Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

IL-4 rapidly produced by V beta 4 V alpha 8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice.

BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

Preparation and analysis of fetal liver extracts.

The aim of this work is to describe the techniques that have been used for preparation and analysis of whole fetal liver extracts destined for in utero transplantation. Nine fetal livers between 12 and 17 weeks of gestation were prepared: cell counts and assessment of the hematopoietic cell viability were performed on cell suspensions. Hepatocytes represented 40 to 80% of the whole cell population. The remaining cells were constituted by hematopoietic cells (mainly erythroblasts), as well as by endothelial cells. The latter expressed CD34 on their surface, interfering with the assessment of CD34+ hematopoietic cells by flow cytometry. Direct visual morphologic control using alkaline phosphatase anti-alkaline phosphatase techniques was needed to differentiate hematopoietic from extra-hematopoietic CD34+ cells. Between 3.0 and 34.6 x 10(6) CD34+ viable hematopoietic cells were collected per fetal liver. Adequate differentiation of these cells into burst-forming units erythroid (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), and colony-forming units granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) has been shown for each sample in clonogeneic cultures. In conclusion, fetal liver is a potential source of hematopoietic stem cells. Their numeration, based on the presence of CD34, is hampered by the expression of this antigen on other cells contained in the liver cell extract, in particular endothelial cells.

Electroencephalogram changes during inhalation with nitric oxide in the pediatric intensive care patient--a preliminary report.

OBJECTIVES: Although endogenous nitric oxide (NO) is an excitatory mediator in the central nervous system, inhaled NO is not considered to cause neurologic side effects because of its short half-life. This study was motivated by a recent case report about neurologic symptoms and our own observation of severe electroencephalogram (EEG) abnormalities during NO inhalation. DESIGN: Blind, retrospective analyses of EEGs which were registered before, during, and after NO inhalation. EEG was classified in a 5-point rating system by an independent electroencephalographer who was blinded to the patients' clinical histories. Comparisons were made with the previous evaluation documented at recording. Other EEG-influencing parameters such as oxygen saturation, hemodynamics, electrolytes, and pH were evaluated. SETTING: Pediatric intensive care unit of a tertiary care university children's hospital. PATIENTS: Eleven ventilated, long-term paralyzed, sedated children (1 mo to 14 yrs) who had EEG or clinical assessment before NO treatment and EEG during NO inhalation. They were divided into two groups according to the NO-indication (e.g., congenital heart defect, acute respiratory distress syndrome). MEASUREMENTS AND MAIN RESULTS: All 11 patients had an abnormal EEG during NO inhalation. EEG-controls without NO showed remarkable improvement. EEG abnormalities were background slowing, low voltage, suppression burst (n = 2), and sharp waves (n = 2) independent of patients' age, NO-indication, and other EEG-influencing parameters. CONCLUSIONS: These preliminary data suggest the occurrence of EEG-abnormalities after application of inhaled NO in critically ill children. We found no correlation with other potential EEG-influencing parameters, although clinical state, medication, or hypoxemia might contribute. Comprehensive, prospective, clinical assessment regarding a causal relationship between NO-inhalation and EEG-abnormalities and their clinical importance is needed.

Signalling of Arabidopsis thaliana response to Pieris brassicae eggs shares similarities with PAMP-triggered immunity.

Insect egg deposition activates plant defence, but very little is known about signalling events that control this response. In Arabidopsis thaliana, oviposition by Pieris brassicae triggers salicylic acid (SA) accumulation and induces the expression of defence genes. This is similar to the recognition of pathogen-associated molecular patterns (PAMPs), which are involved in PAMP-triggered immunity (PTI). Here, the involvement of known signalling components of PTI in response to oviposition was studied. Treatment with P. brassicae egg extract caused a rapid induction of early PAMP-responsive genes. In addition, expression of the defence gene PR-1 required EDS1, SID2, and, partially, NPR1, thus implicating the SA pathway downstream of egg recognition. PR-1 expression was triggered by a non-polar fraction of egg extract and by an oxidative burst modulated through the antagonistic action of EDS1 and NUDT7, but which did not depend on the NADPH oxidases RBOHD and RBOHF. Searching for receptors of egg-derived elicitors, a receptor-like kinase mutant, lecRK-I.8, was identified which shows a much reduced induction of PR-1 in response to egg extract treatment. These results demonstrate the importance of the SA pathway in response to egg-derived elicitor(s) and unravel intriguing similarities between the detection of insect eggs and PTI in Arabidopsis.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

Reactivation of transcription from a vaccinia virus early promoter late in infection.

We have studied the kinetics of RNA synthesis from the vaccinia virus 7,500-molecular-weight gene (7.5K gene) which is regulated by early and late promoters arranged in tandem. Unexpectedly, after a first burst of RNA synthesis early in infection, transcription was reactivated late in infection. Reactivation was not dependent on the location of the promoter in the genome or on the presence of the upstream late regulatory sequences. The mRNA synthesized from the reactivated promoter in the late phase had the same 5' and 3' ends as the molecules transcribed in the early phase. Interestingly, these molecules were efficiently translated despite the absence of the poly(A) leader characteristic of late mRNAs. Reactivation appears to be dependent on virus assembly since it is prevented by rifampin, a specific inhibitor of morphogenesis. Finally, analysis of various other early genes showed that reactivation is not unique to the 7.5K early promoter.

Traitement pharmacologique de l'état de mal réfractaire [Drug treatment of refractory status epilepticus]

Status epilepticus (SE) refractory to benzodiazepines and other antiepileptic agents is managed with intravenous anesthetic compounds, such as thiopental, propofol or midazolam. These drugs display quite different pharmacodynamic and pharmacokinetic properties, but have not been prospectively compared to date. Their use is clearly advocated for the treatment of generalized convulsive SE, whereas partial-complex, or absence SE are generally managed less aggressively, in consideration of their better prognosis. The most important aspect seems to be related to the correct use of these anesthetics in the right context, rather than the choice of one specific compound. An electroencephalographic burst-suppression should be targeted for about 24hour, before progressive weaning of the dosage under EEG monitoring. If this approach proves unsuccessful, the use of other drugs, including inhalational anesthetics, has been described.

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.

" Leishmania major " metacaspase in programmed cell death

Abstract: The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a programmed cell death (PCD) when differentiating into infectious stages and under a variety of stress conditions. In mammalian cells, the principal effector molecules of PCD or apoptosis are caspases. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the L. major genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. Metacaspases are also present in other protozoan parasites such as Trypanosoma and Plasmodium species and are not present in mammalian cells, which make them a possible drug target for the treatment of the parasitic diseases they cause. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we evaluated the functional heterologous complementation of a S. cerevisiae ycal null mutant with the L. major metacaspase (LmjMCA} in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however, LmjMCA did not exhibit any activity with caspase substrates. In contrast, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations and addition of the arginal inhibitor leupeptin. In order to identify the L. major proteins that may function as substrates, inhibitors, or may bind and recruit LmjMCA, a yeast two-hybrid screening with cDNA libraries from different life cycle stages of the parasite was conducted. Proteins putatively involved in PCD were identified as interacting with LmjMCA, however, the interaction of LmjMCA with proteins involved in other physiological processes such as vesicle transport, suggests that LmjMCA could have additional roles in the different life cycle stages of the parasite. Résumé: Plusieurs caractéristiques morphologiques et biochimiques rappelant la mort cellulaire programmée ont été identifiées dans les stades infectieux et sous des conditions de stress, chez le parasite protozoaire humain, Leishmania major. Dans les cellules de mammifères, les caspases sont les molécules effectrices principales impliquées dans la mort cellulaire programmée et l'apoptose. Bien qu'une activité caspase ait été retrouvée dans des parasites en mon` cellulaire, le génome de Leishmania ne contient aucun gène qui pourrait coder pour une caspase. À la place, on retrouve un gène unique codant pour une métacaspase. Une prédiction de la structure secondaire de la métacaspase montre que cette métacaspase a un domaine catalytique contenant la dyade histidine/cystéine présente dans les caspases et les autres métacaspases décrites chez les plantes et la levure. Les métacaspases sont aussi présentes dans d'autres parasites protozoaires tels que Trypanosome et Plasmodium, mais ne sont pas présentes dans les cellules de mammifères, ce qui en fait des cibles intéressantes pour le développement de drogue. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L. major lors de l'induction de ta mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YGA1 dans le programme de mort cellulaire chez la levure et que celte fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNc provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Résumé destiné à un large public: De nos jours, la leishmaniose est la deuxième plus importante maladie parasitaire après la malaria. Malgré les avancées accomplies dans les stratégies de contrôle, près de deux millions de nouveaux cas apparaissent chaque année. Actuellement, la principale stratégie pour faire face à ce problème épidémiologique consiste en un traitement pharmacologique des personnes infectées. Pourtant, seule une dizaine de médicaments, dont la plupart sont toxiques, est disponible pour traiter la leishmaniose et des cas de résistance émergent dans certains pays endémiques. Il devient donc urgent de mettre au point de nouveaux traitements anti-leishmaniens capables d'éliminer le parasite sans effets indésirables sur le patient. Récemment, des caractéristiques morphologiques et biochimiques de la mort cellulaire programmée (MCP) semblables au processus de l'apoptose chez les mammifères ont été décrites dans Leishmania. Cependant, des gènes codant pour des protéines similaires à celles qui sont impliquées dans l'apoptose, comme les caspases, ne se retrouvent pas dans le génome de Leishmanía major. Néanmoins, les espèces de Leishmanía, aussi bien que d'autres parasites protozoaires responsables des trypanosomiases et de la malaria, possèdent des métacaspases qui sont des protéines homologues aux caspases mais qui ne sont pas présentes chez les mammifères. C'est pourquoi, la caractérisation de la métacaspase de Leishmania (LmjMCA) ainsi que ses mécanismes d'activation pourrait être une piste d'investigation intéressante dans l'identification de nouvelles cibles thérapeutiques dans les voies de signalisation de la MCP des parasites protozoaires. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L major lors de l'induction de la mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YCA1 dans le programme de mort cellulaire chez la levure et que cette fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNe provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Resumen destinado al público en general: La leishmaniasis es la segunda enfermedad parasitaria más importante en el mundo actual. Aproximadamente 2 millones de nuevos casos ocurren cada año a pesar de los avances logrados en el desarrollo de nuevos métodos de control. El tratamiento farmacológico de las personas infectadas es actualmente la principal estrategia de control, sin embargo, menos de una decena de medicamentos se encuentran disponibles en el mercado, la mayoría de ellos son tóxicos, y ya empiezan a encontrarse parásitos resistentes en algunos países endémicos para la leishmaniasis. El desarrollo de nuevos medicamentos capaces de eliminar los parásitos sin producir efectos indeseables en los humanos, es una necesidad inminente. Recientemente, algunas de las características morfológicas y bioquímicas de la muerte celular programada (MCP) similares al proceso de la apoptosis en mamíferos, han sido descritas en parasitos de Leishmania. Sin embargo, genes que codifiquen proteínas similares a aquellas involucradas en la apoptosis, como las caspasas, no se encuentran en el genoma de Leishmania major. AI contrario, las especies de Leishmania, así como de los otros parásitos responsables de la tripanosomiasis y de la malaria, poseen metacaspases, proteínas homologas a las caspases pero que no están presentes en las células de mamíferos. La caracterización de la metacaspasa de L. major y de sus mecanismos de activación constituye, por lo tanto, un área de investigación interesante para la identificación de nuevos blancos terapéuticos en el proceso de MCP de los parásitos protozoarios. En la levadura Saccharomyces cerevisiae, la metacaspasa YCA1 ha sido descrita como implicada en la muerte de células envejecidas, células defectuosas en algunas funciones biológicas, y en células expuestas a diferentes tipos de estrés ambiental. En el presente estudio se evaluó la complementación heteróloga funcional de una levadura mutante deficiente en YCA1 con el gen de metacaspase de L. major (LmjMCA) en la MCP inducida por estrés oxidativo. Nuestros resultados muestran que la LmjMCA puede reemplazarla YCA1 en la MCP de la levadura dependiente de su actividad catalítica y que la LmjMCA se auto-procesa similar a las caspasas. Sin embargo, LmjMCA no reconoce los substratos de caspasas sino substratos con una arginina en ta posición P1. Dicha actividad enzimática fue abolida con la inducción de las mutaciones puntuales H147A y C202A en la díada catalítica de LmjMCA y con la adición de leupeptina, un inhibidor con arginina. Con el fin de identificar proteínas que pudieran funcionar como substratos, inhibidores o moléculas modificadoras de LmjMCA, se aplicó el método de doble-híbrido en levadura con bibliotecas de ADNc provenientes de diferentes estadios del ciclo de vida del parásito. Algunas proteínas potencialmente implicadas. en la MCP del parásito fueron identiñcadas interactuando con LmjMCA. La identificación de otras proteínas involucradas en transporte vesicular sugiere que la LmjMCA podría tener una función biológica adicional en los diferentes estadios del ciclo de vida dei parásito.

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.

Propagation velocity kinetics and repolarization alternans in a free-behaving sheep model of pacing-induced atrial fibrillation.

AIMS: Experimental models have reported conflicting results regarding the role of dispersion of repolarization in promoting atrial fibrillation (AF). Repolarization alternans, a beat-to-beat alternation in action potential duration, enhances dispersion of repolarization when propagation velocity is involved. METHODS AND RESULTS: In this work, original electrophysiological parameters were analysed to study AF susceptibility in a chronic sheep model of pacing-induced AF. Two pacemakers were implanted, each with a single right atrial lead. Right atrial depolarization and repolarization waves were documented at 2-week intervals. A significant and gradual decrease in the propagation velocity at all pacing rates and in the right atrial effective refractory period (ERP) was observed during the weeks of burst pacing before sustained AF developed when compared with baseline conditions. Right atrial repolarization alternans was observed, but because of the development of 2/1 atrioventricular block with far-field ventricular interference, its threshold could not be precisely measured. Non-sustained AF was not observed at baseline, but appeared during the electrical remodelling in association with a decrease in both ERP and propagation velocity. CONCLUSION: We report here on the feasibility of measuring ERP, atrial repolarization alternans, and propagation velocity kinetics and their potential in predicting susceptibility to AF in a free-behaving model of pacing-induced AF using the standard pacemaker technology.

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Comparative Study of Chemoembolization, Loadable Beads: In vitro Drug Release and Physical Properties of DC Bead and Hepasphere, Loaded with Doxorubicin and Irinotecan

Purpose To characterize in vitro the loadability, physical properties, and release of irinotecan and doxorubicin from two commercially available embolization microspheres. Materials and Methods DC Bead (500-700 μm) and Hepasphere (400-600 μm) microspheres were loaded with either doxorubicin or irinotecan solutions. Drug amount was quantified with spectrophotometry, bead elasticity was measured under compression, and bead size and loading homogeneity were assessed with microscopy image analysis. Drug release was measured over 1-week periods in saline by using a pharmacopeia flow-through method. Results Almost complete drug loading was obtained for both microsphere types and drugs. Doxorubicin-loaded DC Beads maintained their spherical shape throughout the release. In contrast, Hepaspheres showed less homogeneous doxorubicin loading and, after release, some fractured microspheres. Incomplete doxorubicin release was observed in saline over 1 week (27% ± 2 for DC beads and 18% ± 7 for Hepaspheres; P = .013). About 75% of this amount was released within 2.2 hours for both beads. For irinotecan, complete release was obtained for both types of beads, in a sustained manner over 2-3 hours for DC Beads, and in a significantly faster manner as a 7-minute burst for Hepaspheres. Conclusions The two drug-eluting microspheres could be efficiently loaded with both drugs. Incomplete doxorubicin release was attributed to strong drug-bead ionic interactions. Weaker interactions were observed with irinotecan, which led to faster drug release.