Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension.

Excessive proliferation of vascular wall cells underlies the development of elevated vascular resistance in hypoxic pulmonary hypertension (PH), but the responsible mechanisms remain unclear. Growth-promoting effects of catecholamines may contribute. Hypoxemia causes sympathoexcitation, and prolonged stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs) induces hypertrophy and hyperplasia of arterial smooth muscle cells and adventitial fibroblasts. Catecholamine trophic actions in arteries are enhanced when other conditions favoring growth or remodeling are present, e.g., injury or altered shear stress, in isolated pulmonary arteries from rats with hypoxic PH. The present study examined the hypothesis that catecholamines contribute to pulmonary vascular remodeling in vivo in hypoxic PH. Mice genetically deficient in norepinephrine and epinephrine production [dopamine beta-hydroxylase(-/-) (DBH(-/-))] or alpha(1)-ARs were examined for alterations in PH, cardiac hypertrophy, and vascular remodeling after 21 days exposure to normobaric 0.1 inspired oxygen fraction (Fi(O(2))). A decrease in the lumen area and an increase in the wall thickness of arteries were strongly inhibited in knockout mice (order of extent of inhibition: DBH(-/-) = alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-)). Distal muscularization of small arterioles was also reduced (DBH(-/-) > alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-) mice). Despite these reductions, increases in right ventricular pressure and hypertrophy were not attenuated in DBH(-/-) and alpha(1B)-AR(-/-) mice. However, hematocrit increased more in these mice, possibly as a consequence of impaired cardiovascular activation that occurs during reduction of Fi(O(2)). In contrast, in alpha(1D)-AR(-/-) mice, where hematocrit increased the same as in wild-type mice, right ventricular pressure was reduced. These data suggest that catecholamine stimulation of alpha(1B)- and alpha(1D)-ARs contributes significantly to vascular remodeling in hypoxic PH.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

Electrical neuroimaging reveals intensity-dependent activation of human cortical gustatory and somatosensory areas by electric taste.

To analyze the neural basis of electric taste we performed electrical neuroimaging analyses of event-related potentials (ERPs) recorded while participants received electrical pulses to the tongue. Pulses were presented at individual taste threshold to excite gustatory fibers selectively without concomitant excitation of trigeminal fibers and at high intensity evoking a prickling and, thus, activating trigeminal fibers. Sour, salty and metallic tastes were reported at both intensities while clear prickling was reported at high intensity only. ERPs exhibited augmented amplitudes and shorter latencies for high intensity. First activations of gustatory areas (bilateral anterior insula, medial orbitofrontal cortex) were observed at 70-80ms. Common somatosensory regions were more strongly, but not exclusively, activated at high intensity. Our data provide a comprehensive view on the dynamics of cortical processing of the gustatory and trigeminal portions of electric taste and suggest that gustatory and trigeminal afferents project to overlapping cortical areas.

A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor gamma-dependent pathway.

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

The cost-effectiveness and public health benefit of nalmefene added to psychosocial support for the reduction of alcohol consumption in alcohol-dependent patients with high/very high drinking risk levels: a Markov model.

OBJECTIVES: To determine whether nalmefene combined with psychosocial support is cost-effective compared with psychosocial support alone for reducing alcohol consumption in alcohol-dependent patients with high/very high drinking risk levels (DRLs) as defined by the WHO, and to evaluate the public health benefit of reducing harmful alcohol-attributable diseases, injuries and deaths. DESIGN: Decision modelling using Markov chains compared costs and effects over 5 years. SETTING: The analysis was from the perspective of the National Health Service (NHS) in England and Wales. PARTICIPANTS: The model considered the licensed population for nalmefene, specifically adults with both alcohol dependence and high/very high DRLs, who do not require immediate detoxification and who continue to have high/very high DRLs after initial assessment. DATA SOURCES: We modelled treatment effect using data from three clinical trials for nalmefene (ESENSE 1 (NCT00811720), ESENSE 2 (NCT00812461) and SENSE (NCT00811941)). Baseline characteristics of the model population, treatment resource utilisation and utilities were from these trials. We estimated the number of alcohol-attributable events occurring at different levels of alcohol consumption based on published epidemiological risk-relation studies. Health-related costs were from UK sources. MAIN OUTCOME MEASURES: We measured incremental cost per quality-adjusted life year (QALY) gained and number of alcohol-attributable harmful events avoided. RESULTS: Nalmefene in combination with psychosocial support had an incremental cost-effectiveness ratio (ICER) of £5204 per QALY gained, and was therefore cost-effective at the £20,000 per QALY gained decision threshold. Sensitivity analyses showed that the conclusion was robust. Nalmefene plus psychosocial support led to the avoidance of 7179 alcohol-attributable diseases/injuries and 309 deaths per 100,000 patients compared to psychosocial support alone over the course of 5 years. CONCLUSIONS: Nalmefene can be seen as a cost-effective treatment for alcohol dependence, with substantial public health benefits. TRIAL REGISTRATION NUMBERS: This cost-effectiveness analysis was developed based on data from three randomised clinical trials: ESENSE 1 (NCT00811720), ESENSE 2 (NCT00812461) and SENSE (NCT00811941).

Endoglycan mediates malignant leukocyte rolling on e-selectin and signals through SYK and the MAPK pathway

Selectins play a key role regulating leukocyte migration into tissues by mediating leukocyte tethering (capture) and rolling on inflamed endothelium and/or on adherent leukocytes or platelets. During leukocyte rolling, endothelial E- or P-selectin bind to glycoprotein ligands carrying sialyl Lewis χ (sLex) determinant. P-selectin glycoprotein ligand-1 (PSGL-1) is a common ligand for L-, P- and E-selectin, which sequentially cooperates with CD44 and E- selectin ligand-1 (ESL-1) to roll on E-selectin. During rolling on endothelial selectins, PSGL-1 and CD44 signal through Src family kinases and Syk, leading to αι_β2 integrin partial activation and slow rolling on intercellular adhesion molecule-1 (ICAM-1). Leukocyte exposure to chemokines then leads to firm adhesion. Little information is available on ligands that mediate malignant leukocyte rolling on E- selectin. We defined these ligands on U937 monoblasts by immunoadsorbtion and immunoblotting using mAb raised against CD43, CD44, PSGL-1, sLex/CLA determinants and E-selectin/IgM chimera. Immunoblotting and blot rolling assays demonstrated that PSGL-1, CD43, CD44 and a -125 kDa sLex/CLA positive ligand contribute to support E-seiectin- dependent rolling. This -125 kDa ligand is endoglycan, a member of the CD34 family of sialomucins. Endoglycan was frequently detected by flow cytometry on primary leukemia, lymphoma and multiple myeloma ceils (in -50% of cases). Endoglycan, immunopurified from U937 cells, as well as endoglycan/IgG chimera efficiently supported E-selectin dependent rolling. Membrane fractionation on sucrose gradient demonstrated that endoglycan is expressed in lipid rafts. We tested the hypothesis that it signals, like PSGL-1 and CD44, through Src kinases and the MAPK pathway. Indeed, endoglycan engagement induced Syk and ERK phosphorylation in a iipid raft-dependent manner. Syk activation was dependent on Src kinase activity. Downstream of Syk, endoglycan activated PI3K and Akt as well as Bruton's tyrosine kinase and p38 MAPK. Thus, endoglycan is a ligand for endothelial selectins which may contribute to regulate leukemia, lymphoma and multiple myeloma cell trafficking and interactions with bone marrow microenvironment. - Les sélectines contrôlent la migration tissulaire des leucocytes en assurant leur capture et leur roulement sur l'endothélium vasculaire enflammé et/ou sur des plaquettes ou des leucocytes adhérant à la paroi vasculaire. Lors du roulement leucocytaire, les sélectines endothéliales (E- et P-sélectine) se lient à des ligands porteurs du saccharide sialyl Lewis χ (sLex). PSGL-1 est un ligand commun des sélectines qui coopère avec CD44 et ESL-1 pour permettre la capture et le roulement des neutrophiles. Lorsque PSGL-1 et CD44 se lient aux sélectines endothéliales, elles induisent la phosphorylation des kinases Src et de Syk conduisant à l'activation partielle de l'intégrine aLp2 et au ralentissement des leucocytes sur les sélectines et ICAM-1. Les chimiokines induisent ensuite l'adhésion ferme des leucocytes. Les ligands des sélectines qui assurent le roulement, sur la E-sélectine, des cellules issues d'hémopathies malignes sont peu connus. Nous avons caractérisé ces ligands en les purifiant avec des anticorps dirigés contre CD43, CD44, PSGL-1, sLex/CLA et en utilisant la chimère E-sélectine/IgM. Des tests d'adhésion ont montré que PSGL-1, CD43, CD44 et une glycoprotéine de ~125 kDa soutiennent les interactions cellulaires dépendant de la E- sélectine. Le ligand de -125 kDa a été identifié comme étant l'endoglycan. Il a été détecté, par cytométrie de flux, sur les cellules leucémiques, les cellules de lymphomes ou de myélome multiple, dans ~50% des cas analysés. Sa forme membranaire, immunopurifiée, ou recombinante (endoglycan/lgG) soutient les interactions cellulaires dépendant de la E- sélectine. Nous avons montré qu'il réside dans les rafts lipidiques membranaires puis avons testé l'hypothèse que l'endoglycan, comme PSGL-1 et CD44, induit une signalisation via les kinases de type Src et la voie des MAPK. Nous avons pu observer que son engagement induit la phosphorylation de Syk et de ERK pour autant que la structure des rafts soit préservée. En aval de Syk, l'endoglycan active la PI3K, Akt, Btk et la MAPK p38. Ces résultats montrent que l'endoglycan est un ligand des sélectines endothéliales qui pourrait participer au contrôle du trafic et des interactions des cellules leucémiques, de lymphomes ou de myélomes multiples avec leur microenvironnement. - Le sang est un élément clé du fonctionnement de notre corps. La circulation sanguine permet la communication et le transfert de molécules et cellules entre divers organes. Lors d'une inflammation aiguë due à une réaction allergique, une infection ou une blessure, on observe un oedème local accompagné de rougeur, de chaleur et souvent de douleurs. Au sein des tissus enflammés, on observe des globules blancs (leucocytes) et diverses molécules inflammatoires qui attirent les leucocytes dans les tissus lésés (chimiokines). Le sang est composé de globules rouges, de plaquettes et de leucocytes spécialisés dans les défenses immunes. Pour atteindre le site d'inflammation, les leucocytes doivent quitter la circulation sanguine. Ils utilisent pour cela des molécules d'adhésion présentes à leur surface qui se lient à d'autres molécules d'adhésion de la paroi sanguine. Leurs interactions permettent aux leucocytes de rouler à la surface du vaisseau sanguin. Lorsqu'ils roulent au voisinage d'un site d'inflammation, les leucocytes sont exposés à des chimiokines qui induisent leur arrêt et les dirigent dans les tissus enflammés. Ce processus physiologique est aussi impliqué dans des pathologies telles que l'infarctus, l'artériosclérose ou la thrombose. Il peut être détourné à des fins moins louables par des cellules cancéreuses pour permettre leur dissémination (métastatisation). Dans ce travail de thèse, nous avons caractérisé une molécule d'adhésion qui soutient l'adhésion des leucocytes aux sélectines endothéliales: l'endoglycan. Nous avons observé que cette molécule d'adhésion est fréquemment exprimée par les cellules malignes de nombreuses maladies du sang comme les leucémies, les lymphomes et le myélome multiple. Nous avons également pu montrer que l'endoglycan envoie des signaux à l'intérieur des cellules malignes lorsqu'elles se lient aux sélectines endothéliales. Ces signaux pourraient jouer un rôle déterminant dans la régulation des interactions des cellules malignes avec leur microenvironnement. Elles pourraient peut-être aussi favoriser leur survie et leur prolifération.

Success of referral for alcohol dependent patients from a general hospital: predictive value of patient and process characteristics.

To assess the effectiveness of a multidisciplinary evaluation and referral process in a prospective cohort of general hospital patients with alcohol dependence. Alcohol-dependent patients were identified in the wards of the general hospital and its primary care center. They were evaluated and then referred to treatment by a multidisciplinary team; those patients who accepted to participate in this cohort study were consecutively included and followed for 6 months. Not included patients were lost for follow-up, whereas all included patients were assessed at time of inclusion, 2 and 6 months later by a research psychologist in order to collect standardized baseline patients' characteristics, process salient features and patients outcomes (defined as treatment adherence and abstinence). Multidisciplinary evaluation and therapeutic referral was feasible and effective, with a success rate of 43%for treatment adherence and 28%for abstinence at 6 months. Among patients' characteristics, predictors of success were an age over 45, not living alone, being employed and being motivated to treatment (RAATE-A score < 18), whereas successful process characteristics included detoxification of the patient at time of referral and a full multidisciplinary referral meeting. This multidisciplinary model of evaluation and referral of alcohol dependent patients of a general hospital had a satisfactory level of effectiveness. Predictors of success and failure allow to identify subsets of patients for whom new strategies of motivation and treatment referral should be designed.