Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-triazole, we used computational structure-based methods to design more potent ligands. This approach yielded highly efficient low molecular weight inhibitors, the most active being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A quantitative structure-activity relationship based on the electrostatic ligand-protein interactions in the docked binding modes and on the quantum chemically derived charges of the triazole ring demonstrated a good explanatory power for the observed activities.

Reduced choroidal neovascularization by AAV-anti-VEGF shRNA delivery

VEGF plays an essential role in ocular angiogenic diseases including the late-stage form of AMD, the primary cause of vision loss in the western world. Over-expression of VEGF leads to development of vasculature emanating from the choroid, invading the subretinal space through breaks in Bruch's membrane. Strategies leading to long-term suppression of inappropriate ocular angiogenesis are required. A panel of 10 shRNAs targeting the coding region of human VEGF165 was tested in HEK293 cells and in the human retinal pigment epithelial cell line, ARPE-19. VEGF knock-down up to 92% was achieved by co-transfecting shRNAexpressing constructs with plasmid encoding the Renilla luciferase gene fused to the VEGF165 sequence. For in vivo delivery of the most potent shRNA cassette, both single-stranded and self-complementary rAAV vectors were packaged in serotype 8 capsids. Intramuscular administration in mice led to localized expression and 96% knock-down of endogenous VEGF. Using eGFP as a marker, efficient gene transfer of retinal pigment epithelial cells, the cells thought to be responsible for the abnormal VEGF production, was obtained by subretinal delivery of rAAV2.8 vectors. The capacity of rAAV-encoded shRNAs to silence endogenous VEGF gene expression was evaluated in the laser-induced murine model of choroidal neovascularization (CNV). In this mouse model of AMD, sizes of the CNV were found to be significantly reduced following rAAV-shRNA subretinal delivery. Thus, our results indicate that gene transfer combining AAV-mediated delivery with triggering of the endogenous RNAi pathway can be used for anti-VEGF therapy and holds great promise for the treatment of AMD.

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Sustained transgene expression using MAR elements.

Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.

Deletion of CREB-Regulated Transcription Coactivator 1 Induces Pathological Aggression, Depression-Related Behaviors, and Neuroplasticity Genes Dysregulation in Mice.

BACKGROUND: Mood disorders are polygenic disorders in which the alteration of several susceptibility genes results in dysfunctional mood regulation. However, the molecular mechanisms underlying their transcriptional dysregulation are still unclear. The transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and the neurotrophin brain-derived neurotrophic factor (BDNF) have been implicated in rodent models of depression. We previously provided evidence that Bdnf expression critically rely on a potent CREB coactivator called CREB-regulated transcription coactivator 1 (CRTC1). METHODS: To further evaluate the role of CRTC1 in the brain, we generated a knockout mouse line and analyzed its behavioral and molecular phenotype. RESULTS: We found that mice lacking CRTC1 associate neurobehavioral endophenotypes related to mood disorders. Crtc1(-/-) mice exhibit impulsive aggressiveness, social withdrawal, and decreased sexual motivation, together with increased behavioral despair, anhedonia, and anxiety-related behavior in the novelty-induced hypophagia test. They also present psychomotor retardation as well as increased emotional response to stressful events. Crtc1(-/-) mice have a blunted response to the antidepressant fluoxetine in behavioral despair paradigms, whereas fluoxetine normalizes their aggressiveness and their behavioral response in the novelty-induced hypophagia test. Crtc1(-/-) mice strikingly show, in addition to a reduced dopamine and serotonin turnover in the prefrontal cortex, a concomitant decreased expression of several susceptibility genes involved in neuroplasticity, including Bdnf, its receptor TrkB, the nuclear receptors Nr4a1-3, and several other CREB-regulated genes. CONCLUSIONS: Collectively, these findings support a role for the CRTC1-CREB pathway in mood disorders etiology and behavioral response to antidepressants and identify CRTC1 as an essential coactivator of genes involved in mood regulation.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells.

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function

Effects of exercise on the isoelectric patterns of erythropoietin.

OBJECTIVES: Recombinant erythropoietin has a strong impact on aerobic power and is therefore one of the most potent doping agents in endurance sports. The anti-doping control of this synthetic hormone relies on the detection, in the urine, of its isoelectric pattern, which differs from that of the corresponding natural hormone, the latter being typically more acidic than the former. However, a small number of natural urinary patterns, referred to as "atypical patterns," are less acidic than the dominant form. Based on anecdotal evidence, the occurrence of such patterns seems to be related to particular strenuous exercises. This study aimed to demonstrate this relation using a strenuous exercise protocol. DESIGN: Seven athletes took part in a training protocol including a series of supramaximal short-duration exercises. Urine and blood samples were collected throughout the protocols. SETTINGS: World Cycling Center, Aigle, Switzerland, and research laboratories. PARTICIPANTS: Seven top-level athletes (cyclists) were involved in this study. MAIN OUTCOME MEASURES: Erythropoietin (EPO) isoelectric patterns were obtained by submitting blood and urine samples to isoelectric focusing. Additional protein dosages were performed. RESULTS: Supramaximal short-duration exercises induced the transformation of typical urinary natural EPO patterns into atypical ones. None of the obtained atypical patterns fulfilled the 3 criteria mandatory for reporting an adverse analytical finding. Serum EPO patterns were not affected by the exercises that caused the transformation of urinary patterns. CONCLUSION: An exercise-induced transient renal dysfunction is proposed as a hypothetic explanation for these observations that rely on parallel investigations of proteinuria in the same samples.

The role of NFI-C liver regeneration and its potential function as a general regulator of regenerative processes

Collectively, research aimed to understand the regeneration of certain tissues has unveiled the existence of common key regulators. Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed a misregulation of growth factor signaling, in particular that of transforming growth factor ß-1 (TGF-ßl), which led to alterations of skin wound healing and the growth of its appendages, suggesting it may be a general regulator of regenerative processes. We sought to investigate this further by determining whether NFI-C played a role in liver regeneration. Liver regeneration following two-thirds removal of the liver by partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes following injury lead to a rapid, phased proliferation. However, mechanisms controlling the action of liver proliferative factors such as transforming growth factor-ßl (TGF-ß1) and plasminogen activator inhibitor-1 (PAI-1) remain largely unknown. We show that the absence of NFI-C impaired hepatocyte proliferation due to an overexpression of PAI-1 and the subsequent suppression of urokinase plasminogen (uPA) activity and hepatocyte growth factor (HGF) signaling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wildtype mice. The subsequent transient down regulation of NFI-C, as can be explained by a self- regulatory feedback loop with TGF-ßl, may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. Overall, we conclude that NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration. Taken together with NFI-C's actions in other in vivo models of (re)generation, it is plausible that NFI-C may be a general regulator of regenerative processes. - L'ensemble des recherches visant à comprendre la régénération de certains tissus a permis de mettre en évidence l'existence de régulateurs-clés communs. L'étude des souris, dépourvues du gène codant pour le facteur de transcription NFI-C (Nuclear Factor I-C), a montré des dérèglements dans la signalisation de certains facteurs croissance, en particulier du TGF-ßl (transforming growth factor-ßl), ce qui conduit à des altérations de la cicatrisation de la peau et de la croissance des poils et des dents chez ces souris, suggérant que NFI-C pourrait être un régulateur général du processus de régénération. Nous avons cherché à approfondir cette question en déterminant si NFI-C joue un rôle dans la régénération du foie. La régénération du foie, induite par une hépatectomie partielle correspondant à l'ablation des deux-tiers du foie, constitue un modèle de régénération bien établi dans lequel la lésion induite conduit à la prolifération rapide des hépatocytes de façon synchronisée. Cependant, les mécanismes contrôlant l'action de facteurs de prolifération du foie, comme le facteur de croissance TGF-ßl et l'inhibiteur de l'activateur du plasminogène PAI-1 (plasminogen activator inhibitor-1), restent encore très méconnus. Nous avons pu montrer que l'absence de NFI-C affecte la prolifération des hépatocytes, occasionnée par la surexpression de PAI-1 et par la subséquente suppression de l'activité de la protéine uPA (urokinase plasminogen) et de la signalisation du facteur de croissance des hépatocytes HGF (hepatocyte growth factor), un mitogène puissant des hépatocytes. Cela indique que NFI-C agit en premier lieu pour promouvoir la prolifération des hépatocytes au début de la régénération du foie chez les souris de type sauvage. La subséquente baisse transitoire de NFI-C, pouvant s'expliquer par une boucle rétroactive d'autorégulation avec le facteur TGF-ßl, pourrait limiter le nombre d'hépatocytes qui entrent dans la première vague de division cellulaire et/ou inhiber l'initiation de la mitose tardive. L'ensemble de ces résultats nous a permis de conclure que NFI-C agit comme un régulateur de la prolifération des hépatocytes synchrones au cours de la régénération du foie.

Mico-cocktail validation for CYP3A and CYP2D6 assessment using a low dose of midazolam (0.075 mg) and dextromethorphan (2.5 mg)

Background/Aim: Cocktail approach is generally preferred to individual administration of probes in order to characterize the activity of multiple enzymes. However, cocktail strategy has several drawbacks such as drug-drug interactions, tolerability and toxicity. Hence, there is a need to develop cocktails using low doses of probes. Our aim was to investigate whether the simultaneous oral administration of microdoses of midazolam (MDZ) and dextromethorphan (DEM) can be used to assess the simultaneous activities of CYP3A and CYP2D6. Methods: As part of a 5 arm randomized cross-over control trial on the analgesic efficacy of oxycodone, ten healthy young non-smoking males received the following combinations of drugs: Quinidine (Q)+ ketoconazole (K) or Q+placebo (P) or K+P or P+P. In all cases MDZ (0.075 mg) and DEM (2.5 mg) were administrated 1 hour after Q, K or P. CYP2D6 and CYP3A activities were determined after urine collection during 8 hours (ratio DEM/DOR), and a blood sample (EDTA) after 30 min (ratio 1-OH-MDZ/MDZ). DEM and DOR analysis was performed using LC-fluorescence. MDZ and 1-OH-MDZ determination was performed using GC-MS. Allele's variants of CYP2D6 were detected using the AmpliChipTMCYP450 (Roche). Results: CYP2D6 genotype predicted 1 poor (PM), 1 intermediate (IM), 7 extensive (EM) and 2 ultra rapid (UM) metabolizers. A good correlation was obtained between the predicted and the measured phenotypes except for 1 EM phenotyped as UM. Two duplications for alleles *41/*41xN and *1/*2xN were detected and the two volunteers were phenotyped as UM. A potent inhibition of CYP2D6 or CYP3A4 was obtained when Q or K were used. Mean metabolic ratio DEM/DOR in P and K groups were 0.015 (±0.028) and 0.015 (±0.019). It significantly increased in Q and QK groups (0.668 (±0.676) and 0.743 (±1.038)). Mean 1-OH-MDZ/MDZ in P, Q were 2.73 (±1.05) and 2.55 (±1.40) while it significantly decreased in K and QK groups (0.11 (±0.05), 0.10 (±0.05)). Moreover, there were no statistically significant differences between QK and K sessions for CYP3A and between QK and Q for CYP2D6 which indicate that there is no interaction between the two metabolic pathways. Conclusion: Simultaneous assessment of CYP3A and CYP2D6 activities can be obtained by low oral doses (micro-cocktail) of MDZ and DEM. Specific inhibitors such as Q or K modulates selectively CYP2D6 or CYP3A activities.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.

Evidence for an overestimated vulnerability of adolescent rats to develop signs of drug addiction

Drug addiction is a multi-etiological disorder to which some individuals are more vulnerable an others. Whereas converging clinical and epidemiological studies report a peak of drug use ring adolescence, many behavioral traits characterizing teenagers have been proposed to contribute to this vulnerability, including a heightened sensation-seeking, an enhanced impulsivity d a larger influence exerted by peers. By many aspects, juvenile rodents display behavioral traits at resemble those of teenagers. However, the concept of increased vulnerability to drug addiction juvenile rats remains in debate. Indeed, only a few studies directly compared juvenile and adult fdents regarding behavioral predictors of drug abuse. Moreover, some key features of drug diction have never been investigated in juvenile rats yet. For this very reason, we conducted a arge-scale behavioral comparison of adult and adolescent rats with the aim of dissecting their espective behavioral traits and vulnerabilities to drug addiction. We first have shown that juvenile rats exhibited an enhanced motor impulsivity, and a loss of control over reward seeking assessed by a persistent reward taking despite adverse consequences mild electric footshocks]. We also report that juvenile rats displayed a higher anxiety profile, ind we discuss why these behaviors might represent key underpinning mechanisms leading to an enhanced vulnerability to drug abuse. Meanwhile, we collected clear cut observations that do not support such an interpretation. In Articular, juvenile and adult rats displayed identical novelty-induced habituation and preference at are considered to represent two potent predictors of cocaine initiation and compulsive intake, "pre strikingly, juvenile rats were less attracted by cues predicting reward in a Pavlovian utoshaping task, suggesting a lower propensity for cues or context to trigger the reinstatement of a^previously extinguished reward seeking behavior. Finally, using a paradigm assessing schedule- ciuced polydipsia, juvenile and adult rats exhibited similar compulsive drinking, under control conditions and following a chronic cocaine treatment as well. Hence, these observations call for a cautious interpretation of adolescent vulnerability to drug use. In particular, we underlined that even the most compulsive young rats did not consume ärger amounts of cocaine than adults, nor exhibited larger efforts in a cue-induced relapse aradigm, despite a transient increased motivation for lever-pressing. And further, despite a higher ensitivity to the behavioral effects of cocaine, juvenile rats did not differ from adults in their ropensity to constantly prefer saccharin over cocaine in a discrete-choice procedure, even after a ?'Id chronic stress procedure. Altogether, our results shape an objective overview of the juvenile rats' behavior in relation to oth drug and non-drug rewards, suggesting a heterogeneous and task-specific profile. Despite elements potentially underlying a real risk for substance use, adolescent rats do not exhibit a ehavioral repertoire suggesting increased vulnerability for compulsive drug abuse. Our conclusions strongly encourage deeper neurobiological investigations of the developing brain, and also open a debate on a possible overestimation of juvenile rats' and teenager's risk to develop aladaptive behaviors and drug addiction. - L'addiction aux drogues est une pathologie d'origine multifactorielle, à laquelle certains individus sont plus vulnérables que d'autres. De nombreuses études cliniques et épidémiologiques suggèrent une consommation excessive de drogues pendant l'adolescence, et plusieurs explications ont été avancées pour justifier cette tendance, parmi lesquelles on note une augmentation de la recherche de sensation, une impulsivité plus marquée et une plus forte influence de l'entourage. Le rat juvénile présente de nombreuses caractéristiques développementales similaires à l'adolescence humaine. En revanche, la vulnérabilité des rats juvéniles à l'abus de drogue est encore sujette à caution. En effet, peu d'études ont directement comparé des traits de comportements pouvant refléter un accroissement du risque d'abus chez les rats juvéniles par comparaison aux rats adultes. En outre, certaines caractéristiques fondamentales de l'addiction chez l'homme n'ont pas encore été étudiées chez le rat adolescent. Ce travail de thèse s'est donc donné pour objectif de comparer le comportement de rats adultes vis-à-vis de celui de rats adolescents, afin d'évaluer dans quelle mesure ces derniers seraient plus vulnérables à l'abus de drogues. Nos résultats indiquent que les rats juvéniles présentent une augmentation des comportements impulsifs, ainsi qu'une plus grande persistance à rechercher de manière compulsive une récompense en dépit de légers chocs électriques. Les rats juvéniles présentent également un profil anxieux plus élevé, ce qui peut constituer une autre source de vulnérabilité. Cependant, certaines caractéristiques comportementales ne suggèrent pas de vulnérabilité chez les rats juvéniles. Aucune différence entre rats adultes et adolescents n'a été trouvée pour l'habituation et la préférence pour la nouveauté, deux traits prédisant l'initiation et la prise compulsive de drogue. De plus, nous avons montré que les rats adolescents attribuent moins d'intérêt à des stimuli prédisant la disponibilité d'une récompense, suggérant une vulnérabilité plus faible à la rechute induite par les stimuli associés à la prise de drogue. Une étude complémentaire des comportements compulsifs indique une absence de différence entre rats adultes et adolescents, à la fois en condition basale ou après un traitement chronique à la cocaïne. L'étude des comportements de prise de drogue ne va pas non plus dans le sens d'une vulnérabilité des rats adolescents. Bien que les rats compulsifs sélectionnés pendant la période juvénile présentent une plus grande motivation à prendre de la cocaïne, ils ne diffèrent ni dans la quantité de cocaïne consommée, ni dans la rechute induite par les stimuli environnementaux. En dépit d'une sensibilisation comportementale plus importante, les rats adolescents présentent la même préférence que les adultes face à un choix entre une drogue et une récompense alternative, suggérant une résilience à la cocaïne comparable à celle des adultes. Enfin, cette résilience pour la cocaïne n'est pas affectée par un stress chronique lors de l'adolescence. En résumé, cette étude dresse un regard objectif sur les comportements en lien avec une vulnérabilité à l'abus de drogues chez le rat juvénile, suggérant que celle-ci est hétérogène et spécifique au protocole utilisé. En dépit de certains éléments de vulnérabilité, les rats adolescents ne présentent pas d'attirance excessive pour la cocaïne, ni de prédisposition à la consommation compulsive de cette drogue. L'ensemble de ces éléments pourra constituer une base solide pour l'investigation neurobiologique du cerveau en développement, et ouvre un débat sur une possible surestimation de la vulnérabilité des rats juvéniles et de leurs homologues humains aux pathologies psychiatriques telles que l'addiction aux drogues.

Canakinumab (Acz885) Relieves Pain And Controls Inflammation Rapidly In Patients With Difficult-To-Treat Gouty Arthritis : Comparison With Triamcinolone Acetonide

Background : Monosodium urate (MSU) crystals stimulate the productionof interleukin-1b (IL-1b), a potent inflammatory cytokine. Targeted IL-1b blockade with canakinumab, a fully human monoclonal anti-IL-1b antibody, is a novel treatment for gouty arthritis. Its effects on pain and inflammation in acute gouty arthritis flares were compared with triamcinolone acetonide (TA). TA has been shown to be effective in the treatment of acute gouty arthritis flares.Methods : This was an 8-week, dose-ranging, multicenter, blinded, active-controlled trial. Patients _18 to _80 years with an acute gouty arthritis flare, refractory to or contraindicated to NSAIDs and/or colchicine were randomized to one subcutaneous dose of canakinumab (10, 25, 50, 90, or 150 mg; n¼143) or one intramuscular dose of TA (40 mg; n¼57). Primary outcome was pain intensity at 72 hours post dose on VAS scale (0-100 mm). Secondary outcomes included Creactive protein (CRP), serum amyloid A (SAA), and physician's assessment of tenderness, swelling and erythema of target joint at 72 hours, 7 days, 4 and 8-weeks post dose.Results : 191/200 patients completed the study. Canakinumab showed a statistically significant dose response at 72 hours. The 150mg dose group reached superior pain relief compared to TA group starting from 24 hours as previously reported. At 72 hours post dose, 78% of canakinumab 150mg treated patients achieved _75% and 96% achieved _50% reduction in pain from baseline. In contrast, 45% and 61% of patients treated with TA achieved _75% and _50% pain reduction, respectively. Median CRP/SAA levels were normalized by Day 7 for all canakinumab doses above 10mg and remained below the upper limit of normal [(ULN): CRP 3.0 mg/L; SAA 6.7 mg/L)] for rest of the study. In TA group, median CRP levels remained above the ULN throughout the study while median SAA levels decreased below ULN only 28 days after first dose. At 72 hours post dose, canakinumab 150mg group was 3.2 (95% CI, 1.27-7.89) times more likely to have less joint tenderness and 2.7 (95% CI, 1.09-6.5) times more likely to have less joint swelling than TA group (p<0.05). At 72 hours post dose, erythema disappeared in 74.1% of patients receiving canakinumab150mg and 69.6% of patients receiving TA. At 7 days post dose, erythema was absent in 96.3% of canakinumab 150mg treated patients vs. 83.9% of patients receiving TA. The overall incidence of AEs was similar for canakinumab (41%) and triamcinolone acetonide (42%). Serious AEs (canakinumab treatment groups n¼4, TA n¼1) were not considered treatment-related by investigators. No discontinuationsdue to AEs occurred.Conclusions : Canakinumab 150mg provided superior pain relief compared to TA for acute flares in difficult-to-treat gouty arthritis patients. Canakinumab provided rapid normalization of markers of inflammation accompanied by reduction of clinical signs and symptoms of inflammation.Disclosure statement : U.A., V.M., D.R. and P.S. are shareholders and employees of Novartis Pharma AG. A.P. has received research support from Novartis Pharma AG. N.S. has received research support from and acts as a consultant for Novartis Pharmaceuticals Corporation, has served on advisory boards for Novartis, Takeda, Savient, URL Pharma and Enzyme Rx, and is/has been a member of a speakers' bureau for Takeda. A.S. has received consultancy fees from Novartis Pharma AG, Abbott, Wyeth, UCB, Roche, MSD, Pfizer, Essex and Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Multifocal epithelial tumors and field cancerization from loss of mesenchymal CSL signaling.

It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.