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Melatonin Protects CD4+ T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation
Alziana Moreno da Cunha Pedrosa, Ricardo Weinlich, Giuliana Patricia Mognol, Bruno Kaufmann Robbs, João Paulo de Biaso Viola, Ana Campa and Gustavo Pessini Amarante-Mendes
J Immunol April 1, 2010, 184 (7) 3487-3494; DOI: https://doi.org/10.4049/jimmunol.0902961
Alziana Moreno da Cunha Pedrosa
Departamento de Análises Clínica e Toxicológica, Faculdade de Ciências Farmacêuticas, and
Ricardo Weinlich
Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo; Instituto de Investigação em Imunologia, Instituto Nacional de Ciência e Tecnologia, São Paulo; and
Giuliana Patricia Mognol
Divisão de Biologia Celular, Instituto Nacional de Câncer, Rio de Janeiro, Brazil
Bruno Kaufmann Robbs
João Paulo de Biaso Viola
Ana Campa
Gustavo Pessini Amarante-Mendes
Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4+ T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.
The highly evolutionarily conserved hormone melatonin (N-acetyl-5-methoxytryptamine) was first isolated from the bovine pineal gland (1) and subsequently described in algae, protozoa, plants, and a variety of mammals (2). In mammals, melatonin is recognized as a major regulator of seasonal and circadian rhythms by means of its daily nocturnal increase in plasma levels (3). In addition, melatonin presents antioxidant, anti-inflammatory, and immunomodulatory activities (4–6).
Over the past two decades, a substantial body of evidence has demonstrated that melatonin can be synthesized by a number of nonendocrine extrapineal sites, including gut, retina, skin, and the bone marrow (7–9). More recently, it has been shown that stimulated human lymphocytes have the necessary machinery to produce and release large amounts of melatonin (10). Along with the fact that melatonin receptors are present in neutrophils (11), monocytes (12), and lymphocytes (13), it was proposed that melatonin exerts intracrine, autocrine, and paracrine immunomodulatory activities (10).
Indeed, several studies have reported the effect of exogenous melatonin on lymphoproliferation (14), activation (15), differentiation to effector Th1 cells (16), migration (17), and cytokine production by human lymphocytes (18, 19). In particular, endogenous production of melatonin by human lymphocytes has been related to enhanced release of IL-2 and upregulation of IL-2R (20), suggesting that melatonin may be involved in the clonal expansion of Ag-stimulated human T lymphocytes. Importantly, the effects of melatonin on activated T cell death remain largely unknown.
Activation-induced cell death (AICD), an apoptotic process that occurs after restimulation of T cells via their Ag receptor complex (TCR/CD3), was initially described in T cell hybridomas (21) and later on demonstrated in all major T cell subsets, including Th1, Th2, and Th17 (22, 23). AICD is dependent on the upregulation of Fas ligand (CD95L), which binds to its cognate receptor Fas (CD95), triggering the activation of a caspase-dependent apoptotic pathway (24).
AICD is involved in the maintenance of the T cell homeostasis, at least, in three different ways. First, AICD participates in the deletion of self-reactive T cell clones, avoiding the accumulation of potentially dangerous cells (25). Second, it contributes to the elimination of chronically stimulated cells (26), which can produce harmful levels of cytokines. Finally, AICD is partially responsible for the elimination of an expanded and no longer necessary T cell population after chronic infection (27). In fact, mice or humans who present defects in the CD95/CD95L pathway show accumulation of activated T cells with lymphadenopathy and splenomegaly and generally develop severe autoimmunity (28, 29).
In this study, we found that melatonin protects human and murine T cells from AICD by inhibiting anti-CD3–mediated CD95L upregulation. Melatonin prevented NFAT dephosphorylation induced by TCR/CD3 stimulation, thereby interfering with NFAT translocation to the nucleus. Melatonin also blocked the transactivation of the human CD95L reporter promoter, even when cells were cotransfected with an NFAT construct. Importantly, melatonin failed to inhibit the transactivation of either NFAT-responsive promoter or the human CD95L promoter in cells transfected with a constitutively active form of NFAT. Taken together, our data demonstrated for the first time a role for melatonin in T cell survival by preventing TCR/CD3-mediated NFAT activation of CD95L transcription and subsequent AICD.
Mice, cell lines, and reagents
Spleen and lymph nodes of 6- to 8-wk-old BALB/c mice were used as a T lymphocyte source. Human PBMCs were obtained from healthy donors after written consent. Jurkat cells and DO11.10 T lymphocyte hybridoma were a gift of Dr. Douglas Green (Saint Jude Research Children's Hospital, Memphis, TN). Primary cells were grown at 37°C in 5% CO2 in DMEM supplemented with 10% FCS, 10 mM HEPES, 2 mM l-glutamine, 1 mM sodium piruvate, 100 mM nonessential amino acids, 100 mM vitamins, 10 mM 2-ME, 100 mg/ml streptomycin, and 100 U/ml penicillin. Cell lines were regularly maintained at 37°C in 5% CO2 in RPMI 1640 supplemented with 10% FCS, 10 mM HEPES, 2 mM l-glutamine, 100 μg/ml of streptomycin, and 100 U/ml of penicillin.
Melatonin, Percoll, and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO). Cyclosporine A (CsA) was obtained from LC Laboratories (Woburn, MA). PMA was obtained from Calbiochem (San Diego, CA), and Ficoll-Paque Plus was from GE Healthcare (Chalfont St. Giles, U.K.). RPMI 1640, DMEM, l-glutamine, penicillin, streptomycin, FCS, and PHA, nonessential amino acids, vitamins, and 2-ME were purchased from Life Technologies (Rockville, MD). Ionomycin was obtained from Calbiochem, and anti-CD3 (clone 2C11), anti-CD95L (clone MFL3), and anti-CD14 Abs were purchased from BD Pharmingen (San Diego, CA). Anti–NF-κB and anti–phospho-NF-κB Abs were bought from Cell Signaling Technology (Beverly, MA). Anti-NFAT1 Abs (clone 67.1) were kindly provided by Dr. Anjana Rao (Harvard Medical School, Boston, MA).
Generation of murine T cell blasts
Spleens or lymph nodes were removed aseptically from BALB/c mice and teased into single-cell suspension. RBCs were lysed with ammonium chloride solution, and adherent cells were removed by 2-h incubation in tissue-culture plates. For generation of T cell blasts, 1 × 106 cells were stimulated for 48 h with 1 mg/ml plate-bound anti-CD3 and 1 μg/ml soluble anti-CD28 Abs in flat-bottomed six-well plates in a final volume of 1 ml. Cells were washed and cultured for 4 more d with 100 U/ml recombinant human IL-2 (proleukin, Zodiac Produtos Farmacêuticos, São Paulo, Brazil). Later, dead cells and debris were eliminated by centrifugation over Ficoll-Paque Plus.
Generation of T cell blasts from human PBMCs
PBMCs were purified from healthy donors by Ficoll-Paque density centrifugation. Lymphocytes were isolated from PBMCs by an additional Percoll gradient. In all experiments, no less than 75% of the recovered cells were CD3-positive, and no more than 2% were CD14-positive, as assessed by flow cytometry (data not shown). T cell blasts were generated by stimulating 2 × 106 cells/ml with 1 μg/ml PHA for 16 h, followed by additional 6-d culture in the presence of 30 U/ml recombinant human IL-2, added every 2 d of culture. Percentage of CD3+ T cells was at least 98% in all experiments (data not shown).
Apoptosis induction
DO11.10 hybridoma, murine, and human T cell blasts were induced to apoptosis by different stimuli, including 1 μg/ml plate-bound anti-CD3 Abs, 10 ng/ml PMA plus 1 μM ionomycin, 1 μg/ml soluble anti-Fas Abs, 10 μM Teniposide (VM-26), and 1 μM actinomycin D. In some cases, during these death-inducing stimuli, cells were treated with different concentrations of melatonin, lactacystin, CsA, or N-acetylcysteine.
Assessment of apoptosis and cell death
Apoptosis was estimated by multiple parameters (30). For cell cycle analysis of total DNA content, cells were collected, lysed, and nuclei stained with hypotonic fluorescent solution (50 μg/ml PI, 0.1% Triton X-100, and 0.1% sodium citrate). Samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA), and numbers represent the mean percentage of cells with DNA fragmentation ± SD in triplicate samples. Cell death was also quantified by PI exclusion. Cells were stained with 10 μg/ml PI and analyzed by flow cytometry. Numbers represent the mean percentage of PI-positive cells ± SD in triplicate samples. Some samples were also analyzed morphologically or based in changes of light scattering properties of the dead cells (31).
RT-PCR and quantitative PCR
Total mRNA were extracted by TRIzol (Invitrogen, Carlsbad, CA) from DO11.10 cells and converted to cDNA using Superscript III (Invitrogen) as described by the manufacturer's protocols.
Quantitative PCR was performed using TAQMAN technology (Applied Biosystems, Foster City, CA). CD95L mRNA expression was analyzed by the Gene Expression Assay catalog number Mm00438864_m1, CD95 by Mm01204974_m1, and cellular FLICE-like inhibitory protein (c-FLIP) by Mm01255578_m1, and results were normalized by GAPDH expression, assayed by the Endogenous Controls Assay catalog number 4352932E. The expression values are relative to the amplification of unstimulated cells.
Quantification of CD95L protein expression
CD95L protein expression was detected by flow cytometry as previously described (32) using anti-CD95L.PE Abs (clone MFL3; BD Pharmingen). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
SDS-PAGE and Western blot
Protein samples were resolved as previously described (33). Cells were harvested, washed in ice-cold PBS, lysed directly in SDS sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 2.5% 2-ME), and boiled for 5 min. Samples were resolved under reducing conditions in SDS-polyacrylamide gels. Separated proteins were then blotted onto polyvinylidene difluoride membranes and probed with an appropriate dilution of primary Abs. Reactions were detected with suitable secondary Ab conjugated to HRP (GE Healthcare) using ECL solution (Pierce, Rockford, IL).
Intracellular localization of NFAT1
For intracellular localization of NFAT1, protein cells were attached to cover slips previously coated for 1 h with 2% gelatin and left unstimulated or were stimulated for 16 h at 37°C with 1 μM ionomycin. Fifteen minutes prior to stimulation, 1 mM melatonin or 10 mM CsA was added to cells. Subsequently, cells were fixed in 3% paraformaldehyde, permeabilized with 0.1% Nonidet P-40, and stained with anti-NFAT1.PE Abs and 300 mM DAPI. The cells were photographed under ×100 magnification with a Zeiss Axiovert S100 microscope (Zeiss, Oberkochen, Germany), using red and blue filters. MERGE represents the overlay of red- and blue-filter images.
Promoter transactivation assays
Jurkat cells (3 × 106/600 μl) were electroporated (950 μF, 250 V) in a 0.4-cm Gene Pulser Cuvette (Bio-Rad, Hercules, CA) with GenePulser II electroporator (Bio-Rad). Cells were cotransfected with 4 μg luciferase reporter plasmids [either 3xNFAT/AP-1.luciferase (p3xNFAT), which was constructed by fusion of three distal NFAT element-binding sequences of the IL-2 promoter (34), or hFasLpromoter.luciferase (pHFLP), a plasmid that contains the luciferase gene under the control of a 1.2-kb region of human CD95L promoter (35)] and 0.4 μg RL.TK Renilla luciferase plasmid (pRL-TK), which encodes the Renilla luciferase and is used for normalization of transfection efficiency. In some experiments, a third plasmid (40 μg) was cotransfected: pcDNA5 (empty vector), pcDNA5.NFAT1 or pL.IRES2.NFAT1 (wild-type NFAT1), or pL.IRES2.CA-NFAT1 (constitutively active form of NFAT). Plasmid constructions are described in detail elsewhere (36, 37). After 24 h, cells were washed and stimulated at 37°C for 6 h with 10 nM PMA plus 1 μM ionomycin in the presence or absence of 1 mM melatonin or 10 mM CsA. The next day, cells were harvested and lysed for 15 min at room temperature with 50 μl of 1× passive lysis buffer (Promega, Madison, WI). Crude extracts (10 μl) were analyzed in a Veritas TM Microplate Luminometer (Turner Biosystems, Sunnyvale, CA) using Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were expressed as relative light units.
Experiments were always performed in triplicate and at least three times. Data are presented as mean values ± SD. Statistical analysis of the data was performed using one-way ANOVA and Tukey as a posttest. Differences between experimental groups were considered significant for p ≤ 0.01 or, in some cases, p ≤ 0.05. All statistic tests were performed using Prism version 5 software (GraphPad, San Diego, CA).
Melatonin protects T cells from anti-CD3–induced apoptosis
To evaluate the influence of melatonin on T cell survival, DO11.10 hybridoma cells were stimulated with immobilized anti-CD3 Abs to mimic anti-CD3-induced apoptosis (AICD). Different concentrations of melatonin (0.125–1 mM) were added to anti-CD3–stimulated cells, and, after 18 h of culture, apoptosis was evaluated by cell cycle analysis of DNA content. As shown in Fig. 1A, melatonin inhibited the appearance of subdiploid cells in a dose-dependent manner, reaching almost full protection at the highest dose (1 mM). To exclude the possibility that melatonin, rather than protecting cells from AICD, was converting anti-CD3–induced apoptosis into a necrotic form of cell death, analysis of PI exclusion was also performed. Our data showed that melatonin prevented the appearance of PI-positive cells (Fig. 1B). Importantly, melatonin was not toxic to cells in any concentration used (Fig. 1A, 1B), and the vehicle had no influence on the results (data not shown).
Effect of melatonin on AICD of T cells. A and B, DO11.10 cells were stimulated with or without plate-bound anti-CD3 Abs for 18 h in the presence of different concentrations of melatonin. C, Murine splenic T cell blasts were stimulated with or without plate-bound anti-CD3 Abs for 18 h in the presence or not of 1mM melatonin. D, T cell blasts derived from human peripheral blood leukocytes were stimulated with 10 ng/ml PMA plus 1 μM ionomycin (P+I) in the presence or not of 1 mM melatonin. A, Apoptosis was estimated by cell cycle analysis. Numbers represent the average percentage ± SD of cells with subdiploid DNA content. B–D, Cell death was quantified by PI exclusion. Each set of experiments was performed at least three times. *p ≤ 0.01.
Next, we tested whether melatonin could also protect primary T lymphocytes from AICD. As naive cells are not susceptible to AICD, we generated AICD-sensitive murine T cell blasts in vitro and stimulated these cells with plate-bound anti-CD3 Abs. Melatonin significantly inhibited AICD in murine T cell blasts either derived from spleen (Fig. 1C) or lymph nodes (data not shown). In addition, freshly isolated human T cells obtained from PBMCs were sensitized to AICD by 6-d culture with PHA and IL-2 and killed by incubating with PMA plus ionomycin, in the presence or absence of 1 mM melatonin (Fig. 1D). Again, melatonin was able to significantly protect human T cell blasts from AICD. Altogether, these results demonstrated that melatonin is able to protect human and mouse T lymphocytes from AICD.
Effect of melatonin on cell death is specific for TCR/CD3-mediated apoptosis
To test the specificity of the antiapoptotic effect of melatonin, DO11.10 cells were incubated for 18 h with different apoptogenic stimuli. Interestingly, melatonin suppressed apoptosis induced by the combination of PMA and ionomycin, which activates the same pathways as the TCR/CD3 signaling (Fig. 2A), but did not protect DO11.10 cells from stimulation with 1 μg/ml agonistic anti-CD95 Abs (Fig. 2B), teniposide (VM-26) (Fig. 2C), etoposide, vincristine sulfate, or UVC irradiation (data not shown). Additionally, melatonin synergized with actinomycin D, increasing its proapoptotic effect (Fig. 2D). These data suggest that melatonin specifically prevented TCR/CD3-induced apoptosis and that this protection is upstream of CD95 engagement.
Melatonin protects DO11.10 cells from PMA/ionomycin-induced death but not from other apoptosis-inducing stimuli. DO11.10 cells were stimulated for 18 h with 10 ng/ml PMA plus 1 μM ionomycin (P+I) (A), 1 μg/ml anti-Fas Abs (clone Jo2) (B), 10 μM Teniposide (VM-26) (C), or actinomycin D (ActD) (D) and simultaneously treated or not with 1 mM melatonin. Apoptosis was estimated by cell cycle analysis. Numbers represent the average percentage ± SD of cells with subdiploid DNA content. Figure shows representative data of three independent experiments. *p ≤ 0.01.
Melatonin inhibits anti-CD3–induced CD95L upregulation, an early event of AICD induction
To evaluate whether the melatonin was involved in blocking early or late events of AICD, we stimulated DO11.10 with immobilized anti-CD3 Abs and added melatonin after different time points (0–240 min). Fig. 3 shows that melatonin only protected T cells from death if added up to 120 min after the initiation of anti-CD3 stimulation, suggesting that this molecule acts on early events of AICD.
The addition of melatonin up to 2 h after anti-CD3 stimulation protects DO11.10 cells from AICD. DO11.10 cells were stimulated for 18 h with plate-bound anti-CD3 Abs, and melatonin was added after different periods, as indicated in the graphic. Apoptosis was estimated by Annexin V-FITC staining (A) and cell cycle analysis (B). Numbers represent the average percentage ± SD of cells stained for Annexin V-FITC or with subdiploid DNA content. Figure shows representative data of two independent experiments. *Statistically different from anti-CD3 stimulation, p ≤ 0.01.
Because CD95 and CD95L expression is augmented in T cells after TCR restimulation (24, 38) and the sensitivity to AICD is associated with a reduction in the levels of the caspase-8 inhibitor c-FLIP (39), we examined whether melatonin could alter the expression of one or more of these genes. When DO11.10 cells were stimulated for 4 h with immobilized anti-CD3 Abs in the presence or absence of 1 mM melatonin, we found no effect of melatonin on CD95 and c-FLIP gene expression (Fig. 4A). However, melatonin dramatically suppressed the anti-CD3–mediated upregulation of CD95L at both the mRNA (Fig. 4A) and protein (Fig. 4B) levels.
Melatonin inhibits anti-CD3–induced CD95L expression in DO11.10. A, Total mRNA was extracted from DO11.10 cells stimulated with anti-CD3 Abs for 4 h in the pres-ence or absence of 1 mM melatonin and converted to cDNA. Then, quantitative PCR was per-formed for CD95, FLIP, and CD95L. The expression was nor-malized by GAPDH expression of each sample. B, CD95L protein expression in DO11.10 cells stimulated or not with anti-CD3 in the presence of 1 mM melatonin for 8 h. The expression was measured by flow cytometry using anti–CD95L-PE Abs.
Melatonin blocks NFAT activation
The transcription factor NFAT is a major modulator of CD95L expression. Therefore, we investigated whether the inhibitory effect of melatonin on CD95L was mediated by interference on the NFAT-dependent pathway. Because the first step of NFAT activation is its dephosphorylation by calcineurin (40), we analyzed the effect of melatonin on anti-CD3–induced dephosphorylation of NFAT. As illustrated in Fig. 5A, anti-CD3 stimulation augmented the proportion of dephosphorylated (active)/phosphorylated (inactive) forms of NFAT1, which was dose dependently inhibited by melatonin. This effect was accompanied by a melatonin-induced retention of NFAT1 in the cytosol, as seen by fluorescence microscopy of DO11.10 cells stimulated with anti-CD3 (Fig. 5B). The same results were found when we used ionomycin, a Ca2+ ionophore that activates NFAT1 and induces its translocation to the nucleus without activating other anti-CD3–induced pathways (Supplemental Fig. 1).
Melatonin blocks NFAT activation, which is essential for AICD in DO11.10 cells. A, Western blot for NFAT1. The Ab used detects both the phosphorylated/inactive and nonphosphorylated/active form of NFAT1. Cell extracts were obtained after 1 h of anti-CD3 stimulation in the presence or absence of different concentrations of melatonin. β-actin detection was used as a loading control. B, Immunofluorescence for NFAT1. DO11.10 cells were stimulated for 1 h with anti-CD3 Abs in the presence or absence of 1 mM melatonin and then stained with DAPI and anti–NFAT1-PE Abs. C–F, Jurkat cells were transfected by eletroporation with 0.4 μg pRL-TK and 4 μg p3xNFAT (C–E) or 0.4 μg pRL-TK and 4 μg pHFLP (F). In addition, Jurkat cells were transfected with 40 μg pcDNA5.Vector or pcDNA5.NFAT1 (D) or pL.IRES2.NFAT1 or pL.IRES2.CA-NFAT1 (E, F). Cells were stimulated or not with 10 ng/ml PMA plus 1 μM ionomycin (P+I) in the presence of 1 mM melatonin or 10 mM CsA, and after 6 h, total cell lysates were obtained. Luciferase activity was measured as described and expressed as relative light units relative to the treatment presented in the first graphic column. *p ≤ 0.05.
Using luciferase reporter assays, we confirmed that melatonin interferes with NFAT activation. Jurkat T cells transfected with p3xNFAT reporter plasmid, combined or not with pcDNA5.NFAT1 or the pcDNA5 vector control, and stimulated with PMA plus ionomycin produced different levels of luciferase activity (Fig. 5C, 5D). In every situation, the addition of melatonin resulted in lower luciferase expression (Fig. 5C, 5D). Importantly, melatonin was unable to interfere with PMA plus ionomycin-induced luciferase activity when the p3xNFAT reporter plasmid was cotransfected with the pL.IRES2.CA-NFAT1, containing a constitutively activated form of NFAT1 (Fig. 5E). Finally, we observed a similar effect of melatonin when Jurkat cells were transfected with a reporter plasmid construct containing the luciferase gene under the control of the human CD95L promoter (Fig. 5F).
The effect of melatonin on cell death varies depending on the cell type and the nature of stimuli. According to the literature, melatonin can protect cells from death, can be innocuous, or can even potentiate apoptosis (reviewed in Ref. 41). For instance, injection of melatonin enhances thymic cellularity (42), whereas pinealectomy seems to accelerate thymus involution and consequently lower the cellular immune response (43, 44). In the case of mature T lymphocytes, exogenous melatonin was able to diminish the cytotoxicity of x-ray irradiation (45) or idarubicin (46). However, in some cases, instead of providing protection, melatonin potentiates T cell death, as demonstrated in the case of CD95-induced death in Jurkat cells cultured in glutathione-depleted medium (47). Our own results show that whereas melatonin protects from stimulation with anti-CD3 Abs or with the combination of PMA and ionomycin, it has no effect on anti-CD95 or teniposide-induced cell death and potentiates apoptosis triggered by actinomycin D.
It is well known that TCR/CD3 stimulation results in rapid CD95L upregulation, and CD95L-mediated CD95 oligomerization is believed to be the primary mechanism of AICD (24, 38, 48). We observed that melatonin has no effect on direct CD95 stimulation with the agonistic Ab, suggesting that melatonin prevented AICD prior to CD95 engagement. In fact, melatonin was not able to prevent AICD when added to DO11.10 cells after 2 h of anti-CD3 stimulation, indicating that melatonin blocked early events of TCR/CD3-induced apoptosis.
TCR/CD3 stimulation can be mimicked by the combination of PMA and ionomycin, which can trigger similar biochemical pathways but independently and downstream of TCR/CD3 clustering, activation of Src-family kinases, phosphorylation of ITAMs, and recruitment of specific adaptor or scaffold proteins. As melatonin also blocked PMA/ionomycin-induced apoptosis, it is unlikely that the protection involves interference with one or more of these very early events of TCR/CD3 activation.
Melatonin was shown to bind to calmodulin in a Ca2+-dependent and melatonin receptor-independent way (49, 50). One important target of calmodulin is calcineurin, a phosphatase responsible for the dephosphorylation and activation of the transcription factor NFAT. Notably, NFAT1 is the most abundant NFAT family member in T lymphocytes (51) and a major transactivator of the CD95L promoter (52, 53). NFAT1 is strongly activated after TCR/CD3 stimulation, and the involvement of NFAT1 on CD95L-induced T cells apoptosis is evidenced by studies using CsA, a specific inhibitor of calcineurin-mediated dephosphorylation of NFAT that abolishes CD95L expression and drastically reduces AICD in thymocytes and T cells (52, 54). Although some downstream targets of calmodulin were already shown to be inhibited by melatonin, such as Ca2+/calmodulin-dependent kinase II (55) and calmodulin-dependent phosphodiesterase (56), there are no data in the literature so far implicating melatonin as a modulator of NFAT activation. In this study, we demonstrated for the first time that melatonin prevented, in a dose-dependent fashion, both anti-CD3– and ionomycin-induced dephosphorylation of NFAT1 and its translocation to the nucleus. Using the p3xNFAT or the pHFLP reporter plasmids, we confirmed that melatonin is capable of preventing promoter transactivation induced by the combination of PMA and ionomycin. In addition, melatonin was able to suppress the enhancement of promoter activity induced by enforced expression of an NFAT1 construct. Importantly, transfection of CA-NFAT1 in Jurkat cells induced basal activity of a p3xNFAT promoter that was not blocked by addition of either melatonin or CsA. Noteworthy, CA-NFAT1 was generated by mutations from serine to alanine in the regulatory domain of an NFAT1 molecule that maintains the nuclear localization signal exposed, thereby allowing the protein to migrate to the nucleus even in the absence of calcineurin activity (57). Altogether, our data indicate that melatonin acts upstream of NFAT migration to the nucleus, specifically avoiding NFAT1 dephosphorylation by Ca2+/calmodulin-dependent calcineurin. Interestingly, the fact that melatonin receptor-deficient mice did not show any overt T cell deficiency supports our hypothesis that the effect of melatonin on FasL expression and consequent T cell survival is mediated through direct binding of melatonin to calmodulin and consequent inhibition of NFAT activation.
Because of their weak interaction (58), it was proposed that the binding of melatonin to calmodulin would not be possible at the physiological level of melatonin found in circulation (59, 60). Nevertheless, it is important to consider that melatonin is present at much higher levels in some microenvironments—in particular, at inflammatory sites (61). Indeed, inhibition of calmodulin by melatonin was suggested to be involved in many physiological processes, including microtubule polymerization (62), rat myotube-acethylcoline receptor expression (63), neuronal NO synthase expression (64), estrogen receptor α activation (65), and cytoskeleton rearrangement (66) and also to avoid the progression of scoliosis in mice and humans (67, 68).
The prevention of anti-CD3–mediated CD95L upregulation by melatonin reduces AICD levels in T cells, which may have an important role for the survival of T cell populations and the development and extension of T cell-mediated immune responses. Interestingly, melatonin was shown to increase Ab titers to thymus-dependent Ags and is being tested as a vaccine adjuvant against bacterial and viral infection (69, 70). Also, the death of T cells throughout the Ag-driven clonal expansion in vivo is proportional to the Ag concentration (71) and is counteracted by the presence of TLR ligands through an unknown mechanism independent of Bim, Bcl-2, and Bcl-xL (72). As TLR stimulation leads to a potent proinflammatory response and melatonin level is increased during inflammation, it is reasonable to conceive that melatonin-mediated downregulation of CD95L may play a role in the survival of T cells during an immune response.
Interference with expression of CD95L in T lymphocytes may also have an impact on the survival of APCs during the initiation or maintenance of the immune response. OTII transgenic CD4+ T cells stimulated by OVA-pulsed dendritic cells (DCs) express CD95L and are able to kill OVA-pulsed DCs, but not bystander DCs (73). Danger signals, including TLR ligands, inhibit CD95L expression in CD4+ T cells by upregulating costimulatory molecules (74). In addition, TLR stimulation induces the release of PGE2 by APCs, which in turn prevents TCR/CD3-mediated increase in CD95L and killing of target cells (32).
In light of these new findings, future studies are necessary to investigate if long-term usage of melatonin, such as in patients with sleep disorders and jet-lagged flight crew members, has any effects on T cell survival and development of autoimmune disorders. Indeed, some studies had already shown a correlation between melatonin administration and worsening of collagen-induced arthritis (75) and the development of experimental autoimmune encephalomyelitis (76). On the other hand, melatonin could be useful for treatment of diseases that develop with T cell loss, such as HIV/AIDS. In fact, HIV+ patients with reduced serum melatonin levels presented a more accelerated progression of the disease (77), and in a phase II pilot clinical study, s.c. treatment with IL-2, in combination with melatonin, enhanced CD4+ T cell count in HIV+ patients (78).
We thank Zodiac Produtos Farmacêuticos for providing the rIL-2 (Proleukin, Novartis, East Hanover, NJ).
Disclosures The authors have no financial conflicts of interest.
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and the Brazilian Research Council. A.M.d.P. and R.W. were recipients of fellowships from Fundação de Amparo à Pesquisa do Estado de São Paulo, G.P.A.-M. from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and B.K.R. from the Brazilian Research Council.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
activation-induced cell death
CA-NFAT1
constitutively active form of NFAT
c-FLIP
cellular FLICE-like inhibitory protein
cyclosporin A
dendritic cell
p3xNFAT
3xNFAT/AP-1.luciferase plasmid
pHFLP
hFasLpromoter.luciferase plasmid
propidium iodide
pRL-TK
RL.TK Renilla luciferase plasmid.
Received September 8, 2009.
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You are going to email the following Melatonin Protects CD4+ T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation
Alziana Moreno da Cunha Pedrosa, Ricardo Weinlich, Giuliana Patricia Mognol, Bruno Kaufmann Robbs, João Paulo de Biaso Viola, Ana Campa, Gustavo Pessini Amarante-Mendes
The Journal of Immunology April 1, 2010, 184 (7) 3487-3494; DOI: 10.4049/jimmunol.0902961
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

redpajama_set_name: RedPajamaCommonCrawl


Long-term and short-term exchanges of faculty and staff.
Programmes relating to research, teaching, and/or outreach.
Exchange of students at the undergraduate and/or graduate level.
Organization of training of trainers programmes in key curriculum areas, such as gender mainstreaming, gender budgeting and auditing.
Establishment of short courses to build the capacity of public servants civil society leaders and other professionals aimed at promoting gender equality and women's empowerment in cross-cutting themes ( e.g MGDs, PRSP, good governance democracy knowledge economy, ITC) of interest to both the Cameroon and Rwandan governments.
EdQual (Education Quality)project Research on the use of ICT to support Basic Education and is in partnership with 12 schools.
Delphe project – Girls into Science and Technology in Rwanda Capacity building of young women to participate in supporting the development process in Rwanda by increasing the number of young women offering applied sciences.
Development of teachers who can employ a range of pedagogies to promote community cohesion in both formal and non-formal educational settings.
Child Friendly Schools: Development of an integrated school based model of learning and delivering of essential services for children who are educationally disadvantaged.
Any other kind of co-operation that may prove useful to achieve the joint objectives.


Since the creation of the modern Middle East in the early 20th Century the region has been consumed with both conflict and crisis. Many of these problems stem from the legacies of Ottoman and European colonialism. This course will examine the impact of colonialism, the creation of new nation-states, the division of ethnic and tribal groups, and the experimentation with new and foreign political ideologies. The search for national political identity in a post-colonial world has been influenced and shaped by key regional developments such as the establishment of Israel, the Palestinian refugees, intra-country conflict, the Cold War, and the influence (and at times occupation by) regional and Western powers. In the post-Cold War period a political vacuum emerged which was quickly filled by political Islamists and led to the era known as the 'war on terror'; resulting in the wars in Afghanistan and Iraq. Currently the region remains an area of stark contrasts of under-development and modernity, poverty and wealth, and a mixture of autocratic, theocratic and democratic systems of governance. The majority of the region's population is under 25 years of age and the political, social and economic implications this is going to have on the region and internationally in the next couple of decades is critical. The changing narratives and discourse emerging from years of foreign intervention, corruption and social change offset through globalisation will be the focus of this short course. This course will examine past conflicts and crises in the Middle East ranging from the creation of its modern borders, the Arab-Israeli wars, the 1956 Suez crisis, the Iranian revolution, the rise of political Islam, to the changing real politik in the post-September 11 environment and beyond.


text:
Act I, Scene 7 of 2100 CE.
Outside the rain came down in thundering sheets, filling the streets and soaking anybody unfortunate enough to be caught in the downpour. It was the first of what were predicted to be hundreds such storms that were to regularly ravage the east coast. The millions of tons of water vaporized when the asteroid struck southern California were now returning to earth after more than a year condensing and accumulating in the upper atmosphere. The year of perpetual cloud cover combined with rapidly flooding rivers and lakes were drowning what few hardy crops remained resulting in massive global food shortages, even among the superpowers. The United Nations and other international organizations talked about using the coming showers to irrigate the barren Sahara, converting it to agricultural farmland. But even if the cloud cover broke and photosynthesis could take hold, there would still be insufficient supply to meet current demands, and now in an ironic twist of fate, food was being imported from agricultural centers on the moon and the wealthier colonies in Earth orbit. And there was still not enough to go around.
Displaced millions took refuge in larger cities, leading to unrest and finally most recently riots correlated with high concentrations of refugees: Chicago, New York, Boston, and Cincinnati here; Houston, Atlanta, and Nashville in the South; Seattle and Portland on the Pacific coast. While some of the refugees opted to work for the International Civil Service or enlist in the Peacekeeping Forces, thus dispersing them in droves across other participant member-states, most refused to leave their native country. Housing and job shortages added to the extreme stresses of food shortages, and in desperation many otherwise insipid individuals turned to crime. As if to demonstrate the severity of the crisis, international Peacekeepers were being authorized to assist in policing, the first time such Blue Berets had been seen in the country in a half century. These recent additions to the Peacekeeping Ground Forces were, in fact, reserve units composed of personnel who were in one way or another rendered unsuited for "conventional or direct action," but still capable of fulfilling the basic duties inherent in peacekeeping operations. It amounted to a military police presence composed of men and women wounded too seriously to return to regular duty: an amputated arm, a missing eye, damaged tendons. There was one soldier she had seen with such horrific scarring from third and fourth degree burns that she had been physically startled at the sight of him—and her heart broke. The pain must have been immense, and she could not imagine the psychological trauma at seeing reactions like hers on a daily, possibly an hourly basis. And she had been unable to stop staring. All she could think was, what if that was Emile? What if that was Emile…?
Through the glass doors of the emergency department, she saw one of the military amphibious vehicles used for ambulance duty rumble to a stop under the covered drive. Long streams of water shot off it in every direction, like some grotesque wheeled fountain. Inside the cabin, she saw the paramedic in the passenger seat fiddling with papers and clipboards.
Although working triage in the ER at three in the morning rarely meant true emergency trauma, there were enough patients regularly streaming in to merit a sigh of relief at seeing the paramedic's lack of alarm or urgency. The driver rounded the vehicle and stood under the covered drive a moment chatting with his partner before the paramedic from the passenger side motioned that he was going to smoke and disappeared into the downpour. The driver shook himself off and came in through the double set of sliding doors.
"You okay?" a voice said at her side.
She whirled around, throwing her arms out in a startled gasp, in the process knocking over a clipboard and some papers.
Nikki regarded her from behind black-rimmed glasses with an amused smile. "Jumpy?"
"Just tired," Julia said, bending down to gather the papers.
"Right," Nikki said knowingly. "Off with your head in the clouds, dreaming about some boy."
Before Julia could respond, a third voice chimed in. "I don't usually get called a boy, but I'll take it."
A hint of a smile touched Julia's lips. She recognized the voice as Ted's, one of the emergency paramedics. "Hi, Ted," she said, replacing the papers. "I didn't recognize you through the rain."
Ted smiled at her with a toothy grin. For as long as Julia had worked at the hospital, Ted had been here, having apparently transferred from service with the Navy as a corpsman to civilian work an ambulance paramedic. He was competent and knew exactly what to say to put anyone at ease. He was also one of the funniest and craziest people she had ever met. "I damn near didn't recognize myself," he said, shaking water off his jacket onto the floor.
"Oh damnit, Ted!" Nikki huffed, grabbing a towel from under the desk. "Here! Don't come in here and start causing trouble."
Ted shrugged sheepishly, toweling off his wet mop of hair and then his rain coat. "Oh Nikki, Nikki dearest, when will you stop this charade and admit you love me?"
"I don't know how you stand him," the senior nurse fumed and stormed off.
They watched her go a heartbeat before Ted said, "So, little bunny, what's this about some boy? I thought it was you and me for life."
Despite herself, Julia blushed at the nickname. When she first met Ted, she had accidentally stumbled into a conversation she had completely misunderstood. To this day she still did not understand just what he had been talking about—something about a giant marshmallow rabbit—but whatever the origin, she had wound up with the silly nickname little bunny which only he called her. To be fair, he had given everyone in the hospital a nickname at some point, but hers was the only one he used consistently. It made her feel a little weird…
Good weird.
"Oh, it's just Nikki," she said with a shrug. Had she been thinking about Emile? She felt silly at the thought. "I haven't seen you in a while," she said, changing the topic. "What have you been up to?"
"Oh, you know," he said, lounging towards her across the desk. "I went down to Nags Head to visit my brother. Did some surfing, a lot of drinking—you know." He smiled, charming. "Too bad about the weather, huh?"
"Yeah," she said, and smiled. "How was Nags Head?"
"Oh, it was awesome. Lots of that stuff," he said, waving towards the doors, "but it's still a cool place to visit. They've done some pretty awesome work on the dikes. Have you seen them?"
Julia shook her head. "I've never been."
"What?" Ted said in mock-surprise.
Out of the corner of her eye she saw a few patients in the waiting area look over, startled by his sudden outburst.
"Dude, we have to go! You would totally love it there," he said, still waving his hands.
To the patients he probably looked and sounded like a crazy person. But then, he kind of was…
"Julia, Julia," he insisted, "we have to go. Like tomorrow or something!"
Even in this serious, sterile environment, his energy was infectious. She laughed aloud. "Ted, you're crazy."
"Maybe—probably," he said, and his tone was at once mock-business formal. "But seriously, we need to go. Say you're going to go with me."
She laughed. "Ted…"
"Say it! Say it!"
"You got to say it!"
"Oh-kay!" she said between bursts of laughter. She was laughing so hard her cheeks hurt and she gasped for breath. "Okay, we'll go. Just stop making a scene, you crazy!"
As if only now becoming aware of the nearby patients, he flashed them that big disarming grin and waved. "It's okay, folks, I work here," he called, pointing to his jacket. "No real need for alarm."
"Ted, you're scaring them," she said in a hushed tone, trying to stifle more laughter.
He turned back, still grinning ear to ear. "I know. But they're at a hospital—in the ER!—so I'm probably not the worst thing that's happened to them today."
She covered her mouth against more laughter, but not before garnering more unhappy looks from the patients.
"So…" he said, "we're going, right?"
"Ted!" This time it was Nikki, calling from the opposite side of the waiting room where she had just emerged from the treatment area. "Get out of here!"
"Right?" he said, ignoring the other nurse.
"TED!"
"Okay, yes, yes," Julia said, fighting to stop another fit. "Now go, before Nikki kills you."
Ted smile, every-charming, but before he could open his mouth to say another silly thing, he was nearly tackled by Nikki. Amid a flurry of sarcastic comments and love overtures aim at Nikki, she ushered him from the desk and out the doors.
As the doors shut out the imminent argument outside, one of the patients stood and approached the desk. "Young lady," she said, addressing Julia," you really should tell your boyfriend not to act like that. He is very disruptive."
Julia, still giggling, smiled at the older lady. "Oh, no. He's not my boyfriend. He's just a friend—a coworker," she said, correcting herself.
"Oh, really?" the woman said skeptically. "Well…you should still keep your relationships out of the workplace."
Julia just smiled back. She was clearly misinformed, but there no reason to bother trying to explain further. "Yes, ma'am," she said politely. "Thank you."
Only later, after her shift ended and she was aboard the light rail home that Emile came to her thoughts again. She saw the disfigured Blue Beret patrolling along the length of the train, passing silently between cars and ignoring the revulsion which filled the railcars in his wake.
Continues with Landon.
Nov 24, 2019 Leave a Comment on Hospital2100 CE
Tags 2100 CE, human conflict, loneliness
Equanimous Equitability
The violence of late republic, Part 2: Punitive Factionalism


Issue 5 / OCT16 is out! It is another diamond of an issue. Contains news of the CSC/T Final, WOC, WTOC, Jircs, JHIs, VHIs, JEC, NAOC, O in RSA, RAFO on Tour, Austrians raiding Dumfries & Galloway, and oodles more of the usual Competitions, columns, cartoons, chat and comment. Don't miss it!
Issue 4 (aka AUG16) contains the UK Club Rankings and Regional rankings, coverage of the SELOC Sprint weekend, Harvester Relays, Swiss O Week, Welsh 5 Days - Croeso, EYOC, Lagganlia and Deeside JROS Junior tours, JWOC in Switzerland, Saunders Lakeland MM, plus part 2 of our new Coaching Column by Geoff Ellis, and all the usual columns and competitions.
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Issue 3 is a sensational delight. It contains coverage of BOC Long and Relays, BOC Middle and Sprints, O in Singapore, a History of the Combined Harvesters and Harvester Relays, JOK Chasing Sprint, a new variation on an old theme - Know your Grade A Controller, Jukola, TioMila and much more - including all the usual columns and competitions, and the start of a new Coaching column! Did you miss it? Make sure your sub is up to date!


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text:
Love Thy Fans Pt. 2
Imagine, if you aren't already, that you're a successful game developer and you have your game on the market, and you want to get some feedback from your customers because you're good at what you do and you want to make the best game you can. When you set out to canvas your current audience, you quickly discover that their suggestions contradict each other. What do you do?
Well first off, don't be surprised. Remember, not everyone will enjoy your games the same way. One person will derive their most fun from a game by setting limits on themselves and accomplishing challenges, whereas another person will get the most fun from a game by making their avatar wear the most fashionable clothing they can find. Thus, one person suggesting changes to maximize one area, will find their suggestions either explicitly or implicitly contradicting suggestions from another.
The next thing to realize is that your game cannot be everything, to everyone. It is impossible, and to attempt this is to water down your game so much it has lost all tastiness. It can certainly be a lot to some, but it can't be everything. Part of this is due to the amount of time, money and manpower you have available to develop things, but part of it has to do with simply contradictory things. Do you make an easy mode for Sekiro, or do you not? Attempting to split the difference on things like that makes everyone dissatisfied, so you have to pick one or the other.
So you have to be selective with what feedback to actually implement. Who do we take more seriously?
Your actual hardcore fans. These hardcore fans are emotionally invested in your success, and really want to see you make a great game. Feedback from casual players can be useful, but along the lines of bug reports and how to make any tutorial make more sense. Be very wary of taking gameplay balancing or feedback of new systems from casual players, since they often don't have as good of a grasp of the big picture.
Again, your hardcore fans won't all agree with each other since they'll like different things about your game, but immediately it's a smaller section of feedback to wade through. Instead, you subdivide the feedback again.
The long-time developer of Defense of the Ancients and DotA 2, Icefrog kept in contact with the absolute highest skilled players of the game for balancing testing. Since DotA is a game about competitive player versus player, the top players had a far more accurate idea of what was strong, what was weak, and what was outright broken than Icefrog knew himself. Numerous posts on the forums about so-and-so being overpowered, Icefrog please nerf, would correctly go ignored, as after various minor tweaks to completely different systems lead to different play, and those in-game heroes or items decried as overpowered just fell out of favor. Nothing really different about them, they were still good at what they did, but those situations in which they shined just didn't happen nearly as often if at all.
Those players on the forums were just as hardcore of fans as the top-skilled players were, but relying on them for feedback on balancing would have been a terrible idea. Too often, they'll want something nerfed into the ground or buffed into heaven, to simply scratch an emotional itch they had regarding some dissatisfaction. Still, they're your hardcore fans, you have to treat them well. They can still give good feedback, just don't weigh it as heavily as the skilled players. In many cases, these middling or poor-skilled players can give a wide panoply of suggestions that the top-skilled players didn't even think to cover which are still valuable goals. Is there something about the UI which could honestly be improved? The average-skilled player who puts in a lot of time can give effective advice on that. The top-skilled player may not even remember it most of the time, when they've focused on wrangling out the rules for a competitive advantage as much as they can instead.
Something to keep in mind during all of this though, is that the vision of the game your hardcore players want probably will be a slightly different game than what you've intended. Still, love thy fans. They're the reason you can feed your family. Treat them well, and it'll be hard to drive them away.
Posted by Student in Blue at 8:00 AM No comments:
Love Thy Fans
I've touched on this briefly, but now is as good a time as any to go more in depth on the subject.
Love your hardcore fans, developer, for they will see you through the rough times. Appealing to the casual audiences may bring lots of dough, but make one misstep and you'll be having some really rough times.
What do I mean by this?
Look, it's pretty clear to anyone without a god complex that not every game you make will be a success. When those games aren't a large enough success to recoup the amount of work that went into them, you start having financial troubles. This is very obvious.
Sufficiently large enough games take an extraordinary amount of work to complete. Practically every game actually worth $20 or more, has had years to percolate. So to recover from a disastrous game puts a lot of stress on a studio, because you need a lot of time in order to release a new game that addresses what the players hated.
Video games generally aren't a hugely profitable industry anyway, and when you have a good success of a game it's quite easy for that money to fly away.
Your fans who are hardcore supporters of you and your studio though, will forgive your inevitable missteps. They'll buy your crappy game, and be eager to buy your next game because they have faith that the next one will be better. In fact, it's actually hard to accidentally alienate a hardcore fan. You have to very purposefully treat them like dirt in order to drive away the most fanatical. Love them, because they love you.
Really, dealing with fans is just an extension of normal interpersonal relationships. Fairweather fans are fairweather friends, and you can never rely on them. Just appreciate them when they're there, but never make plans around them. They're just there for the experience of it all, not because they want to actively contribute and make it their own.
This division between the fairweather and the fanatical extends even further in unexpected ways when you start conversing with your audience, which I'll write more on next week.
Posted by Student in Blue at 11:02 PM No comments:
Let's talk AI.
An aspect of gaming that has been there for almost as long as there's been games, Artificial Intelligence fleshes out the world in MMOs, provides training dummies for competitive PvP games, and for many single player games is the entire reason they exist.
Yet, AI in the gaming sense is not AI in the academic sense. Let's get this perfectly clear, because that's a rather important distinction. In games, the Artificial is clearly artificial, and the Intelligence is a misnomer, whereas the academic AI definition seeks to make Artificial Intelligence indistinguishable from regular human intelligence. There's a good reason for that.
Remember, what is the game being made for? One would hope it would be for the enjoyment of its audience. If the only one enjoying themselves are the creator, that's simply a mockery, not a game. Academic AI isn't made for the audience to enjoy themselves, even people can certainly derive that via wonder at the advancement of technology.
Game AI is for the enjoyment of the user however, and as such often means intentionally flawed AI, very often due to being direct competition for the player. A perfect AI in an MMO would realize he has no chance winning against the player character whatsoever, and as such would run away immediately upon spotting a player. This might be fun for awhile, until the AI realizes that the only winning move is not to play, and simply decides not to respawn. Suddenly fun times are over.
Even not going as far as that, an AI with no intentional flaws has an absurd advantage over human players in that there is hardly any reaction time. Gauging a threat and determining a course of action happens over the span of ~16ms, versus a human reacting blindly out of reflex taking ~160ms, ten times slower than the computer. A machine won't mentally tire from stress or fatigue either, making random "dumb" mistakes. So gaming AI will often have artificial limitations set on their artificial intelligence.
It could be said that part of the reason why is so that it'll be more fair. This is a mistake of what the goal is. Rather, the reason why gaming AI will often have artificial limitations is because it is more enjoyable to play against, not because it is fair. Sometimes it's really fun to smack around very dumb AI, as Valve found out when they were making Left 4 Dead and discovered that mowing down hordes of mindless zombie AI was actually really fun, instead of small amounts of tactically placed enemies. Sometimes it's really fun to challenge yourself and play against very unfair AI as it becomes a tightrope of trying to stay alive while discovering the AI's Achilles heel, and you search for the Achilles heel because you know full well it's impossible to win a straight out fight.
Put in other words, AI from an academic standpoint is meant to be like a human, whereas AI from a gaming standpoint is meant to be a puzzle for the player to try and solve.
So don't be worried if your AI isn't perfect. The real question is, is it fun?
Posted by Student in Blue at 8:00 AM 5 comments:
Labels: Design
Ross from Accursed Farms makes a case as to why Games as a Service is fraud.
It's interesting seeing his side of things because it's very firmly rooted in the player side of things. And I could very easily see why players could view Games as a Service (GaaS) as something shady. However he also only comprehends GaaS on the player end, missing exactly what it means to a developer. And really, it's not so much that Games as a Service is fraud, but rather it's the methodology du jour of fraudsters, just as licensed shovelware and obnoxious game 'collections' like Action 52 used to be. But licensed games, game collections, and even GaaS isn't the cause of fraud, it's just the low-hanging fruit being used.
Ross correctly begins with trying to define exactly what GaaS is, and amusingly goes to Wikipedia before realizing that they aren't very helpful in figuring out what exactly it is. Rather, the concept of what exactly goods and services are, come into play here. Goods being something you own, tangible or intangible, such as a hammer, an ebook, or an intellectual property. Services instead are... services that you pay people for, such as getting a haircut.
Games used to treated as goods, fullstop. The developer made the game, handed it off to the publisher, and promptly forgot about it until either making a sequel or translating it for another country. Otherwise, the code was very static, and any bugs found, <i>remained</i>. Because that's what goods are. You make the good, sell it, and it's no longer your problem unless you're legally liable for injury.
That started changing when the internet became more widespread, patches could realistically be made and applied to all current copies of a game, and more importantly reoccurring server costs started becoming a thing thanks to all of these popular internet-based games. Patches mean you can push off some of the minor bug fixing until later, and even delaying certain game content until it's actually good and ready to be played, instead of rushing it out the door in a buggy mess because the publisher is knocking down your door asking firmly why it's not ready yet.
Approaching the game like this is not treating the game as goods anymore though. Instead of a one-and-done, fire-and-forget, you have to baby the program long after it is released, feeding it, growing it, and maintaining it. The maintenance costs man-hours, and any associated servers cost money. Those are not free. You're servicing the game. It has become a service.
Ross makes an analogy in the video where he compares Games as a Service to a rest stop in a mountainous park area, and that when the state loses money and shuts down the rest stop it somehow denies complete access to the mountains and forests. This is a stretch of an analogy.
If looking to strictly compare services, the service provided by the rest stop are strictly of maintenance of the rest area, and they have nothing to do with the mountain and forest. And what happens when the service for the rest stop is halted? It is the same as with a game hosted on the internet, the whole thing breaks down and you can't use it anymore. The only difference is that the rest stop will break down over a much longer period of time than pulling the plug on a server, requiring years for nature to reclaim building and paved areas. The rest stop is not gatekeeping you from enjoying the nature.
A more accurate analogy would be a theme park, where you have bought a lifetime pass. An online-only game, much like a shuttered theme park, becomes completely unavailable when it closes down despite having paid upfront for a lifetime pass.
Now with all of this said, games as a whole are rather odd, and probably don't rightly belong squarely as a traditional "good" nor a traditional "service". Often it's a bit of a weird combination of the two, and we really don't have a good word for it as far as I know. Computers are still a relatively young concept compared to, say, a hammer and a haircut after all.


text:
Loving Eleanor
February 15, 2016 March 8, 2016 hopewellslibraryoflife
I admit it, I approached this one cautiously. Eleanor Roosevelt is a heroine of mine. I've read and re-read Blanche Weisen Cook's two volume biography (vol. 1 and vol. 2 and have suggested that Doris Kearns Goodwin's No Ordinary Time be required reading for everyone in the public school universe. I'm an Eleanor Fan, plain and simple.
Like everyone, I was more than a little surprised when the Lorena Hickok letters hit the E.R. scene. Lorena was as openly gay as a person could be in her day (i.e. the 1920s—1950s for the most part). Eleanor had had a horrible childhood, but was rescued by Madame Marie Souvestre, eccentric headmistress of Allenwood, a girls school in London. Souvestre originally opened a school in France with her female partner. After their relationship floundered she moved to England and ran Allenswood. By the time of this novel's opening, Eleanor had ended conjugal relations with FDR following his love affair with Lucy Mercer, and was openly living on the family's Springwood Estate (aka "Hyde Park") with a lesbian couple.
Loving Eleanor was so beautifully told—holding to the manners, language and mores of the story's own times that I laughed at my own apprehension. This is not a seedy sex book with a "romance" label slapped on it. This is a love story, pure and simple. I adored it, even though I'm not a fan of Hick herself.
source: Corbis
Hick complained endlessly, magnified every perceived slight and was insanely jealous of nearly everyone in E.R.'s life. That said, she, along with Louis Howe, created the Eleanor Roosevelt we know today. Personally, though, I give much more credit to Louis than to Hick—of whom E.R. was clearly tired long before Hick tired of her.
The Roosevelts had such a complicated post-Lucy marriage that they epitomize the idea that no one knows a marriage but the two people in it. In Loving Eleanor we see the fictional idea of FDR's "Left Hook" at work seemingly spitefully destroying a few of Eleanor's happiest relationships. Yet Eleanor, like many another wronged wife even today, simply embraced her husband's other women and welcomed them at least outwardly. As salacious as the Eleanor-Hick relationship still is, I find it more a distraction for E.R. than a great love "affair". That she loved Hick and cared for her is without doubt, but I have always found the passion a bit one-sided. I think to her dying day that E.R.'s only great love was her father, Elliott Roosevelt, brother of Theodore.
Source: FDR Library
Both Hick and Eleanor today would have been seen for what they were—abused and abandoned children of alcoholic fathers and absent mothers. I believe both most likely experienced some level of incest—Eleanor's grandmother had to have multiple locks installed on Eleanor's bedroom door to protect her from her alcoholic uncles who still lived at home. Hick, whose step-mother threw her out, probably suffered some degree of this from her father and perhaps more abuse from men in whose homes she toiled as the "hired girl". Each woman had the characteristic lack of trust of such children which made their relationships so difficult and made it very hard for them to feel and express real love. In the last Hick did much better. Eleanor later regretted the way she had raised her children largely for this reason.
This is the sort of novel I want to write. Breathing life into great people in history and making them "real." I have one such in the works and, if it can be a fraction as well done as this, I will be supremely happy.
Loving Eleanor by Susan Wittig Albert.
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2 thoughts on "Loving Eleanor"
Kate W says:
I'll allow myself a little time between this and White Houses but very much looking forward to it.
I hope you like it. White Houses' author certainly can write though!