Tim-3 has been linked to several inflammatory diseases by regulation on both adaptive and innate immunities. Here, we assessed the augmented expression of Tim-3 in brain tissue of ischemia-reperfusion mice and PBMCs of ischemic stroke (IS) patients. The augmented expression of Tim-3 significantly correlated with abnormal lipid levels. In vitro studies showed that plasma from ischemic stroke patients induced Tim-3 expression in THP- 1 cells. More importantly, our results revealed a significant correlation of Tim-3 expression on CD4 + T cells with systemic IL-17 in patients with ischemic stroke. Consistently, we also found a positive correlation of Tim-3 expression on CD14 + monocytes and serum TNF-a in IS patients. Collectively, augmented expression of Tim-3 may play an important role in the pathogenesis of ischemic stroke by regulation of proinflammatory cytokines. Further studies will give us new insights on the pathogenesis of ischemic stroke and potentially provide a new target at the medical therapy.

Dietary vitamin E, highly expressed in palm oil, exists as either tocopherols or tocotrienols. Evidence indicates that vitamin Es maybe potent cancer preventive agents. In this study, the y- and O- isoforms of vitamin E were found to he the most effective at cancer cell growth inhibition, with the tocotrienols being more effective than the tocopherols in androgen-independent PC-3 prostate cancer cells. To assure that these compounds were selective toward cancer cells, the growth arrest of PrEC normal prostate cells was compared to PC-3 cells. At concentrations of -30 iM dietary, y-vitamin Es showed no signficant growth arrest on PrEC cell growth, hut selectively inhibited growth in the PC-3 cancer cells. Moreover y-Tocotrienol demonstrated a greater potential to inhibit growth in cancer cells at these lower concentrations than did y-Tocopherol. Two independent pathways important in carcinogenesis were tested: PPAR y and NFicB. The PPAR y was up regulated by both dietary y-vitamin Es by the modulation of the endogenous ligand 15-S-HETE, while NFicB was only regulated by y-Tocotrienol. The modulation of NFicB was confirmed by the down regulation of the pro-Apoptotic proteins clAP, xIAP, and BcL-2 which potentiate apoptosis and are down stream effectors of NFicB.

No description available.

Les cellules progénitrices endothéliales formant des colonies (EFCFs) sont issues decellules CD34+ de la moelle osseuse humaine. Peu de données concernent l’expression dufacteur tissulaire (FT) lors de cette différenciation endothéliale. Outre son rôle dansl’initiation de la génération de thrombine, le FT est impliqué dans l’angiogenèse.Nous montrons que les cellules CD34+ expriment le FT mais non ses isoformes. LesECFCs expriment peu de FT à l’état basal. En revanche, leur stimulation par le TNF-α induitune augmentation de l’expression de FT, et la génération de microparticules pro-coagulantes.Nous avons analysé les modifications fonctionnelles induites par cette stimulation. Nosrésultats montrent que l’expression de FT par les ECFCs est responsable d’une activité procoagulante majeure, alors que les propriétés angiogéniques ne semblent pas affectées.L’expression du tissue factor pathway inhibitor (TFPI) a été évaluée, ainsi que la capacité desmicroparticules issues de ECFCs à générer des métalloprotéinases (MMP2-, MMP-9).Une évaluation de la stabilité chromosomique des cb-ECFCs durant leur expansion aété réalisée, mettant en évidence des anomalies de nombre, mais pas d’anomalies destructures. Les conséquences de ces résultats en termes de thérapie cellulaire appliquées auxpathologies cardio-vasculaires sont discutées. Enfin, nous évoquons la possibilité deconsidérer l’expression de FT comme un marqueur de différenciation cellulaire. / Endothelial colony-forming cells (ECFCs) can be obtained from human bone marrowCD34+ cells. In spite of the essential role of the tissue factor (TF) in coagulation triggeringand angiogenesis, its expression during endothelial differentiation is not established. We showthat CD34+ cells express TF, but not TF splicing forms. ECFCs express a small amount of TFat baseline level. In contrast, ECFCs express TF high levels of TF on response to TNF-α andcan generated highly pro-coagulant microparticles. We have examined the functionalproperties induced by TNF-α stimulation. TF expression confers to ECFCs a strong thrombingeneration capacity without influencing their non-coagulant properties. We have examinedthe co-expression of the tissue factor pathway inhibitor (TFPI) and the ability of ECFCs togenerate microparticles producing metalloproteins (MMP-2, MMP-9).We have performed an evaluation of cb-ECFCs chromosomal stability during theirexpansion. We found quantitative but no structural chromosomal abnormalities. Theconsequences of our observations in the use of cell therapy in cardiovascular diseases arediscussed. We conclude that TF expression may be considered as cell differentiation marker

This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

Les sphingolipides sont une famille de lipides membranaires dotés d'un rôle structural, influant sur les propriétés de la bicouche lipidique, mais ils agissent aussi comme des molécules effectrices au rôle essentiel dans de nombreux aspects de la biologie cellulaire. Les sphingolipides céramide, sphingosine et S1P ont des effets opposés: le céramide et la sphingosine inhibent la prolifération et promeuvent la réponse apoptotique à différents stimulus de stress, la S1P est un stimulateur de la prolifération et de la survie cellulaires. Le céramide peut être produit par la voie de synthèse de novo, et l'hydrolyse de la sphingomyéline membranaire catalysée par les sphingomyélinases. Ces deux voies peuvent être activées par la cytokine pro-inflammatoire TNFa, capable d’induire une perte musculaire et jouant un rôle crucial dans le développement de la cachexie. Nous avons fait l'hypothèse que le céramide, ou un de ses métabolites, peuvent être des médiateurs de la perte musculaire tumeur-induite. Nous avons examiné le rôle du céramide dans l'atrophie induite in vitro par le TNFa chez les myotubes, en utilisant des analogues de céramide et des inhibiteurs du métabolisme sphingolipidique. L'apport de céramides exogènes est capable de reproduire l'effet atrophique du TNFa, ce qui suggère que le céramide peut participer à l'atrophie musculaire. Pour vérifier si les céramides sont les médiateurs de l'atrophie induite par le TNFa, nous avons analysé l'effet d'inhibiteurs ciblant différentes étapes du métabolisme: l'inhibition de la voie de synthèse de novo est incapable de rétablir la taille des myotubes en présence de TNFa, alors que les inhibiteurs de sphingomyélinase neutre suppriment l'atrophie TNFa-induite. L’accumulation de céramide et de sphingosine augmente l’effet pro-atrophique, tandis que la S1P a un effet protecteur. Ces observations montrent que, dans les myotubes, le céramide, ou un métabolite produits par la voie de la sphingomyélinase neutre en réponse à une stimulation par le TNFa, participent à l'atrophie des cellules. Pour évaluer le rôle in vivo des sphingolipides, nous avons traité la souris BalbC porteuse d’un carcinome C26 avec myriocine, inhibiteur de la synthèse de novo, capable d'induire une déplétion du muscle en sphingolipides, Ce traitement protège partiellement les souris contre la perte de poids corporel et de poids des muscles induite par la tumeur, sans affecter la taille de celle-ci. De plus, la myriocine réverse significativement la perte de taille des fibres musculaires et réduit l'expression des atrogenes, ce qui montre qu'elle protège le muscle contre l'atrophie. Ces résultats suggèrent fortement que le céramide, ou un métabolite sphingolipidique en aval, est impliqué dans l'atrophie musculaire tumeur-induite. La voie des sphingolipides apparaît donc comme une nouvelle cible potentielle d'interventions pharmacologiques visant à protéger le tissu musculaire contre l'atrophie. / The sphingolipids are a family of membrane lipids with only a structural role, influencing lipid bilayer properties, but they also act as effector molecules with essential roles in many aspects of cell biology. The sphingolipids ceramide, sphingosine and S1P have shown opposite effects: whereas ceramide and sphingosine usually inhibit proliferation and promote apoptotic responses to different stress stimuli, S1P is known to stimulate cell growth, and promote cell survival. Ceramide can be produced through the de novo synthesis pathway, and by membrane sphingomyelin hydrolysis catalyzed by sphingomyelinases. Both pathways can be activated by the pro-inflammatory cytokine TNFa. Because this cytokine has been shown to promote muscle loss and seems to be crucial in the development of cachexia, we hypothesized that the formation of ceramide, or a metabolite, can be involved in tumor-induced muscle wasting. We investigated the role of ceramide in the in vitro atrophic effects of TNFa on differentiated C2C12 myotubes, by using cell permeant ceramides and inhibitors of sphingolipid metabolism. We observed that TNFa atrophic effects, as evaluated by the reduction in myotube area, are mimicked by exogenous ceramides, supporting the idea that ceramide can participate in muscle atrophy. To verify if ceramide is a mediator of TNFa-induced atrophy, and to identify the metabolites potentially involved, we analyzed the effects of drugs able to block sphingolipid metabolism at different steps: the inhibition of de novo synthesis pathway was unable to restore myotube size in the presence of TNFa whereas the inhibitors of neutral sphingomyelinases reversed TNFa-induced atrophy. Moreover, an accumulation of ceramide and sphingosine induced pro-atrophic effects, whereas sphingosine-1-phosphate had a protective effect. These observations establish that in C2C12 myotubes, ceramide or other downstream metabolites such as sphingosine, produced by the neutral sphingomyelinase pathway in response to TNFa stimulation, participate in cell atrophy. To evaluate the in vivo role of sphingolipids, we treated BalbC mice carrying C26 adenocarcinoma woth Myriocin, an inhibitor of the de novo pathway of ceramide synthesis, that is able to deplete muscle tissue in all sphingolipids, was administered daily to the animals. This treatment partially protected animals against tumor-induced loss of body weight and muscle weight, without affecting the size of tumors. Moreover, myriocin treatment significantly reversed the decrease in myofiber size associated with tumor development, and reduced the expression of atrogenes Foxo3 and Atrogin-1, showing that it was able to protect against muscle atrophy. These results strongly suggest that ceramide, or a downstream sphingolipid metabolite, is involved in tumor-induced muscle atrophy. The sphingolipid pathway thus appears as a new potential target of pharmacological interventions aiming at protecting muscle tissue against atrophy.

Le muscle squelettique fait preuve d'une remarquable plasticité en réponse aux changements physiologiques, comme l’activité physique, et aux situations pathologiques. Il subit notamment une atrophie sévère lors de la cachexie qui accompagne diverses pathologies chroniques comme le cancer, le SIDA, etc. L’atrophie musculaire est aussi une composante de la sarcopénie qui survient lors du vieillissement normal, et se caractérise par un déclin de la force et de la masse musculaire. L'atrophie musculaire, qui entraîne une augmentation de la mortalité et diminue l’efficacité des traitements, constitue donc un problème de santé majeur.La fonte musculaire se caractérise par une altération de l’équilibre entre synthèse et dégradation protéiques dans les fibres adultes. Des taux particulièrement élevés de cytokines circulantes, dont le TNFα, qui affectent l’homéostasie du muscle via différentes voies de signalisation, semblent être à l’origine de l'atrophie. Les mécanismes de la réponse atrophique musculaire à ces taux circulants élevés sont cependant mal définis. Le TNFα a des effets complexes. Il peut activer de multiples voies de signalisation, parmi lesquelles l'induction de la synthèse de sphingolipides, et plus particulièrement de céramides, par la voie de novo et par l'activation des sphingomyélinases. Au niveau musculaire, les céramides sont connus pour leurs effets sur la signalisation de l'insuline, sur l'apoptose et sur la différenciation myogénique. Par contre, leur implication dans le cadre de l'atrophie n'avait jamais été prise en compte. L’objectif de ce travail a été dans un premier temps de démontrer le rôle des céramides dans l’atrophie. Dans un deuxième temps, nous avons caractérisé la voie de signalisation par laquelle l’augmentation intramusculaire de céramide induite par le TNFα aboutit à une chute de la synthèse protéique, couplée à une augmentation de la protéolyse. Dans ce but, nous avons mis au point des modèles in vitro d'atrophie, impliquant des myotubes traités par des concentrations physiologiques de TNF. Nous avons en parallèle étudié un modèle in vivo de cachexie induite chez la souris par l'implantation d’un adénocarcinome C26. L’analyse des sphingolipides nous a permis de montrer l’augmentation des taux de céramides concomitante à l’atrophie générée in vitro et in vivo. Le rôle des céramides dans l’atrophie a été démontré par l’effet protecteur des inhibiteurs de leur synthèse, dans les modèles in vitro et in vivo. Nous montrons de plus dans un modèle in vitro que les effets atrophiques des céramides sont dus à l’inhibition de la voie de signalisation Phospholipase D/mTOR/Akt. Nos résultats nous ont permis de prouver le rôle des sphingolipides dans le contrôle de l’homéostasie protéique du muscle. La modulation du métabolisme des sphingolipides apparaît donc comme une nouvelle cible thérapeutique prometteuse dans le traitement de la perte musculaire associée à diverses pathologies. / Skeletal muscle demonstrated a remarkable plasticity in response to physiological changes, such as physical activity, and pathological situations. He suffered such severe atrophy during cachexia that accompanies various pathologies such as cancer, AIDS, etc.. Muscle atrophy is also a component of sarcopenia that occurs during normal aging, and is characterized by a decline in strength and muscle mass. Muscle atrophy, which leads to increased mortality and decreased treatment efficacy, thus constitutes a health problem majeur.La muscle wasting is characterized by an impaired balance between protein synthesis and degradation in adult fibers. Particularly high levels of circulating cytokines, including TNF, which affect muscle homeostasis via different signaling pathways appear to be the cause of atrophy. Mechanisms of muscle atrophy response to these elevated circulating levels are however unclear. TNFa has complex effects. It may activate multiple signaling pathways, including the induction of the synthesis of sphingolipids, especially ceramides, for the de novo pathway and the activation of sphingomyelinases. In muscle, ceramides are known for their effects on insulin signaling on apoptosis and myogenic differentiation. By cons, their involvement in the context of atrophy was never taken into account. The objective of this work was firstly to demonstrate the role of ceramides in atrophy. In a second step, we characterized the signaling pathway by which increased intramuscular ceramide induced by TNF leads to a fall in protein synthesis, coupled with an increase in proteolysis. For this purpose, we have developed in vitro models of atrophy involving myotubes treated with physiological concentrations of TNF . We studied in parallel an in vivo model of cachexia induced in mice by implantation of adenocarcinoma C26. Analysis of sphingolipids we showed increased levels of ceramide concomitant atrophy generated in vitro and in vivo. The role of ceramide in atrophy has been demonstrated by the protective effect of inhibitors of their synthesis in the in vitro and in vivo. We show further in an in vitro model that atrophic effects of ceramides are due to inhibition of phospholipase D signaling pathway / mTOR / Akt. Our results allowed us to prove the role of sphingolipids in the homeostatic control of muscle protein. Modulation of sphingolipid metabolism appears to be a promising new therapeutic target in the treatment of muscle wasting associated with various pathologies.

Introduction: Current medical treatment for individuals diagnosed with schizophrenia (SCHZ) primarily relies on the inhibition of the dopamine D2 receptor that has been shown to be supersensitive in these patients. Treatment occurs through the use of antipsychotic medication which leads to a number of debilitating dose-dependent side effects, such as weight gain, agranulocytosis, and seizures. Patients diagnosed with SCHZ have also been shown to have increased inflammation in their central nervous system (CNS), particularly within specific brain regions such as the prefrontal cortex and hippocampus. This is in large part due to the interaction between a pro-inflammatory cytokine called tumor necrosis factor-alpha (TNFa) and microglia, which are resident CNS defense cells. TNFa is a cell-signaling protein, regulates a variety of immune cells, and is involved in the acute phase reaction of inflammation. Upon activation by TNFa secretion, microglial cells switch from being anti-inflammatory (M2) to pro-inflammatory (M1), thereby resulting in neuroinflammation as well as synaptic loss and neuronal death. In this project, we hypothesized oral administration through the diet of a novel TNFa modulator (PD2024) developed by P2D Biosciences, Inc. (Cincinnati, OH) would significantly reduce microglia activation in rats neonatally treated with Polyinosinic:polycytidylic acid (poly I:C). Methods and Results: To test our hypothesis, four groups (Neonatal Poly I:C/TNFa, Neonatal Poly I:C/Control, Neonatal Saline/TNFa, and Neonatal Saline/Control) were intraperitoneally injected with either poly I:C or saline during postnatal days (P)5-7. Poly I:C is an immunostimulant that mimics neonatal infection in humans, which also has been found to be a factor for the development of SCHZ later in life. Between days (P)30-(P)60, the Neonatal Poly I:C/TNFa and Neonatal Saline/TNFa groups were orally administered PD2024 through the diet. After (P)60, brain tissue was evaluated by immunohistochemistry (IHC) and confocal microscopy. Immunohistochemistry was used to label microglial cells in the prefrontal cortex and hippocampus with a green fluorescent dye attached to Iba1, a protein that specifically binds to these cells. Upon completion of IHC, tissue was evaluated using a confocal microscope and then analyzed with NIH ImageJ software. Analysis parameters included cell count, sampled cell body fluorescence, and overall image fluorescence. The results obtained showed a significant decrease in microglia activation for the Poly I:C/TNFa group when compared to the Poly I:C/Control group, as well as similarities in activation levels with the Saline/Control group. These results were demonstrated in both sampled cell body fluorescence and overall image fluorescence measurements. Conclusion: This data supports the hypothesis that PD2024 is successful in reducing microglia activation through the modulation of TNFa. Therefore, treatment with a TNFa modulator such as PD2024 alongside of current antipsychotic medication could mediate neuroinflammation and reduce the dose-dependent side effects. This approach could be a promising therapeutic treatment option for those diagnosed with schizophrenia, as well as potentially for other neurocognitive and behavioral disorders.

Die vorliegende publikationsbasierte Dissertationsschrift erörtert auf der Basis experimenteller Untersuchungen im Tiermodell die Auswirkungen von Stress auf die Zytokinproduktion und depressionsähnliches Verhalten. Außerdem wird getestet, ob die Blockade des Zytokins Tumornekrosefaktor-α (TNF‑α) eine Möglichkeit zur antidepressiven Intervention darstellt. Einleitend werden die Zusammenhänge von Stress, Zytokinen und Depression referiert sowie das hypothetische Modell erläutert, das den publizierten Untersuchungen zugrunde liegt. Es wird hypothetisiert, dass Stress zur Erhöhung der Produktion proinflammatorischer Zytokine führt und dass die vermehrte Zytokinproduktion depressive Verhaltensweisen hervorruft. Aus dieser Annahme leitet sich die Möglichkeit ab, durch Blockade der Wirkung des proinflammatorischen Zytokins TNF‑α antidepressive Effekte zu erzielen. In den beiden Arbeiten „The impact of social isolation on immunological parameters in rats“ (Archives of Toxicology) und „Stress-induced cytokine changes in rats“ (European Cytokine Network) wurde der Einfluss von sozialer Isolation, chronischem, milden und akutem Stress auf die Zytokinproduktion untersucht. In diesen Untersuchungen führten die verschiedenen Stressarten zu einer Modulation der Produktion proinflammatorischer Zytokine. Die dritte Publikation „Antidepressant effects of TNF‑α blockade in an animal model of depression“ (Journal of Psychiatric Research) berichtet von einem Experiment, in dem untersucht wurde, ob der TNF‑α-Inhibitor Etanercept antidepressive Effekte aufweist. Tatsächlich zeigte sich unter Etanercept ein Rückgang des depressionsähnlichen Verhaltens im forced swim test (FST) analog zu Verhaltensänderungen durch das in Tierversuchen als Standard-Antidepressivum geltende Imipramin. Die Autoren schlussfolgern, dass das Zytokinsystem durch Stress moduliert wird und so in die pathophysiologische Entwicklung einer Depression involviert sein könnte. Zytokininhibitoren könnten eine neue Klasse der Antidepressiva bei Therapieresistenz werden, wenn sich die Ergebnisse dieser Tierversuche in Studien an Probanden und an Patienten replizieren lassen.