Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-DL-methyl-tryptophane (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Aldosterone deficiency adversely affects pregnancy outcome in mice

Circulating aldosterone levels are increased in human pregnancy. Inadequately low aldosterone levels as present in preeclampsia, a life-threatening disease for both mother and child, are discussed to be involved in its pathogenesis or severity. Moreover, inactivating polymorphisms in the aldosterone synthase gene have been detected in preeclamptic women. Here, we used aldosterone synthase-deficient (AS(-/-)) mice to test whether the absence of aldosterone is sufficient to impair pregnancy or even to cause preeclampsia. AS(-/-) and AS(+/+) females were mated with AS(+/+) and AS(-/-) males, respectively, always generating AS(+/-) offspring. With maternal aldosterone deficiency in AS(-/-) mice, systolic blood pressure was low before and further reduced during pregnancy with no increase in proteinuria. Yet, AS(-/-) had smaller litters due to loss of fetuses as indicated by a high number of necrotic placentas with massive lymphocyte infiltrations at gestational day 18. Surviving fetuses and their placentas from AS(-/-) females were smaller. High-salt diet before and during pregnancy increased systolic blood pressure only before pregnancy in both genotypes and abolished the difference in blood pressure during late pregnancy. Litter size from AS(-/-) was slightly improved and the differences in placental and fetal weights between AS(+/+) and AS(-/-) mothers disappeared. Overall, an increased placental efficiency was observed in both groups paralleled by a normalization of elevated HIF1α levels in the AS(-/-) placentas. Our results demonstrate that aldosterone deficiency has profound adverse effects on placental function. High dietary salt intake improved placental function. In this animal model, aldosterone deficiency did not cause preeclampsia.

3,4-Dehydrodebrisoquine, a novel debrisoquine metabolite formed from 4-hydroxydebrisoquine that affects the CYP2D6 metabolic ratio

Considerable unexplained intersubject variability in the debrisoquine metabolic ratio (urinary debrisoquine/4-hydroxydebrisoquine) exists within individual CYP2D6 genotypes. We speculated that debrisoquine was converted to as yet undisclosed metabolites. Thirteen healthy young volunteers, nine CYP2D6*1 homozygotes [extensive metabolizers (EMs)] and four CYP2D6*4 homozygotes [poor metabolizers (PMs)] took 12.8 mg of debrisoquine hemisulfate by mouth and collected 0- to 8- and 8- to 24-h urines, which were analyzed by gas chromatography-mass spectrometry (GCMS) before and after treatment with beta-glucuronidase. Authentic 3,4-dehydrodebrisoquine was synthesized and characterized by GCMS, liquid chromatography-tandem mass spectrometry, and (1)H NMR. 3,4-Dehydrodebrisoquine is a novel metabolite of debrisoquine excreted variably in 0- to 24-h urine, both in EMs (3.1-27.6% of dose) and PMs (0-2.1% of dose). This metabolite is produced from 4-hydroxydebrisoquine in vitro by human and rat liver microsomes. A previously unstudied CYP2D6*1 homozygote was administered 10.2 mg of 4-hydroxydebrisoquine orally and also excreted 3,4-dehydrodebrisoquine. EMs excreted 6-hydroxydebrisoquine (0-4.8%) and 8-hydroxydebrisoquine (0-1.3%), but these phenolic metabolites were not detected in PM urine. Debrisoquine and 4-hydroxydebrisoquine glucuronides were excreted in a highly genotype-dependent manner. A microsomal activity that probably does not involve cytochrome P450 participates in the further metabolism of 4-hydroxydebrisoquine, which we speculate may also lead to the formation of 1- and 3-hydroxydebrisoquine and their ring-opened products. In conclusion, this study suggests that the traditional metabolic ratio is not a true measure of the debrisoquine 4-hydroxylation capacity of an individual and thus may, in part, explain the wide intragenotype variation in metabolic ratio.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Repetitive transcranial magnetic stimulation of the dorsolateral prefrontal cortex affects divided attention immediately after cessation of stimulation

Transcranial magnetic stimulation has evolved into a powerful neuroscientific tool allowing to interfere transiently with specific brain functions. In addition, repetitive TMS (rTMS) has long-term effects (e.g. on mood), probably mediated by neurochemical alterations. While long-term safety of rTMS with regard to cognitive functioning is well established from trials exploring its therapeutic efficacy, little is known on whether rTMS can induce changes in cognitive functioning in a time window ranging from minutes to hours, a time in which neurochemical effects correlated with stimulation have been demonstrated. This study examined effects of rTMS on three measures of executive function in healthy subjects who received one single rTMS session (40 trains of 2 s duration 20 Hz stimuli) at the left dorsolateral prefrontal cortex (DLPFC). Compared to a sham condition one week apart, divided attention performance was significantly impaired about 30-60 min after rTMS, while Stroop-interference and performance in the Wisconsin Card Sorting Test was unaffected after rTMS. Repetitive TMS of the left DLPFC, at stimulation parameters used in therapeutic studies, does not lead to a clinically relevant impairment of executive function after stimulation. However, the significant effect on divided attention suggests that cognitive effects of rTMS are not limited to the of acute stimulation, and may possibly reflect known neurochemical alterations induced by rTMS. Sensitive cognitive measures may be useful to trace those short-term effects of rTMS non-invasively in humans.

Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.

The IKK inhibitor BMS-345541 affects multiple mitotic cell cycle transitions

The IkappaB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFkappaB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKalpha also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of Aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS-345541 function could be useful for cell cycle control studies and may provide valuable clues for the design of future therapeutics.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Low extracellular (ECF) glucose affects the neurochemical profile in severe head-injured patients

Glucose (Gluc) is the main energy source for the brain. After severe head-injury energy demand is massively increased and supply is often decreased. In pilot microdialysis studies, many patients with severe head-injury had undetectable glucose concentrations, probably reflecting changes in metabolism and/or reduced supply. We therefore investigated whether patients with low ECF glucose (criterion: < 50 microM for > or = 5 hrs), LOWgluc, differ from patients with higher glucose levels (NORMALgluc) We also tested the interrelationships between other parameters such as lactate, glutamate, K+, brain O2 and CO2, ICP, CPP, and CBF in these two groups. We found that patients with low ECF glucose, LOWgluc, have significantly lower lactate concentrations than patients with "normal" glucose, NORMALgluc, levels do. Spearman correlations between glucose and most other parameters were similar in both patient groups. However, glutamate correlated positively with glucose, lactate, brain CO2 and negatively with brain O2 in the NORMALgluc patient group, whereas glutamate did not significantly correlate with any of these parameters in the LOWgluc group. There was also no correlation between outcome and the dialysate glucose. The results indicate that low ECF glucose is almost always present in severe head-injury. Moreover, the lack of correlation between low glucose and outcome, however, suggests that other energy substrates, such as lactate, are important after TBI.