Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.

Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation.

BACKGROUND: Toll-like receptors (TLRs) are essential components of the immune response to fungal pathogens. We examined the role of TLR polymorphisms in conferring a risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. METHODS: We analyzed 20 single-nucleotide polymorphisms (SNPs) in the toll-like receptor 2 gene (TLR2), the toll-like receptor 3 gene (TLR3), the toll-like receptor 4 gene (TLR4), and the toll-like receptor 9 gene (TLR9) in a cohort of 336 recipients of hematopoietic-cell transplants and their unrelated donors. The risk of invasive aspergillosis was assessed with the use of multivariate Cox regression analysis. The analysis was replicated in a validation study involving 103 case patients and 263 matched controls who received hematopoietic-cell transplants from related and unrelated donors. RESULTS: In the discovery study, two donor TLR4 haplotypes (S3 and S4) increased the risk of invasive aspergillosis (adjusted hazard ratio for S3, 2.20; 95% confidence interval [CI], 1.14 to 4.25; P=0.02; adjusted hazard ratio for S4, 6.16; 95% CI, 1.97 to 19.26; P=0.002). The haplotype S4 was present in carriers of two SNPs in strong linkage disequilibrium (1063 A/G [D299G] and 1363 C/T [T399I]) that influence TLR4 function. In the validation study, donor haplotype S4 also increased the risk of invasive aspergillosis (adjusted odds ratio, 2.49; 95% CI, 1.15 to 5.41; P=0.02); the association was present in unrelated recipients of hematopoietic-cell transplants (odds ratio, 5.00; 95% CI, 1.04 to 24.01; P=0.04) but not in related recipients (odds ratio, 2.29; 95% CI, 0.93 to 5.68; P=0.07). In the discovery study, seropositivity for cytomegalovirus (CMV) in donors or recipients, donor positivity for S4, or both, as compared with negative results for CMV and S4, were associated with an increase in the 3-year probability of invasive aspergillosis (12% vs. 1%, P=0.02) and death that was not related to relapse (35% vs. 22%, P=0.02). CONCLUSIONS: This study suggests an association between the donor TLR4 haplotype S4 and the risk of invasive aspergillosis among recipients of hematopoietic-cell transplants from unrelated donors.

Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'- monophosphate responsive element-binding protein CREB in pituitary cells.

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.

Innate immune response to poxvirus vectors

Summary : A large body of evidence indicates that the innate immune system plays a key role in host response to viral infection. Recently, Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptor receptors (NLRs) have emerged as key innate immune sensors of microbial products, eliciting intracellular signaling and leading to the production of chemokines, cytokines and interferons (IFNs) that shape innate immune responses and coordinate the development of adaptive immunity. Poxviruses are currently developed as vaccines vectors for infectious diseases such as HIV, tuberculosis and malaria. Modified vaccinia virus Ankara (MVA) and New York vaccinia virus (NWAC) are attenuated, replication deficient strains of poxvirus. The mechanisms underlying innate immune responses to MVA and NYVAC are poorly characterized. Thus, the objectives of the project were to determine the innate immune profile stimulated by poxviruses in innate immune cells and to evaluate the impact of modifications in the viral genome on MVA and NYVAC immunogenicity. MVA stimulated the production of abundant amounts of chemokines and IFNß but low levels of cytokines by human macrophages. In contrast, NYVAC weakly stimulated the production of all mediators. Interestingly, MVA and NYVAC strongly stimulated innate immune responses in vivo and in human whole blood, suggesting that a soluble factors}, possibly a complement component, was required for optimal activation of innate immune cells by poxviruses. Modified MVA and NYVAC produced by single or multiple deletions of viral genes targeting crucial pathways of host innate immunity, and mutant poxviruses with limited replication capacity, increased the production of pro-inflammatory molecules by human whole blood. Gene expression profiling in human macrophages confirmed the increased immunologic stimulatory capacity of modified poxviruses. The pathways activated by MVA and NYVAC in innate immune cells were described by analysing the response of knockdown or shRNA transduced macrophages with impaired expression of TLRs and their adaptors (MyD8$ and TRIF), RLRs (RIG-I, MDA-5 and the adaptor IPS-1) and the NALP3 inflammasome composed óf the NLR NALP3, caspase-1 and ASC. These experiments revealed a critical role for TLR2-TLR6-MyD88 in the production of tFNß-independent chemokines and of MDA-5-IPS-1 in the production of IFNß and IFNßdependent chemokines. The transcription of the iL1b gene encoding for the IL-1ß cytokine was initiated through TLR2-MyD88, whereas the maturation and the secretion of IL-1ß were controlled by the NALP3 inflammasome. Finally, we analyzed the role of macrophage migration inhibitory factor (MIF), a mediator of inflammation and innate immune responses, in MVA infection. We observed that MVA infection increased MIF production by innate immune cells and that MIF deficiency impaired macrophage and dendritic cell responses (ie migration, maturation, cytokine and IFN production) to MVA infection in vitro and in vivo. Moreover, MIF-deficiency resulted in delayed anti-MVA specific antibody production in mice immunized with the virus. In conclusion, we demonstrate. that poxviruses can be modified genetically to improve their immunogenicity. We also report the first comprehensive analysis of poxvirus sensing by innate immune cells, showing that the TLR, RLR and NLR pathways play specific and coordinated roles in regulating cytokine, chemokine and IFN response to poxvirus infection. Finally, we show that MIF is an integral host component involved in innate and adaptive immune responses to MVA infection. The present findings provide important information relevant to the study of the pathogenesis of poxvirus infections and allow a better understanding of the immunogenic potential of vaccine vectors, which is required for the development of optimized modìfied pox-vaccine vectors.

cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

 During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis.

OBJECTIVES: The aim of our study was to evaluate the role of cell-membrane expressed TLRs and the signaling molecule MyD88 in a murine model of OA induced by knee menisectomy (surgical partial removal of the medial meniscus [MNX]). METHODS: OA was induced in 8-10weeks old C57Bl/6 wild-type (WT) female (n=7) mice and in knockout (KO) TLR-1 (n=7), -2 (n=8), -4 (n=9) -6 (n=5), MyD88 (n=8) mice by medial menisectomy, using the sham-operated contralateral knee as a control. Cartilage destruction and synovial inflammation were evaluated by knee joint histology using the OARSI scoring method. Apoptotic chondrocytes and cartilage metabolism (collagen II synthesis and MMP-mediated aggrecan degradation) were analyzed using immunohistochemistry. RESULTS: Operated knees exhibited OA features at 8weeks post-surgery compared to sham-operated ones. In menisectomized TLR-1, -2, -4, and -6 deficient mice, cartilage lesions, synovial inflammation and cartilage metabolism were similar to that in operated WT mice. Accordingly, using the same approach, we found no significant protection in MyD88-deficient mice in terms of OA progression as compared to WT littermates. CONCLUSIONS: Deficiency of TLRs or their signalling molecule MyD88 did not impact on the severity of experimental OA. Our results demonstrate that MyD88-dependent TLRs are not involved in this murine OA model. Moreover, the dispensable role of MyD88, which is also an adaptor for IL-1 receptor signaling, suggests that IL-1 is not a key mediator in the development of OA. This latter hypothesis is strengthened by the lack of efficiency of IL-1β antagonist in the treatment of OA.

Chlamydiaceae and Chlamydia-like organisms in free-living small mammals in Europe and Afghanistan.

Few data are available on the occurrence of chlamydial infections in wild small mammals. We investigated the significance of free-living small mammals as reservoirs or transmission hosts for microorganisms of the phylum/class Chlamydiae. We obtained 3,664 tissue samples from 911 animals in Switzerland, Germany, Austria, the Czech Republic, and Afghanistan. Samples included internal organs (n = 3,652) and feces (n = 12) from 679 rodents (order Rodentia) and 232 insectivores (order Soricomorpha) and were tested by three TaqMan® real-time PCRs specific for members of the family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia spp. and Waddlia spp. Only one of 911 (0.11%) animals exhibited a questionable positive result by Chlamydiaceae-specific real-time PCR. Five of 911 animals were positive by specific real-time PCR for Parachlamydia spp. but could not be confirmed by quantitative PCR targeting the Parachlamydia acanthamoebae secY gene (secY qPCR). One of 746 animals (0.13%) was positive by real-time PCR for Waddlia chondrophila. This result was confirmed by Waddlia secY qPCR. This is the first detection of Chlamydia-like organisms in small wildlife in Switzerland. Considering previous negative results for Chlamydiaceae in wild ruminant species from Switzerland, these data suggest that wild small mammals are unlikely to be important carriers or transport hosts for Chamydiaceae and Chlamydia-like organisms.

Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling.

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg(677)Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-alpha production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-kappaB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-kappaB by human TLR2Arg(677)Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.

Calcineurin signaling as a negative determinant of keratinocyte cancer stem cell potential and carcinogenesis.

Calcineurin is the only known serine-threonine phosphatase under calcium-calmodulin control and key regulator of the immune system. Treatment of patients with calcineurin-inhibitory drugs like cyclosporin A and FK506 to prevent graft rejection dramatically increases the risk of cutaneous squamous cell carcinoma, which is a major cause of death after organ transplants. Recent evidence indicates that suppression of calcineurin signaling, together with its impact on the immune system, exerts direct tumor-promoting effects in keratinocytes, enhancing cancer stem cell potential. The underlying mechanism involves interruption of a double negative regulatory axis, whereby calcineurin and nuclear factors of activated T-cell signaling inhibits expression of ATF3, a negative regulator of p53. The resulting suppression of keratinocyte cancer cell senescence is of likely clinical significance for the many patients under treatment with calcineurin inhibitors and may be of relevance for other cancer types in which altered calcium-calcineurin signaling plays a role.

Presence of an oligodendroglioma-like component in newly diagnosed glioblastoma identifies a pathogenetically heterogeneous subgroup and lacks prognostic value: central pathology review of the EORTC_26981/NCIC_CE.3 trial.

Glioblastoma (GBM) is a morphologically heterogeneous tumor type with a median survival of only 15 months in clinical trial populations. However, survival varies greatly among patients. As part of a central pathology review, we addressed the question if patients with GBM displaying distinct morphologic features respond differently to combined chemo-radiotherapy with temozolomide. Morphologic features were systematically recorded for 360 cases with particular focus on the presence of an oligodendroglioma-like component and respective correlations with outcome and relevant molecular markers. GBM with an oligodendroglioma-like component (GBM-O) represented 15% of all confirmed GBM (52/339) and was not associated with a more favorable outcome. GBM-O encompassed a pathogenetically heterogeneous group, significantly enriched for IDH1 mutations (19 vs. 3%, p = 0.003) and EGFR amplifications (71 vs. 48%, p = 0.04) compared with other GBM, while co-deletion of 1p/19q was found in only one case and the MGMT methylation frequency was alike (47 vs. 46%). Expression profiles classified most of the GBM-O into two subtypes, 36% (5/14 evaluable) as proneural and 43% as classical GBM. The detection of pseudo-palisading necrosis (PPN) was associated with benefit from chemotherapy (p = 0.0002), while no such effect was present in the absence of PPN (p = 0.86). In the adjusted interaction model including clinical prognostic factors and MGMT status, PPN was borderline nonsignificant (p = 0.063). Taken together, recognition of an oligodendroglioma-like component in an otherwise classic GBM identifies a pathogenetically mixed group without prognostic significance. However, the presence of PPN may indicate biological features of clinical relevance for further improvement of therapy.

Hypolipidemic therapeutics and muscle metabolism

Summary : Lipid metabolism disorders, leading to obesity and cardiovascular diseases, are a major public health issue worldwide. These diseases have been treated by drugs and surgery, leading to tremendous costs and secondary morbidity. The aim of this thesis work is to investigate the mechanisms of actions of a new, micronutrition-based, approach to prevent obesity and cardiovascular diseases. This specific combination of micronutrients, Lipistase, incorporated into any dietary ail can be used in the daily food. Micronutrients are substances used by the living organism in small quantities to maintain physiological homeostasis. However, the human body is not able to produce them and has to obtain them from dietary sources. The combination of micronutrients investigated here, is composed of 26 compounds including trace elements, vitamins, minerals, ails and plant extracts, known to have individually a beneficial effect on lipid metabolism regulation. These specific micronutrients are used for the first time in a combinatorial mode targeting several metabolic pathways for better homeostasis control as opposed to a single target treatment, either chemical or natural. Short and long term studies, in different mouse strains, showed a significant decrease in plasma triglycerides, body weight gain and body fat mass in animals that were fed with a standard diet containing Lipistase. Additionally, a greatly reduced fat accumulation was observed in adipose tissue and liver of Lipistase-treated animals, while lipid and glucose utilization by skeletal muscle was enhanced. Moreover, the size of atherosclerotic plaques was significantly reduced in mice whose masher was treated during pregnancy and suckling, without showing any adverse effect. Finally, Lipistase has been shown to increase longevity by 20%. The control mice that did not receive Lipistase in their diet did not show all these beneficial effects. These micronutrients are used at the lowest dosage ever reported for treating Lipid disorders, resulting in far much lower costs as well as probably a higher safety. This is the first approach being very suitable for an effective large scale prevention policy for obesity and cardiovascular diseases, like iodine in dietary salt has been for goiter. Résumé : Les dysrégulations du métabolisme des lipids, à l'origine d'obésité et de maladies cardiovasculaires, sont un problème de santé publique majeur et mondial. Ces maladies impliquent des traitements médicamenteux et chirurgicaux dont le coût la morbidité secondaire sont très important. Le but de ce travail de thèse est d'étudier les mécanismes d'action d'une nouvelle approche préventive, basée sur la micronutrition. Cette combinaison spécifique de micronutriments, Lipistase, peut être incorporée dans n'importe quelle huile alimentaire et utilisée dans l'alimentation quotidienne. Les micronutrirnents sont des substances essentielles, à très faibles doses, pour le maintien de l'homéostasie physiologique des organismes vivants. Cependant, étant incapable de les synthétiser, le corps humain est dépendant en cela de l'apport alimentaire. La combinaison de micronutriments que nous avons étudié contient 26 composants, incluant des extraits de plantes, des huiles, des vitamines, des métaux et des minéraux, tous connus pour avoir individuellement des effets bénéfiques sur la régulation du métabolisme des lipides. Ces micronutriments spécifiques sont utilisés pour la première fois en mode combinatoire, ciblant ainsi plusieurs voies métaboliques pour un meilleur control de l'homéostasie, par opposition monothérapies chimiques ou naturelles. Des expériences de court et long terme, avec divers modèles de souris, ont montré une diminution significative des taux de triglycérides plasmatiques, de la prise de poids et de la masse graisseuse corporelle chez les animaux qui ont reçu Lipistase dans la nourriture standard. Une accumulation significativement moins importante des graisses a été observée dans le tissu adipeux et hépatique des souris traitées, alors que l'utilisation des lipides et glucose a été favorisée dans le muscle. En outre, la taille des plaques d'athérosclérose aété significativement réduite chez les souris dont la mère a été traitée pendant la grossesse et l'allaitement, sans montrer aucun effet indésirable. Enfin, les souris traitées par Lipistase ont vécu 20% plus longtèmps. Les souris contrôles qui n'ont pas reçu Lipistase dans la nourriture n'ont montré aucun de ces effets bénéfiques. Ces micronutriments sont utilisés au dosage le plus faible jamais rapporté pour le traitement des maladies du métabolisme lipidique, permettant ainsi un coût plus faible et surtout une meilleure sécurité. C'est une approche adéquate pour une politique de prévention de santé publique à large échelle de l'obésité et des maladies cardiovasculaires. C'est en cela et sous bien d'autres aspects, une première dans la prise en charge des maladies du métabolisme lipidique et pourrait même être pour ces dernières ce que l'iode du sel de cuisine a été pour le goitre.

Inhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate.

Astrocytes are responsible for the majority of the clearance of extracellular glutamate released during neuronal activity. dl-threo-beta-benzyloxyaspartate (TBOA) is extensively used as inhibitor of glutamate transport activity, but suffers from relatively low affinity for the transporter. Here, we characterized the effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na(+)-glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na(+) concentration (Na(+)(i)) changes that enable real time monitoring of transporter activity. Na(+)(i) was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na(+)-sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 muM, caused small alterations of Na(+)(i). TFB-TBOA inhibited the Na(+)(i) response evoked by 200 muM glutamate in a concentration-dependent manner with IC(50) value of 43+/-9 nM, as measured on the amplitude of the Na(+)(i) response. The maximum inhibition of glutamate-evoked Na(+)(i) increase by TFB-TBOA was >80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na(+)(i) elevations caused by the application of d-aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts