesse Prascovie Ouvarov, 1885-1917 (veuve du précédent)
 Dimitri Anoutchine en est le directeur adjoint. Il est aussi président à partir de 1890 de la société des amateurs des sciences naturelles, d'anthropologie et d'ethnographie.

Membres
Parmi les membres, on peut distinguer, outre les précédents : Alexandre Artemiev, l'archimandrite Antonin Kapoustine, Vassili Klioutchevski, Nikolaï Kostomarov, Ivan Machkov, Sergueï Soloviov, Apollinary Vasnetsov, Ivan Zabéline.

Littérature
 Compte rendu de la Commission impériale archéologique, document numérique de la Bibliothèque de l'Université de Heidelberg

Source

Article connexe
 Société impériale russe d'archéologie

Centre ou institution archéologique
Archéologie en Russie

redpajama_set_name: RedPajamaWikipedia


text:
COVID-19 Remembrance: A Wife Carries On Her Late Husband's Legacy
Published March 8, 2021 at 10:59 AM CST
Joshua Terry, Joelle Wright Terry , Marshall Terry and Micah Terry. (Courtesy of Joelle Wright Terry)
The U.S. vaccine rollout appears to be helping slow COVID-19 deaths, which have dropped about 10% in the past two weeks. But waves of grief continue for millions of Americans who have lost someone they loved.
We remember the life of a man who died of COVID-19 earlier in the pandemic: Marshall Terry III of Clinton Township, Michigan. His wife, Joelle Wright Terry, a COVID-19 survivor herself, tells us about his love for music and God and how she is carrying on her husband's legacy.

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About to Get Inexpensive Freight Prices From a new Dependable Global Airlift Provider?
You may have heard often the phrase that world has started to become a scaled-down position. Clearly it isn't, the main world stays on the exact measurements, yet the techniques of transport in addition to delivery that many of us have available today causes it to become very uncomplicated to have possessions from side on the globe to the other along with bare minimum publicity.
Sanctioned assistance that individuals might effectively consider for provided. All of us create a reserving for some courier organization to build up our opportunities (either web based, about the cellphone or simply by way of a stop by at their depot). The courier then finds our doorsteps and often the package deal is definitely removed. The next idea may well is that that is actually brought, conceivably tens of hundreds of kilometer after kilometer away.
There is usually a fine chance that a parcel has undertaken a few journeys to start its vacation destination, using a number of methods about vehicles.
Pertaining to example, should you live in any agricultural section of the ENGLAND, you package can offer first been recently transported in the regional center and maybe perhaps stored there overnight. These kinds of day it could have also been whisked off of to your flight terminal together with rich in a new freight planes. According to size of the main package this could proceed by beach but opportunities can go suggests surroundings while its quicker and generally much less expensive.
After the program reaches it has the destination place there is certainly yet some sort of lot associated with floor in order to make right up. If the exact getaway is usually the exact place as the airport in that case it could only just stop up gonna a sending depot just where it escapes for shipment the then day. However if the actual location can be further also it may include a long road or even rail vacation ahead regarding the idea, or perhaps maybe possibly even one other vacation just by oxygen.
You may be competent to realize that there will be quite a few pretty special considering that should require place to make certain the parcel does possibly not overlook it's actual after that truck, particularly consignment can be with a time period sensitive shipping and delivery (that can it be has so that you can be at its destination by way of certain time). So, the earth might seem including it is becoming a scaled-down destination, nonetheless this is only appreciate it to agencies and individuals that work tirelessly to generate it occur!


Chocolate Covered Dried Fruit and Nuts. Chocolate covered in milk, white and dark chocolate coating.
NOTE: SHIPPING RESTRICTIONS APPLY: DURING WARM WEATHER CHOCOLATE GIFT PACKS CAN BE SHIPPED MON/ TUES/ WED, OVERNIGHT OR 2-DAY AIR ONLY on chocolate products starting May 1st through October 15th. Occasionally, weather can be unpredictable. If the weather in delivery area is above 80 degrees during any month of the year we suggest the package be expedited.


Section 721.1. What does this part cover?
12 CFR § 721.1 - What does this part cover?
§ 721.1 What does this part cover?
This part authorizes a federal credit union (you) to engage in activities incidental to your business as set out in this part. This part also describes how interested parties may request a legal opinion on whether an activity is within a federal credit union's incidental powers or apply to add new activities or categories to the regulation. An activity approved in a legal opinion to an interested party or as a result of an application by an interested party to add new activities or categories is recognized as an incidental powers activity for all federal credit unions. This part does not apply to the activities of corporate credit unions.


text:
Фантасмаго́рия (от  — призрак и  — публично выступаю):

Происходит от , образованного из др.-греч. φάντασμα «явление, призрак» и λέω «говорю». С XVIII века. Изначально — название аттракциона (волшебный фонарь).

Фантасмагория — искусство изображать призраки,  видения или воздушные картины посредством зеркальных отражений.
 Фантасмагория (искусство) — нагромождение причудливых образов, видений, фантазий; хаос, сумбур, гротеск.
 Фантасмагория (представление) — жанр театрального представления в Европе в XVIII—XIX веках, в котором при помощи «волшебного фонаря» на заднем плане демонстрировались пугающие образы: скелеты, демоны, привидения.
 Фантасмагория (кино) — поджанр кинофантастики, представляющий фильмы о чём-то совершенно нереальном, изображающий причудливые видения, бредовые фантазии.
 Фантасмагория (мультфильм) — немой короткометражный мультфильм, Франция, 1908 год. Режиссёр —  Эмиль Коль.
 Фантасмагория (система целеуказания) — российская авиационная станция целеуказания для противорадарных ракет Х-58 и Х-25 МПУ.

Phantasmagoria
 Phantasmagoria (игра) — компьютерная игра, разработана Sierra On-Line, 1995 год.
 Phantasmagoria: A Puzzle of Flesh — компьютерная игра, сиквел Phantasmagoria.
 Phantasmagoria (Touhou Project) — объединенные этим названием игры в серии, обладающие нестандартным геймплеем: скролл-шутеры на двух игроков (Phantasmagoria of Dim. Dream, Phantasmagoria of Flower View).
 Phantasmagoria (группа) — японская группа, играющая в стиле visual kei.
 Phantasmagoria (альбом Curved Air) — 3-й студийный альбом британской прог-рок-группы Curved Air, 1972 год.
 Phantasmagoria (альбом Limbonic Art) — 7-й студийный альбом норвежской симфо-блэк-метал-команды Limbonic Art.
 Phantasmagoria — альбом рок-группы The Damned.
 Phantasmagoria (песня) — песня канадской металл-команды Annihilator из альбома Never, Neverland.
 Fantasmagoria (песня) — песня пауэр-метал-команды Emerald Sun из альбома Regeneration.
 Phantasmagoria in Two (песня) — песня американского исполнителя Тима Бакли из альбома Goodbye and Hello 1967 года.


Deliverables should be a knowledge worker's best friend but, like all good friendships, the relationship can get complicated. One of the most direct ways to improve the quality of knowledge work is to spend more time and effort to define appropriate deliverables.
A consulting report prepared by the consultant or consulting team containing recommendations to the client sponsor.
A list of recommended job offers to prospective employees prepared by a campus recruiting team and handed to the hiring manager.
Production Release 1.0 of a new application system installed and operating on the companies intranet.
A project work plan and business case delivered to a steering committee for approval.
In the world of industrial work, process outputs are well-defined. This gives organizations a straightforward process improvement path. Holding the outputs constant allows process improvement to proceed by identifying and eliminating activities that don't add value to the output; to break down, reorder, and redesign process steps to create more standard outputs with less input; and so on.
This path isn't available to knowledge work practices. The goal is not to produce well-defined, standardized outputs. Deliverables are the closest analogue but their value depends on how well they match the unique needs of their users. No one is interested in a spreadsheet full of someone else's data; no teacher is likely to value a copy of a paper you've submitted to another class. Understanding what aspects and features of a knowledge work deliverable are most valuable to its intended user is key to focusing efforts on producing the desired deliverable.
Some knowledge work processes produce obvious and natural deliverables. A campus recruiting effort generates a list of candidates to extend offers to. There may be a variety of associated supporting documents (e.g., interview notes, assessments), but the list is the deliverable and is a natural outgrowth of the process.
Other knowledge work processes generate more amorphous or less obvious outputs. For example, what's the deliverable for a typical project status meeting? Shared understanding among the project team? Agreement about the state of the project between the team and the project sponsor?
Consider the typical status report as a more artificial than natural deliverable and ask how much value resides in this particular item. If you can define the deliverable more effectively, is the world a better place?
The better we can define deliverables, the better and more effective we can make our knowledge work. Rather than ignore the end product, we need to be systematic in extracting as much as we can about the expected deliverable that can guide our effort to create it. There are three productive paths to explore in using the deliverable to drive your knowledge work.
Does this deliverable need to be exactly correct whatever the time or cost, as good as possible given a particular deadline/budget, or the best you can come up with on the phone? Each deliverable demands a different approach; each requires a conversation (and perhaps a negotiation) with the end user. The history of systems development is littered with examples of failing to follow this path.
We've been conditioned by one hundred years of industrial experience to value uniformity. In knowledge work, the value of uniformity is to free time and attention for the essential uniqueness we've defined. The folks in marketing may believe that corporate identify standards are about branding. For a knowledge worker, they let you ignore formatting decisions to focus on the content that matters.
There was an joke that was old even in my early consulting days that you knew the design phase was complete when the budget ran out. Possibly the biggest challenge for managing knowledge work is determining how to recognize that the deliverable is done. Think of the unfinished doctoral dissertations and manuscripts of the great American novel gathering dust on a shelf or lost in a lonely directory of a disk drive. Too many knowledge-work efforts fail simply because no one thinks about how to recognize what "done" will look like.
Focusing on deliverables means working backwards with the end in mind. Knowledge work is valuable to the extent that it produces end results, i.e. deliverables, that meet the unique requirements of a particular customer or end user. Time invested in understanding what that deliverable should look like will yield the greatest return in defining and focusing the activities that contribute to creating that particular deliverable at the right time and place.
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Running a business is a winding road full of ups and downs, highs and lows, and usually a roadblock or two (or twelve).
If you find yourself stuck in a particular area, spinning your wheels and you're just not sure how to move forward, I'm here to break down the barriers and start making things happen for you in your business.
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Anyone can start and run a business, but it's not easy. The only thing standing between you and your dream is clearly-defined hard work.
It was such a relief to have found a mentor who honestly wants to help and see you succeed and is willing to encourage and give resources to do it!
I was super overwhelmed by the process of backing up and storing the thousands of files I have on my computer from being a portrait photographer but Adam helped me get everything organized and created a system that made me feel secure and safe!
Looking to level up your game in a specific area of your business or photography? Sign up for more information and pricing on Mentor Sessions today!


Opengear (www.opengear.com), a leading provider of critical infrastructure management solutions offering advanced console servers, remote management, monitoring, and cellular out-of-band products, today announced that Secure-24 has deployed Opengear CM4100s and IM4200s to remotely manage critical infrastructure.
Secure-24 is a SAP-certified Hosting and Cloud Provider with a broad SAP Basis Competency Center. Secure-24 is also an Oracle Gold Partner for hosting Oracle Enterprise Performance Management (EPM) solutions, such as Oracle E-Business Suite, PeopleSoft, JD Edwards, and Hyperion. Using Opengear, Secure-24 manages world-class data centers in Michigan and Arizona.
Opengear provides Secure-24 a direct serial management connection to all of its geographically distributed data center servers and equipment. Opengear provides "remote hands," enabling visibility and control using cost-effective out-of-band access.


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Plant a Tree for Janet
Janet S. (McCauley) Hollcraft
April 30, 1943 ~ December 6, 2020 (age 77)
Janet Sue (McCauley) Hollcraft was born, April 30, 1943, a Midwestern farmer's daughter in the small town of Cassville, Indiana; the youngest child of four to Russell and Eva (Merrell). She spent summers playing in a nearby creek, family pond and eating homegrown watermelon chilled in the cattle watering trough, and also learn the value of hard work around the farm raising cattle, hogs, chickens and harvesting corn in the fields owned by the family. And yet, the big city life was drawing her to a future as a wife, mother and friend.
After high school, she moved to Indianapolis and went to work at a local machine shop. Every week, a self-described "dirty old rag man" would visit the machine shop to deliver and pick up soiled uniforms and shop rags. And each week, this dirty old rag man would thrust a clipboard at her and ask for her name and phone number with a wink and a smile. Week after week, Jan smiled back and applied her signature to indicate delivery as needed. Finally, after many repeated requests, she gave her phone number to the guy and began the adventure of her life.
Janet and John Kenneth Hollcraft (Kenny) married in 1963. Over the years, Janet became a successful small business owner, real estate agent and school bus driver for Perry Township for 23 years. She and Kenny brought into the world, loved and raised seven children and were blessed with 15 grandchildren and two great-grandchildren.
She will be remembered as the first person to lend a hand to a loved one or friend in need. She was the type of Grandma where the grandkids were always welcome at her home at any time. She hosted every holiday and family gathering and loved her children and their kids to gather around her. She was a big fan of Elvis Presley and never passed up a rooster decoration for her home.
Janet was preceded in death by her parents, three siblings Russell McCauley, Ida Pearl Pfifer, Emma Jean Richie and love of her life Kenny, who crossed over to heaven in 1994. Janet, 77, joined her beloved Kenny in heaven on December 6, 2020. She is survived and eternally loved by her seven children and their spouses Anthony, Christopher (Heather Redding), John and Jody, Phil and Eileen, Andrew (Robin Broderick), Anna (Jim) Harris, and Matthew (Kate Schiferl) of Saratoga, CA as well as 15 grandchildren and two great-grandchildren.
"Love me tender, love me sweet. Never let me go. You have made my life complete. And I love you so." Elvis Presley
Visitation will be at N.F. Chance Funeral Home, 504 National Ave, Indianapolis, on December 9, from 4:00 PM to 8:00 PM. Funeral Mass will be at St. Roch Catholic Church on December 10, 2020 at 10:00 AM, followed by a funeral procession for burial at Calvary Cemetery immediately following. Memorial contributions can be made to St Jude Hospital at Stjude.org or (800)-478-5833.
To send flowers to the family or plant a tree in memory of Janet S. (McCauley) Hollcraft, please visit our floral store.
You can still show your support by sending flowers directly to the family, or by planting a memorial tree in the memory of Janet S. (McCauley) Hollcraft


The financial landscape for nonprofit organizations across the country is changing. Traditional sourcing of funds are flat or declining, while the need for the community-based services and economic opportunity tools nonprofits offer is increasing. Nonprofits are facing many challenges including: meeting payroll, funding goals and covering fixed expenses. Without an evolution in the way nonprofit organizations acquire and use financial resources, fewer people will benefit from their services and fewer nonprofits will survive.
Recognizing this, and inspired by existing social enterprise models within our network, NeighborWorks America has acted to equip the nearly 250 local nonprofit organizations in the NeighborWorks network with training, technology and technical assistance to create and sustain mission-oriented revenue streams.
NeighborWorks America partnered with the Stanford Social Innovation Review on a special supplement issue, "NeighborWorks America Leading Innovation," to introduce this comprehensive framework of business and culture change. The first part of this effort focused on a path to business sustainability tailored for nonprofit organizations engaged in providing homeownership services in their communities. This pilot, called the Sustainable Homeownership Program, has seen tremendous results for the NeighborWorks network.
Results show that participating nonprofits reduced the costs of serving customers, increased the number of customers served and increased the percentage of costs covered by earned revenue and leveraged new technologies to grow more efficiently.
At the center of this effort is a commitment to a social enterprise paradigm. The goal is not to transform NeighborWorks nonprofit organizations into commercial enterprises solely focused on bottom line results. Instead, our goal is to create a mission-driven model for robust community development that incorporates enhanced appetite for risk and clearer understanding of how the work that is done and the people that are doing it contribute to more resilient communities and organizations.
The techniques and strategies presented in the supplement can be applied across the NeighborWorks network and be among the tools organizations will use to identify how their lines of business and service can be transformed into revenue generating sources of mission-based capital. These approaches can also help the organizations analyze new areas of service to enter and how that entry will help build stronger communities and stronger organizations.
The Heat Squad is part of the sustainable business strategy of NeighborWorks Western Vermont.
Our blog offers examples of how community development organizations can benefit from becoming sustainable businesses.
Forbes highlights how one organization rebranded to increase awareness, stand out from the competition and increase the services they offer.
Our acting vice president of national homeownership programs explains how social enterprise is going to rebuild homeownership.


text:
Q: swift more efficient way to search array for match of different types I am using Swift 3 to find a GKGraphNode in an array that has a x,y position that matches the target x,y position.  Below is the init for the nodes.  You can see each node has a vector_int2 coordinate called gridPosition.  I create an array of these nodes to match my map's hexagonal grid.
class HexGraphNode : GKGraphNode {
var gridPosition: vector_int2!

required init(gridPosition: vector_int2) {
    super.init()

    self.gridPosition = gridPosition

}

required init?(coder aDecoder: NSCoder) {
    fatalError("init(coder:) has not been implemented")
}

}
I need to be able to search this array to find the node that occupies some specific grid coordinate.  I am using the function below.  This function works fine when the map is small like under 500 nodes.  However, as the array grows, the program slows down to the point of being unacceptable.  I need a more efficient way to search the array and find the matching node without starting from index zero each time.  I have looked for other postings, but they always seem to compare objects of the same type.  In this case I need to compare a property of the object (node.position) to a different type (vector_int2 which contains an x,y integer).
func node(atGridPosition position: vector_int2) -> NodeType {

    var result: NodeType!

    for node in self.nodes as! [NodeType] {

        if node.gridPosition.x == position.x && node.gridPosition.y == position.y {
            result = node

            break
        }
    }

    return result
}

Any help would be greatly appreciated.

A: If the grid positions of your nodes don't change dynamically, you could keep a dictionary of nodes rather than (or in addition to) you array.  If you have a fixed (or maximum) width or height for your grid, the x,y coordinates can be converted to a single integer value to use as a key in the dictionary.  Otherwise you could use a two level dictionary.
Accessing the nodes then would be an O(1) operation rather than O(n/2).

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Our guest rooms are the perfect farm stay for guests wanting a night in a country town. There is nothing like a breakfast of fresh scones and jam while watching the sunrise over the green hills.
See where we pasteurise and bottle our Living Milk and make our renowned Bodalla Bush Tucker Cheddar cheeses.
See where we churn our Living Milk into wholesome ice cream, so fresh it's only available at our cafe. Tastings straight from the churner at 11am Fridays.
Just 40 mins drive from Batemans Bay, you can spend all day relaxing in Bodalla and our surrounding area.


text:
Andrews has no choice – he must resign says Walsh
Andrews has no choice –…
Victorian Premier Daniel Andrews has finally, irrevocably, irretrievably, confirmed what most of us have known for a long time – his government is a farce.
The Nationals leader Peter Walsh said Daniel Andrews, the man who demanded – and got – emergency powers allowing him to govern any way he likes, without being answerable to anyone, including Parliament, this afternoon faced the Hotel Quarantine Inquiry and 27 times told the hearing he "could not recall".
"The Premier has delivered an absolute insult to all those who are now grieving loved ones as a direct result of this Labor Government's failures.
"Daniel Andrews' disastrous mismanagement of hotel quarantine killed more than 700 Victorians and has ripped the soul out of small businesses and communities.
"Daniel Andrews today proved he is unworthy of office. Andrews and his COVID Eight must resign."
Mr Walsh said the Andrews Labor government spent the better part of 100 million taxpayer dollars botching its hotel quarantine program that caused our crippling second wave of COVID-19 – yet not one Minister, department official or the Premier himself can say who was responsible for managing it.
He said while other states have successfully got the virus under control, Victoria remains the nation's pariah state because of the bad decisions made by the Andrews Labor Government.
"This farcical, botched handling of its shambolic hotel quarantine program is just one of many reasons Victorians have lost confidence in Daniel Andrews and his Labor Government," Mr Walsh added.
"Victorians deserved the truth today, but instead we got more lies and more cover-ups from arrogant Andrews," he said.
"The amnesia virus, which has swept through the ranks of Victoria's most senior elected officials, leaves us with just one conclusion – the millions and millions of your dollars, spent to drive our state into crisis mode and an unparalleled lockdown with curfews, protests and massive fines; and had police trampling on free speech – was not authorised by anyone in the Andrews Labor government.
"So, who had the power; and approval, to spend such a vast sum of money to create such a shambles it has left Victoria a national pariah, locked out for months from the rest of the country?" he said.
"For Mr Andrews to stand there on television, watched by all Victoria – and probably much of Australia – and blithely deflect the direct question from Counsel assisting the inquiry, Rachel Ellyard, if he had made the decision, or did he know who did, to staff the most crucial part of the state's COVID-19 defence was an insult to all those who have died, and their families, as a result of that fateful and flawed strategy."
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Музе́й «Ку́рский солове́й»  — музей, посвященный символу Курского края — курскому соловью, расположенный в Курске.

Музей был открыт 4 июня 2007, в Соловьиный день по русскому народному календарю, при Курском Областном Центре детского и юношеского туризма, краеведения и экскурсий..

В настоящее время собрание музея содержит более 1000 предметов и произведений, в том числе более 200 единиц подлинников различных документов, и более 800 ксерокопий стихов, песен, сказок, рассказов про соловья, современных цветных фотографий по тематике музея, тематических подборок материалов.

Экспозиция
 фотографии курского соловья, сделанные в 2007 году курским фотохудожником Виктором Викторовым и в 1960-е годы 15-летним Гришей Кокоровцом (ныне — Григорий Александрович Кокоровец, фотограф Курского госуниверситета), которая удостоена диплома Международной выставки детского и юношеского творчества в Монреале;
 материалы о Соловье-разбойнике, так как он издревле считается обитателем курского края, как это показывает в своих трудах курский археолог Ю. А. Липкинг;
 буквенные записи трелей курского соловья курского писателя Владимира Конорева (у него их получилось около 900 разновидностей); видеозапись встречи с ним сделана 01.12.2007;
 рассказы «Мой соловей», «Тётёнь» курского писателя Владимира Конорева, книги-подарки других курских поэтов и писателей, воспевающих соловья;
 старинные рушники, на которых соловей запечатлён в вышивке и кружеве;
 исторические данные о происхождении фразеологизма «Курский соловей»;
 диски и пластинки с пением соловья, пластинка «Танго соловья»;
 материалы о людях, кого на их родине называют курскими соловьями (о Надежде Плевицкой, Иване Суржикове, Ирине Стародубцевой, сёстрах Толмачёвых, Владимире Неведрове);
 материалы о курском фирменном поезде «Соловей»;
 глиняные «фирменные» соловьи курских мастеров-глинщиков;
 материалы о соловьиной переписи в Москве, Курске;
 фирменные футболки, значки, конфеты «Соловушек перезвон», мороженое «Соловушка», бальзам «Курский соловей» (больше не выпускается…), даже выращенные Л. Н. Ерёминой и замаринованные в баночках огурцы «Соловей»;
 о фестивале бардовской песни в Курске «Соловьиная трель», марафоне «Курский соловей», зимних автогонках «Курский соловей», празднике дружбы и партнёрства «Курский соловей», детском исполнительском конкурсе «Курский соловей»;
 настоящее гнездо соловья и другие материалы.

Примечания

Музеи Курска


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Posted on May 27, 2018 July 4, 2018 by 3Tblkadm
CryptoKitties raises money for Seattle Children's Hospital from Blockchain Bidding
Block.News May 27, 2018
A blockchain-based charity auction has raised more than $15,000 for a children's hospital located in Seattle, Washington, CryptoKitties said in its Medium post.
Kitties for a Cause a charitable initiative launched by Bella's Kitty Den (BKD), earned 21.6 ether tokens, or more than $15,000 at that time, in less than three weeks after the auction started on April 6.
At present, 21.6 ether tokens would amount to nearly $13,000.
Proceeds will go to Seattle Children's Hospital, a children's hospital in the Laurelhurst neighborhood of Seattle that has been ranked as one of best hospitals in the United States.
Aside from the ethereum raised, another $180 were donated to the hospital directly through the campaign that was supposed to run for a month.
Users were able to bid on 370 different CryptoKitties donated to Bella's Kitty Den, according to Axiom Zen spokesperson Yasmine Nadery.
Axiom Zen is the parent firm of CryptoKitties which specializes in new venture creation, building products and companies at the cutting edge of technology. It helped draw attention to the campaign until the bidding ended on April 15.
Nadery said Bella, a 10-year-old fan of the blockchain-based platform, and her father started Bella's Kitty Den as a business learning experience for the fourth grader and an opportunity to earn some money for her studies.
The initiative originally started as a way "to do good" with the kitties owned by Bella.
"One day while playing, I said we should give away some of our Kitties since we have so many of them. Dad asked who we should give them to. I told him kids in the hospital who are hurting. So with help from my Dad and others from the CryptoKitties community, we have created the Kitties For A Cause (K4C) fund," Bella said on CryptoKitties' Medium post.
"This event was created by a CryptoKitties community member, and we were in awe of the initiative this young girl took. When she and her father told us about it, we jumped at the opportunity to partner with them partner with them and our community creators to spread the word about Bella's cause," she added.
Nadery said CryptoKitties hopes these donations would inspire charitable undertakings in the future.
"Seeing something like this be inspired by our game is all the more reason for us to keep moving forward with our vision of bringing a billion people to the blockchain," she said.
Launched in 2017, CryptoKitties is a blockchain-based virtual game developed by Axiom Zen which allows users to purchase, collect, breed, and sell different types of virtual cats.