G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

Role of dietary carbohydrates and macronutrients in the pathogenesis of nonalcoholic fatty liver disease.

PURPOSE OF REVIEW: The prevalence of nonalcoholic fatty liver disease is increasing worldwide and there is strong evidence that dietary factors play a role in its pathogenesis. The present review aims to provide a better understanding of how carbohydrates and other macronutrients may affect the disease. RECENT FINDINGS: The effects of carbohydrates on the development of nonalcoholic fatty liver disease differ depending upon the carbohydrate type; high-glycemic index foods are related to increased hepatic fat in both rodents and humans. Similarly, simple carbohydrates, such as fructose, stimulate hepatic de-novo lipogenesis and decrease lipid oxidation, thus leading to increased fat deposition. The underlying mechanisms may involve the activation of transcription factors. Fat intake broadly leads to hepatic fat deposition in rodents but few data are available on humans. Both carbohydrates and fat trigger inflammatory factors, which are closely related to metabolic disorders and nonalcoholic fatty liver disease. Lifestyle interventions appear to be the most appropriate first-line treatment for nonalcoholic fatty liver disease. SUMMARY: There is strong evidence that the diet may affect the development of nonalcoholic fatty liver disease. Although simple carbohydrates are clearly shown to have deleterious effects in humans, the role of fat remains controversial. Further studies will be required to evaluate the effects of macronutrient composition on the development of nonalcoholic fatty liver disease.

Role of ATH5 in the specification of retinal ganglion cells and expression and cell compartmentalization of EFEMP1, a protein associated with Malattia Leventinese

ABSTRACT : The development of the retina is a very complex process, occurring through the progressive restriction of cell fates, from pluripotent cell populations to complex tissues and organs. In all vertebrate species analyzed so far, retinal differentiation starts with the generation of retinal ganglion cells (RGC)s. One of the documented key essential events in the specification of RGCs is the expression of ATHS, an atonal homolog encoding a bHLH transcription factor. Despite the putative role of master regulator of RGC differentiation, the mechanism of integrating its functions into a coherent program underlying the production of this subclass of retinal neurons has not yet been elucidated. By using chromatin immunoprecipitation combined with microarray (ChIP-on-chip) we have screened for ATH5 direct targets in the developing chick retina at two consecutive periods: E3.5 (stage HH22) and E6 (stage HH30), covering the stages of progenitor proliferation, neuroepithelium patterning, RGC specification, cell cycle exit and early neuronal differentiation. In parallel, complementary analysis with Affymetrix expression microarrays was conducted. We compared RGCs versus retina to see if the targets correspond to genes preferentially expressed in RGCs. We also precociously overexpressed ATH5 in the retina of individual embryo, and contralateral retina vas used as a control. Our integrated approach allowed us to establish a compendium of ATH5-targets and enabled us to position ATH5 in the transcription network underlying neurogenesis in the retina. Malattia Leventinese (ML) is an autosomal, dominant retinal dystrophy characterized by extracellular, amorphous deposits known as drusen, between the retinal pigment epithelium (RPE) and Bruch's membrane. On the genetic level, it has been associated with a single missense mutation (R345W) in a widely expressed gene with unknown function called EFEMP1. We determined expression patterns of the EFEMP1 gene in normal and ML human retinas. Our data shown that the upregulation of EFEMP1 is not specific to ML eye, except for the region of the ciliary body. We also analyzed the cell compartmentalization of different versions of the protein (both wild type and mutant). Our studies indicate that both abnormal expression of the EFEMP1 gene and mutation and accumulation of EFEMP 1 protein (inside or outside the cells) might contribute to the ML pathology. Résumé : 1er partie : L'ontogenèse de la rétine est un processus complexe au cours duquel des cellules progénitrices sont engagée, par vagues successives, dans des lignées où elles vont d'abord être déterminées puis vont se différencier pour finalement construire un tissu rétinien composé de cinq classes de neurones (les photorécepteurs, les cellules horizontales, bipolaires, amacrines et ganglionnaires) et d'une seule de cellules gliales (les cellules de Muller). Chez tous les vertébrés, la neurogenèse rétinienne est d'abord marquée par la production des cellules ganglionnaires (RGCs). La production de cette classe de neurone est liée à l'expression du gène ATH5 qui est un homologue du gène atonal chez la Drosophile et qui code pour un facteur de transcription de la famille des protéines basic Helix-Loop-Helix (bHLH). Malgré le rôle central que joue ATH5 dans la production des RGCs, le mécanisme qui intègre la fonction de cette protéine dans le programme de détermination neuronale et ceci en relation avec le développement de la rétine n'est pas encore élucidé. Grâce à une technologie qui permet de combiner la sélection de fragments de chromatine liant ATH5 et la recherche de séquences grâce à des puces d'ADN non-codants (ChIP-on-chip), nous avons recherché des cibles potentielles de la protéine ATH5 dans la rétine en développement. Nous avons conduit cette recherche à deux stades de développement de manière à englober la phase de prolifération cellulaire, la détermination des RGCs, la sortie du cycle cellulaire ainsi que les premières étapes de la différentiation de ces neurones. Des expériences complémentaires nous ont permis de définir les patrons d'expression des gènes sélectionnés ainsi que l'activité promotrice des éléments de régulation identifiés lors de notre criblage. Ces approches expérimentales diverses et complémentaires nous ont permis de répertorier des gènes cibles de la protéine ATH5 et d'établir ainsi des liens fonctionnels entre des voies métaboliques dont nous ne soupçonnions pas jusqu'alors qu'elles puissent être associées à la production d'une classe de neurones centraux. 2ème partie : Malattia Leventinese (ML) est une maladie génétique qui engendre une dystrophie de la rétine. Elle se caractérise par l'accumulation de dépôt amorphe entre l'épithélium pigmentaire et la membrane de Bruch et connu sous le nom de drusen. Cette maladie est liée à une simple mutation non-sens (R345W) dans un gène dénommé EFEMP1 qui est exprimé dans de nombreux tissus mais dont la fonction reste mal définie. Une étude détaillée de l'expression de ce gène dans des rétines humaines a révélé une expression à un niveau élevé du gène EFEMP1 dans divers tissus de l'oeil ML mais également dans des yeux contrôles. Alors que l'accumulation d'ARN messager EFEMP1 dans les cellules de l'épithélium pigmentaire n'est pas spécifique à ML, l'expression de ce gène dans le corps cilié n'a été observée que dans l'oeil ML. Nous avons également comparé la sécrétion de la protéine sauvage avec celle porteuse de la mutation. En résumé, notre étude révèle que le niveau élevé d'expression du gène EFEMP1 ainsi que l'accumulation de la protéine dans certains compartiments cellulaires pourraient contribuer au développement de pathologies rétiniennes liées à ML.

Cannabis dependence in Swiss adolescents: An exploration of the role of anxiety, coping styles and psychosocial difficulties

This naturalistic cross-sectional study explores how and to what extent cannabis dependence was associated with intrapersonal aspects (anxiety, coping styles) and interpersonal aspects of adolescent functioning (school status, family relationships, peer relationships, social life). A convenience sample of 110 adolescents (aged 12 to 19) was recruited and subdivided into two groups (38 with a cannabis dependence and 72 nondependent) according to DSM-IV-TR criteria for cannabis dependence. Participants completed the State-Trait Anxiety Inventory (STAI-Y), the Coping Across Situations Questionnaire (CASQ), and the Adolescent Drug Abuse Diagnosis (ADAD) interview investigating psychosocial and interpersonal problems in an adolescent's life. Factors associated with cannabis dependence were explored with logistic regression analyses. The results indicated that severity of problems in social life and peer relationships (OR = 1.68, 95% CI = 1.21 - 2.33) and avoidantcoping (OR = 4.22, 95% CI = 1.01 - 17.73) were the only discriminatory factors for cannabis dependence. This model correctly classified 84.5% of the adolescents. These findings are partially consistent with the "self-medication hypothesis" and underlined the importance of peer relationships and dysfunctional coping strategies in cannabis dependence in adolescence. Limitations of the study and implications for clinical work with adolescents are discussed.

The role of potassium in inflammasome activation by bacteria.

Many Gram-negative bacteria possess a type III secretion system (TTSS( paragraph sign)) that can activate the NLRC4 inflammasome, process caspase-1 and lead to secretion of mature IL-1beta. This is dependent on the presence of intracellular flagellin. Previous reports have suggested that this activation is independent of extracellular K(+) and not accompanied by leakage of K(+) from the cell, in contrast to activation of the NLRP3 inflammasome. However, non-flagellated strains of Pseudomonas aeruginosa are able to activate NLRC4, suggesting that formation of a pore in the cell membrane by the TTSS apparatus may be sufficient for inflammasome activation. Thus, we set out to determine if extracellular K(+) influenced P. aeruginosa inflammasome activation. We found that raising extracellular K(+) prevented TTSS NLRC4 activation by the non-flagellated P. aeruginosa strain PA103DeltaUDeltaT at concentrations above 90 mm, higher than those reported to inhibit NLRP3 activation. Infection was accompanied by efflux of K(+) from a minority of cells as determined using the K(+)-sensitive fluorophore PBFI, but no formation of a leaky pore. We obtained exactly the same results following infection with Salmonella typhimurium, previously described as independent of extracellular K(+). The inhibitory effect of raised extracellular K(+) on NLRC4 activation thus reflects a requirement for a decrease in intracellular K(+) for this inflammasome component as well as that described for NLRP3.

Role of vitreoretinal surgery in maximizing treatment outcome following complications after proton therapy for uveal melanoma.

L'utilisation de faisceaux de protons accélérés dans le traitement des mélanomes de l'uvée a été utilisée pour la première fois en Suisse (et par ailleurs en Europe) en 1984. Depuis, la protonthérapie a constamment évolué avec des logiciels toujours plus performants et précis pour devenir à l'heure actuelle le traitement de référence pour ce type de tumeurs. Ainsi, jusqu'à ce jour, l'Institut Paul Scherrer à Villigen a traité plus de 7000 cas de tumeurs oculaires. Mais la protonthérapie, aussi efficace soit-elle avec un taux de guérison de plus de 98%, comporte malheureusement un certain nom bre d'effets secondaires et indésirables pouvant parfois mener le patient jusqu'à l'énucléation secondai re. De la simple dermatite actinique à l'hémorragie intravitréenne massive, les complications induites sont pour la plupart bien connues et documentées mais leurs prises en charge, notamment sur un organe préalablement irradié diffèrent. Alors que nous avons beaucoup de recul sur la protonthérapie, la gestion de ses complications reste propre à chaque centre de soin et n'est que très peu documentée. Les complications majeures de la protonthérapie qui ont nécessité une prise en charge par le chirurgien vitrorétinien représentent souvent un défi majeur. Bien que rares, puisqu'elles ne représentent que 2% de notre collectif, celles-ci peuvent avoir de lourdes conséquences. Pa r exemple, une hémorragie intravitréenne massive, complication la plus fréquente dans notre série, compromet l'observation de la tumeur au fond d'oeil et empêche le bon suivi oncologique. La chirurgie vitrorétinienne a alors pour mission, de restaurer la transparence des milieux, élément indispensable à l'ophtalmologue pour le suivi clinique, iconographique et radiologique des mélanomes de l'uvée. Secondairement, cette chirurgie permet parfois d'augmenter l'acuité visuelle de l'oeil malade. La chirurgie vitrorétinienne est un précieux atout pour l'oncologue et permet d'éviter une énucléation secondaire. Elle participe ainsi à la prise en charge globale du patient atteint de mélanome de l'uvée.

Hypotensive effect of human factor XII active fragment in conscious normotensive rats: role of bradykinin.

The active fragment derived from factor XII (factor XIIf) was purified from human plasma and administered intravenously to normotensive conscious rats. Factor XIIf-mediated hypotension was dose-dependent and augmented by pretreatment with captopril, an inhibitor of the bradykinin-processing enzyme kininase II. These results therefore suggest that factor XIIf-mediated hypotension is due to the formation of bradykinin.

A Pneumocystis jirovecii pneumonia outbreak in a single kidney-transplant center: role of cytomegalovirus co-infection.

Pneumocystis jirovecii pneumonia (PCP) and cytomegalovirus (CMV) infection represent possible complications of medical immunosuppression. Between 2005 and 2010, non-human immunodeficiency virus (HIV) PCP patients admitted to a nephrology unit were analyzed for outcome, CMV comorbidity, and patient-to-patient contacts prior to PCP. In contrast to 2002-2004 (no cases) and 2008-2010 (10 cases), a PCP outbreak of 29 kidney-transplant recipients and one patient with anti-glomerular basement membrane disease occurred between 2005 and 2007. None of the patients were on PCP chemoprophylaxis. In four PCP patients, the genotyping data of bronchoalveolar lavage specimen showed an identical Pneumocystis strain. PCP cases had a higher incidence of CMV infection (12 of 30 PCP patients) and CMV disease (four patients) when compared to matched PCP-free controls (p < 0.05). Cotrimoxazole and, if applicable, ganciclovir were started 2.0 ± 4.0 days following admission, and immunosuppressive medication was reduced. In-hospital mortality was 10% and the three-year mortality was 20%. CMV co-infection did not affect mortality. CMV co-infection more frequently occurred during a cluster outbreak of non-HIV PCP in comparison to PCP-free controls. Here, CMV awareness and specific therapy of both CMV infection and PCP led to a comparatively favorable patient outcome. The role of patient isolation should be further investigated in incident non-HIV PCP.

Tasosartan, enoltasosartan, and angiotensin II receptor blockade: the confounding role of protein binding.

Tasosartan is a long-acting angiotensin II (AngII) receptor blocker. Its long duration of action has been attributed to its active metabolite enoltasosartan. In this study we evaluated the relative contribution of tasosartan and enoltasosartan to the overall pharmacological effect of tasosartan. AngII receptor blockade effect of single doses of tasosartan (100 mg p.o. and 50 mg i.v) and enoltasosartan (2.5 mg i.v.) were compared in 12 healthy subjects in a randomized, double blind, three-period crossover study using two approaches: the in vivo blood pressure response to exogenous AngII and an ex vivo AngII radioreceptor assay. Tasosartan induced a rapid and sustained blockade of AngII subtype-1 (AT1) receptors. In vivo, tasosartan (p.o. or i.v.) blocked by 80% AT1 receptors 1 to 2 h after drug administration and still had a 40% effect at 32 h. In vitro, the blockade was estimated to be 90% at 2 h and 20% at 32 h. In contrast, the blockade induced by enoltasosartan was markedly delayed and hardly reached 60 to 70% despite the i.v. administration and high plasma levels. In vitro, the AT1 antagonistic effect of enoltasosartan was markedly influenced by the presence of plasma proteins, leading to a decrease in its affinity for the receptor and a slower receptor association rate. The early effect of tasosartan is due mainly to tasosartan itself with little if any contribution of enoltasosartan. The antagonistic effect of enoltasosartan appears later. The delayed in vivo blockade effect observed for enoltasosartan appears to be due to a high and tight protein binding and a slow dissociation process from the carrier.

Définir le risque du carcinome spinocellulaire: rôle de la clinique et du rapport anatomopathologique [Risk of cutaneous squamous cell carcinomas: the role of clinical and pathological reports].

The history of most cutaneous squamous cell carcinomas (CSCC) is limited to the skin. However, about 4% of these malignancies are at risk of metastasis and can be life-threatening. This risk is determined by clinical and histological elements which are individually recognized, but so far staging systems allow us neither to assess a risk score, nor to adopt a standardized therapeutical approach. This article reviews prognostic factors for CSCC, and underlines the need for the clinician to have all clinical and histological elements available, in order to try to define the best therapeutical strategy for each case, following up-to-date recommendations.

Oestrogen replacement treatment and the risk of endometrial cancer : an assessment of the role of covariates

The relationship between oestrogen replacement treatment and the risk of endometrial cancer was analysed in a case-control study of 158 histologically confirmed incident cases below the age of 75 and 468 controls in hospital for acute, non-neoplastic, non-hormone-related conditions conducted in the Swiss Canton of Vaud in 1988-1992. Overall, 60 (38%) cases vs. 93 (20%) controls had ever used oestrogen replacement treatment: the corresponding multiple logistic regression relative risk (RR) was 2.7 (95% confidence interval, CI: 1.7-4.1). The risk was directly related to duration of use, and rose to 5.1 (95% CI: 2.7-9.8) for > 5 year-use. The RR was still significantly elevated 10 or more years after stopping use (RR = 2.3, 95% CI: 1.2-4.5). When the role of covariates was considered, a significant interaction was observed with body mass index (RR for long-term oestrogen use = 6.0 for lean or normal weight women vs. 2.4 for overweight women). There was also a hint of a negative interaction with oral contraceptive (OC) use, since the RR for oestrogens was higher (or restricted) to women who had never used OC (RR = 5.4, for long-term oestrogen use), as compared with those who had used OC, who showed no significant evidence of association with oestrogens (RR = 0.9 for long-term use). There was no significant interaction with cigarette smoking. Thus, this study confirms the presence of a strong association between oestrogen replacement treatment and endometrial cancer risk, since in the late 1980s or early 1990s about 25% of cases could be attributed to oestrogen replacement treatment in this Swiss population. Further, it confirms the presence of significant negative interactions of oestrogen use with obesity, and, possibly, with OC as well.

THE ROLE OF SPINDLES FOR SLEEP: MANIPULATING OSCILLATORY ACTIVITY IN THE THALAMOCORTICAL NETWORK

Modern urban lifestyle encourages the prolongation of wakefulness, leaving less and less time for sleep. Although the exact functions of sleep remain one of the biggest mysteries in neuroscience, the society is well aware of the negative consequences of sleep loss on human physical and mental health and performance. Enhancing sleep's recuperative functions might allow shortening sleep duration while preserving the beneficial effects of sleep. During sleep, brain activity oscillates across a continuum of frequencies. Individual oscillations have been suggested to underlie distinct functions for sleep and cognition. Gaining control about individual oscillations might allow boosting their specific functions. Sleep spindles are 11 - 15 Hz oscillations characteristic for light non-rapid-eye-movement sleep (NREMS) and have been proposed to play a role in memory consolidation and sleep protection against environmental stimuli. The reticular thalamic nucleus (nRt) has been identified as the major pacemaker of spindles. Intrinsic oscillatory burst discharge in nRt neurons, arising from the interplay of low-threshold (T-type) Ca2+ channels (T channels) and small conductance type 2 (SK2) K+ channels (SK2 channels), underlies this pacemaking function. In the present work we investigated the impact of altered nRt bursting on spindle generation during sleep by studying mutant mice for SK2 channels and for CaV3.3 channels, a subtype of T channels. Using in vitro electrophysiology I showed that nRt bursting was abolished in CaV3.3 knock out (CaV3.3 KO) mice. In contrast, in SK2 channel over-expressing (SK2-OE) nRt cells, intrinsic repetitive bursting was prolonged. Compared to wildtype (WT) littermates, altered nRt burst discharge lead to weakened thalamic network oscillations in vitro in CaV3.3 KO mice, while oscillatory activity was prolonged in SK2-OE mice. Sleep electroencephalographic recordings in CaV3.3 KO and SK2-OE mice revealed that reduced or potentiated nRt bursting respectively weakened or prolonged sleep spindle activity at the NREMS - REMS transition. Furthermore, SK2-OE mice showed more consolidated NREMS and increased arousal thresholds, two correlates of good sleep quality. This thesis work suggests that CaV3.3 and SK2 channels may be targeted in order to modulate sleep spindle activity. Furthermore, it proposes a novel function for spindles in NREMS consolidation. Finally, it provides evidence that sleep quality may be improved by promoting spindle activity, thereby supporting the hypothesis that sleep quality can be enhanced by modulating oscillatory activity in the brain. Le style de vie moderne favorise la prolongation de l'éveil, laissant de moins en moins de temps pour le sommeil. Même si le rôle exact du sommeil reste un des plus grands mystères des neurosciences, la société est bien consciente des conséquences négatives que provoque un manque de sommeil, à la fois sur le plan de la santé physique et mentale ainsi qu'au niveau des performances cognitives. Augmenter les fonctions récupératrices du sommeil pourrait permettre de raccourcir la durée du sommeil tout en en conservant les effets bénéfiques. Durant le sommeil, on observe des oscillations à travers un continuum de fréquences. Il a été proposé que chaque oscillation pourrait être à l'origine de fonctions spécifiques pour le sommeil et la cognition. Pouvoir de contrôler les oscillations individuelles permettrait d'augmenter leurs fonctions respectives. Les fuseaux sont des oscillations de 11 à 15 Hz caractéristiques du sommeil à ondes lentes léger et il a été suggéré qu'elles jouent un rôle majeur pour la consolidation de la mémoire ainsi que dans la protection du sommeil contre les stimuli environnementaux. Le nucleus réticulaire du thalamus (nRt) a été identifié en tant que générateur de rythme des fuseaux. Les bouffées oscillatoires intrinsèques des neurones du nRt, provenant de l'interaction de canaux calciques à bas seuil de type T (canaux T) et de canaux potassiques à faible conductance de type 2 (canaux SK2), sont à l'origine de la fonction de générateur de rythme. Dans ce travail, j'ai étudié l'impact de la modulation de bouffées de nRT sur la génération des fuseaux pendant le sommeil en investiguant des souris génétiquement modifiées pour les canaux SK2 et les canaux CaV3.3, un sous-type de canaux T. En utilisant l'électrophysiologie in vitro j'ai démontré que les bouffées du nRT étaient abolies dans les souris knock-out du type CaV3.3 (CaV3.3 KO). D'autre part, dans les cellules nRT sur-exprimant les canaux SK2 (SK2-OE), les bouffées oscillatoires intrinsèques étaient prolongées. Par rapport aux souris wild type, les souris CaV3.3 KO ont montré un affaiblissement des oscillations thalamiques en réponse à un changement des bouffées de nRT, alors que l'activité oscillatoire était prolongée dans les souris SK2-OE. Des enregistrements EEG du sommeil dans des souris de type CaV3.3 KO et SK2-OE ont révélé qu'une réduction ou augmentation des bouffées nRT ont respectivement affaibli ou prolongé l'activité des fuseaux durant les transitions du sommeil à ondes lentes au sommeil paradoxal. De plus, les souris SK2-OE ont montré des signes de consolidation du sommeil à ondes lentes et un seuil augmenté pour le réveil, deux mesures qui corrèlent avec une bonne qualité du sommeil. Le travail de cette thèse propose que les canaux CaV3.3 et SK2 pourrait être ciblés pour moduler l'activité des fuseaux. De plus, je propose une fonction nouvelle pour les fuseaux dans la consolidation du sommeil à ondes lentes. Finalement je suggère que la qualité du sommeil peut être améliorée en promouvant l'activité des fuseaux, soutenant ainsi l'idée que la qualité du sommeil peut être améliorée en modulant l'activité oscillatoire dans le cerveau.