In vivo over-expression of interleukin-10 increases resistance to focal brain ischemia in mice.

Early studies showed that the administration of the anti-inflammatory cytokine interleukin-10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO-related molecular mechanisms. IL10 was over-expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase 3 compared with wild type mice. Under basal conditions, anti-inflammatory factors such as nerve growth factor and GSH were up-regulated and the pro-inflammatory cytokine IL1beta was down-regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro-inflammatory cytokines, including tumor necrosis factor-alpha, interferon-gamma, and IL1beta. Our data indicate that constitutive IL10 over-expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Age-specific variation of resistance to oxidative stress in the greater flamingo (Phoenicopterus ruber roseus)

Birds exhibit exceptional longevity and are thus regarded as a convenient model to study the intrinsic mechanisms of aging. The oxidative stress theory of aging suggests that individuals age because molecules, cells, tissues, organs, and, ultimately, animals accumulate oxidative damage over time. Accumulation of damage progressively reduces the level of antioxidant defences that are expected to decline with age. To test this theory, we measured the resistance of red blood cells to free radical attack in a captive population of greater flamingo (Phoenicopterus ruber roseus) of known age ranging from 0.3 to 45 years. We observed a convex relationship with young adults (12-20 years old) having greater resistance to oxidative stress than immature flamingos (5 months old) and old flamingos (30-45 years old). Our results suggest that the antioxidant detoxifying system must go through a maturation process before being completely functional. It then declines in older adults, supporting the oxidative theory of aging. Oxidative stress could hence play a significant role in shaping the pattern of senescence in a very long-lived bird species.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Molecular mechanisms of penicillin-induced killing and penicillin tolerance in Streptococcus gordonii

Abstract The main thesis topic relates to the 'molecular mechanisms of penicillin-induced bacterial death. Indeed, bacteria have developed two principal mechanisms to escape the killing effect of ß-lactam antibiotics: resistance and tolerance. Resistant bacteria are characterized by their ability to grow in the presence of drug concentrations higher than the one inhibiting the growth of susceptible members of the same species. Hence, resistant bacteria have an increased minimal inhibitory concentration (MIC) of the drug. Nevertheless, when exposed to antibiotic concentrations exceeding their new MIC, resistant bacteria remain sensitive to the antibiotic killing effect. In contrast, tolerant bacteria have an unchanged MIC. However, they have a considerably increased ability to survive drug-induced killing, even at concentrations exceeding their MIC by several orders of magnitude. In other words, in the presence of the antibiotic, tolerant bacteria become persister cells which stop growing but are not killed. In the present thesis, it is shown that the survival phenotype of a tolerant Streptococcus gordonii strain depends on two components belonging to sugar metabolism pathways. First, the transcription factor CcpA which mediates a global regulatory mechanism allowing bacteria to utilize the most efficient sugar source for their growth. We show that the inactivation of the ccpA gene leads to a partial loss of penicillin tolerance both in vitro and in a rat model of experimental endocarditis. Second, the Enzyme I of the phosphotransferase system which is involved in the uptake and phosphorylation of sugars. Here, we -show that a single nucleotide mutation in ptsI, the gene encoding the Enzyme I, is sufficient to confer a fully tolerant phenotype in S. gordonii both in vivo and in vivo. The mutation results in a radical proline to arginine substitution in the C-terminal domain of the protein, probably leading to a decrease in its homodimerization and subsequent activity. Taken together our results prove that tolerance is a global survival mechanism linked to sugar metabolism. We hypothesize that, in the presence of the antibiotic, the already altered metabolic processes of the tolerant strain are completely inactivated. Hence, bacteria may enter in a dormant state and become insensitive to the bactericidal effect of ß-lactams, which depends on actively dividing cells. This thesis manuscript also contains two other side-projects. The first one establishes that the ability to form a biofilm is not a requisite for the successful establishment of endocarditis due to S. gordonii. The second one characterizes the S. gordonii a-phosphoglucomutase gene, and shows that its inactivation results in a loss of in vitro fitness and in vivo virulence. Résumé Le sujet principal de cette thèse concerne les mécanismes moléculaires de la mort bactérienne induite par la pénicilline. En effet, les bactéries ont développé deux mécanismes principaux pour échapper à l'effet bactéricide des ß-lactamines : la résistance et la tolérance. Les bactéries résistantes sont caractérisées par leur capacité de croître en présence de concentration d'antibiotiques plus élevées que celles inhibant la croissance des organismes sensibles de la même espèce. Les bactéries résistantes ont donc une augmentation de leur concentration minimale inhibitrice (CMI) à l'antibiotique. Néanmoins, quand elles sont exposées à des concentrations dépassant leur nouvelle CMI, elles restent sensibles à l'effet bactéricide. Au contraire, les bactéries tolérantes ont une CMI inchangée. Toutefois, elles ont une très importante capacité à survivre à l'effet bactéricide des ß-lactamines, ceci même à des concentrations excédant leur CMI de plusieurs ordres de grandeur. En d'autres termes, en présence de l'antibiotique, les bactéries tolérantes deviennent des cellules persistantes qui arrêtent leur croissance mais ne sont pas tuées. Dans la présente thèse, il est montré que le phénotype de survie d'un Streptococcus gordonii tolérant dépend de deux composants appartenant aux voies du métabolisme des sucres. Premièrement, le facteur de transcription CcpA qui contrôle un système global de régulation permettant à la bactérie d'utiliser les sources de sucre les plus efficaces pour sa croissance. Il est montré que l'inactivation du gène ccpA résulte en la perte partielle de la tolérance à la pénicilline aussi bien in vitro que dans un modèle d'endocardite expérimentale chez le rat. Deuxièmement, l'Enzyme I du système de phosphotransfert impliqué dans l'import et la phosphorylation des sucres. Nous montrons qu'une mutation ponctuelle d'un nucléotide dans ptsl, le gène codant pour l'Enzyme I, suffit à complètement conférer un phénotype tolérant chez S. gordonii aussi bien in vitro qu'in vivo. La mutation induit la substitution radicale d'une proline en une arginine dans le domaine C-terminal de la protéine, résultant probablement en une diminution de sa capacité d'homodimérisation et donc d'activité. Dans leur ensemble, nos résultats prouvent que la tolérance est un mécanisme global de survie lié au métabolisme des sucres. Nous présentons l'hypothèse que, en présence de l'antibiotique, les processus métaboliques déjà altérés de la souche tolérante deviennent complètement inactifs. En conséquence, les bactéries entreraient dans un état dormant nonréplicatif, devenant ainsi insensibles à l'effet bactéricide des ß-lactamines qui nécessite des cellules en cours de division active. Le manuscrit de cette thèse contient également deux projets secondaires. Le premier montre que la capacité de former un biofilm n'est pas un prérequis pour le succès de l'initiation de l'endocardite à S. gordonii. Le second caractérise le gène de l'a-phosphoglucomutase de S. gordonii et montre que son inactivation résulte en une perte de fitness in vitro et de virulence in vivo.

Mechanisms of CYR61 mediated breast cancer metastasis to the lung

CYR61 (Cysteine-rich angiogenic inducer 61) is a matricellular protein that regulates cell proliferation, adhesion, migration and cell survival through interaction with various types of integrin cell adhesion receptors. At tissue level it is implicated in the regulation of embryonic development, wound healing and angiogenesis. CYR61 has also been involved in cancer progression, however its role appears to be diverse and complex depending on the cancer type and stage. Its contribution to metastasis formation is still unclear. Previous findings reported by our laboratory demonstrated that CYR61 cooperates with avßs integrin to promote invasion and metastasis of cancers growing in a pre-irradiated microenvironment. In this work, we used an orthotopic model of breast cancer to show for the first time that silencing of CYR61 in breast cancer cells suppresses lung metastasis formation. Silencing of MDA-MB-231 reduced both local growth and lung metastasis formation of tumor cells implanted in a pre-irradiated mammary fat pad. CYR61 silencing in tumors growing in non-irradiated mammary fat pads did not impact primary tumor growth but decreased lung metastasis formation. The effect of CYR61 on spontaneous lung metastasis formation during natural cancer progression was further examined by using an experimental model of metastasis. Results from these experiments indicate that CYR61 is critically involved in promoting cancer cells entry into lung parenchyma rather than later steps of colonization. In vitro experiments showed that CYR61 promotes tumor cell spreading, migration and transendothelial migration. CYR61 also supported colony formation under anchorage-independent condition and promotes resistance to anoikis through the involvement of ß1 and ß3 integrin. These results indicate that CYR61 promotes lung metastasis of breast cancer by facilitating extravasation into lung parenchyma through enhanced motility, transendothelial migration and resistance to anoikis. - CYR61 (Cysteine-rich angiogenic inducer 61) est une protéine matricellulaire qui régule la prolifération, l'adhérence, la migration et la survie des cellules par son interaction avec différents types de récepteurs d'adhésion cellulaire de la famille des intégrine. Au niveau des tissus, CYR61 est impliquée dans la régulation du développement embryonnaire, de la cicatrisation et de l'angiogenèse. CYR61 a également été impliquée dans le cancer, mais son rôle semble être divers et complexe en fonction du type du cancer et de son stade. Son rôle dans la formation des métastases n'est pas encore clair. Des résultats antérieurs rapportés par notre laboratoire ont montré que CYR61 coopère avec l'intégrine avß5 pour favoriser l'invasion et la métastase de tumeurs se développant dans un micro-environnement pré-irradié. Dans ce travail, nous avons utilisé un modèle orthotopique de cancer du sein pour démontrer pour la première fois que l'extinction (silencing) du gène CYR61 dans le cancer du sein réduit la formation de métastases pulmonaires. L'extinction de CYR61 dans la lignée cellulaire de cancer du sein humain MDA-MB- 231 réduit à la fois la croissance local ainsi que la formation de métastases pulmonaires à partir de cellules implantés dans les coussinets adipeux mammaires pré-irradié. L'extinction de CYR61 dans des tumeurs grandissant dans les coussinets adipeux mammaires non irradiées n'a pas d'incidence sur la croissance tumorale primaire mais réduit la formation des métastases pulmonaires. Par la suite nous avons examiné l'effet de CYR61 sur la formation de métastases pulmonaires en utilisant un modèle expérimental de métastase. Les résultats de ces expériences indiquent que CYR61 est impliquée de manière cruciale dans les étapes précoces de la formation de métastases, plutôt que dans les étapes tardives de colonisation du poumon. Des expériences in vitro ont montré que CYR61 favorise l'étalement, la migration et la transmigration endothéliale des cellules tumorales. CYR61 favorise également la formation de colonies dans des conditions indépendante de l'ancrage et la résistance à l'anoïkis par l'engagement des intégrines ß1 et ß3. Ces résultats indiquent que CYR61 favorise les métastases pulmonaires du cancer du sein en facilitant l'extravasation dans le parenchyme pulmonaire grâce à la stimulation de la motilità, de la migration transmigration endothéliale et de la résistance à l'anoïkis.

Experimental cutaneous Leishmaniasis: a powerful model to study in vivo the mechanisms underlying genetic differences in Th subset differentiation.

The murine model of infection with Leishmania major has allowed the demonstration in vivo of the importance CD4+ T cell subsets, distinguishable by the pattern of cytokines they produce, on the outcome of infectious diseases. Genetically determined resistance and susceptibility to infection with this parasite are the result of the development of Th1 and Th2 response, respectively. In this short paper, we present some results obtained in our group pertaining to the analysis of the mechanisms, operational during the early phase of this infection, responsible for the maturation of these functionally distinct CD4+ responses.

Resistance of Candida spp. to antifungal drugs in the ICU: where are we now?

Current increases in antifungal drug resistance in Candida spp. and clinical treatment failures are of concern, as invasive candidiasis is a significant cause of mortality in intensive care units (ICUs). This trend reflects the large and expanding use of newer broad-spectrum antifungal agents, such as triazoles and echinocandins. In this review, we firstly present an overview of the mechanisms of action of the drugs and of resistance in pathogenic yeasts, subsequently focusing on recent changes in the epidemiology of antifungal resistance in ICU. Then, we emphasize the clinical impacts of these current trends. The emergence of clinical treatment failures due to resistant isolates is described. We also consider the clinical usefulness of recent advances in the interpretation of antifungal susceptibility testing and in molecular detection of the mutations underlying acquired resistance. We pay particular attention to practical issues relating to ICU patient management, taking into account the growing threat of antifungal drug resistance.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Enhanced Botrytis cinerea resistance of Arabidopsis plants grown in compost may be explained by increased expression of defense-related genes, as revealed by microarray analysis

Composts are the products obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant cultivation. There is a small but increasing number of reports that suggest that foliar diseases may be reduced when using compost, rather than standard substrates, as growing medium. The purpose of this study was to examine the gene expression alteration produced by the compost to gain knowledge of the mechanisms involved in compost-induced systemic resistance. A compost from olive marc and olive tree leaves was able to induce resistance against Botrytis cinerea in Arabidopsis, unlike the standard substrate, perlite. Microarray analyses revealed that 178 genes were differently expressed, with a fold change cut-off of 1, of which 155 were up-regulated and 23 were down-regulated in compost-grown, as against perlite-grown plants. A functional enrichment study of up-regulated genes revealed that 38 Gene Ontology terms were significantly enriched. Response to stress, biotic stimulus, other organism, bacterium, fungus, chemical and abiotic stimulus, SA and ABA stimulus, oxidative stress, water, temperature and cold were significantly enriched, as were immune and defense responses, systemic acquired resistance, secondary metabolic process and oxireductase activity. Interestingly, PR1 expression, which was equally enhanced by growing the plants in compost and by B. cinerea inoculation, was further boosted in compost-grown pathogen-inoculated plants. Compost triggered a plant response that shares similarities with both systemic acquired resistance and ABA-dependent/independent abiotic stress responses.

Twofold cost of reproduction: an increase in parental effort leads to higher malarial parasitaemia and to a decrease in resistance to oxidative stress.

Parental effort is usually associated with high metabolism that could lead to an increase in the production of reactive oxidative species giving rise to oxidative stress. Since many antioxidants involved in the resistance to oxidative stress can also enhance immune function, an increase in parental effort may diminish the level of antioxidants otherwise involved in parasite resistance. In the present study, we performed brood size manipulation in a population of great tits (Parus major) to create different levels of parental effort. We measured resistance to oxidative stress and used a newly developed quantitative PCR assay to quantify malarial parasitaemia. We found that males with an enlarged brood had significantly higher level of malarial parasites and lower red blood cell resistance to free radicals than males rearing control and reduced broods. Brood size manipulation did not affect female parasitaemia, although females with an enlarged brood had lower red blood cell resistance than females with control and reduced broods. However, for both sexes, there was no relationship between the level of parasitaemia and resistance to oxidative stress, suggesting a twofold cost of reproduction. Our results thus suggest the presence of two proximate and independent mechanisms for the well-documented trade-off between current reproductive effort and parental survival.

Study of simple sequence repeat markers from coffee expressed sequences associated to leaf miner resistance

The objective of this work was to identify expressed simple sequence repeats (SSR) markers associated to leaf miner resistance in coffee progenies. Identification of SSR markers was accomplished by directed searches on the Brazilian Coffee Expressed Sequence Tags (EST) database. Sequence analysis of 32 selected SSR loci showed that 65% repeats are of tetra-, 21% of tri- and 14% of dinucleotides. Also, expressed SSR are localized frequently in the 5'-UTR of gene transcript. Moreover, most of the genes containing SSR are associated with defense mechanisms. Polymorphisms were analyzed in progenies segregating for resistance to the leaf miner and corresponding to advanced generations of a Coffea arabica x Coffea racemosa hybrid. Frequency of SSR alleles was 2.1 per locus. However, no polymorphism associated with leaf miner resistance was identified. These results suggest that marker-assisted selection in coffee breeding should be performed on the initial cross, in which genetic variability is still significant.

Inhibition of tumor angiogenesis by non-steroidal anti-inflammatory drugs: emerging mechanisms and therapeutic perspectives.

Chronic intake of non steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of developing gastrointestinal tumors, in particular colon cancer. Increasing evidence indicates that NSAID exert tumor-suppressive activity on pre-malignant lesions (polyps) in humans and on established experimental tumors in mice. Some of the tumor-suppressive effects of NSAIDs depend on the inhibition of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxane, which is highly expressed in inflammation and cancer. Recent findings indicate that NSAIDs exert their anti-tumor effects by suppressing tumor angiogenesis. The availability of COX-2-specific NSAIDs opens the possibility of using this drug class as anti-angiogenic agents in combination with chemotheapy or radiotherapy for the treatment of human cancer. Here we will briefly review recent advances in the understanding of the mechanism by which NSAIDs suppress tumor angiogenesis and discuss their potential clinical application as anti-cancer agents.

Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.