Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2018-09-12T18:08:44Z No. of bitstreams: 1 2017 - Julia Dias Villas Boas.pdf: 1378660 bytes, checksum: 2a1537e1b92056b0327c8e50b73feb34 (MD5) / Made available in DSpace on 2018-09-12T18:08:44Z (GMT). No. of bitstreams: 1 2017 - Julia Dias Villas Boas.pdf: 1378660 bytes, checksum: 2a1537e1b92056b0327c8e50b73feb34 (MD5) Previous issue date: 2017-04-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / The horse has a natural predisposition for the sport, however, its use in competitions can result in stress related problems that impair its sporting performance and especially its health. In this way it is fundamental not only to understand how the different risk and resilience factors to different stressors influence the response to stress, but also to develop strategies that can prevent or minimize the deleterious effects of stress. In this sense, acupuncture is an ancient technique of Traditional Chinese Medicine that has been used in the treatment and prevention of stress-related diseases. The present study proposed the use of two models of stress: one physical (physical exercise) and another psychological (startle model) to verify the reactivity to the stress of athletes horses. In addition, it was also evaluated if horses of different sporting modalities present different psychological stress responses and if acupuncture can alter the responses to physical stress. In the experiment 1, 16 Thoroughbred race horses were submitted to a exercise in the field of high intensity and short duration (12 m / s, 4 min). The RR intervals for analysis of the Heart Rate Variability were acquired through the Polar Equine ? heart rate monitor and blood samples were collected before and immediately after 2h, 4h, 6h, and 24h after exercise. The exercise promoted autonomic alterations in the sympatho-vagal balance since it significantly increased the low frequency component (LF), the heart rate and the LF / HF ratio, and decreased the high frequency component (HF) (p <0.01). There was an increase in hematocrit, plasma proteins, glucose and plasma lactate immediately after exercise (p <0.001). There was an increase (p <0.01) in serum cortisol values after 30 minutes, returning to baseline values after 60 min. However, no significant difference was observed in plasma cytokines IL-1? and IL-6 between moments after exercise and baseline. In experiment 2, horses of the experimental group 1 after exercise were randomly divided into two groups: CTL (C2): Control (without manipulation) and ACUP (C2)): animals submitted to weekly sessions of acupuncture at points VG1, C7, VG20 and B52 for 10 weeks. After the treatment period the animals repeated the same exercise and the same parameters were analyzed. Acupuncture reduced the LF / HF ratio, promoting a faster recovery of the animals, showing no influence on the other parameters analyzed. In the experiment 3, 24 equines were used, from three equestrian modes: Polo (PSI) (n = 9), Dressage (Brazilian Horse Riding) (n = 6) and Endurance (n=6) were subjected to the experimental model of startling through the abrupt opening of an umbrella. The results showed that endurance horses presented a significantly less intense startle-induced autonomic response than Polo and Dressage horses (lower LF / HF ratio at the time of the jump), paradoxically Enduro horses have cortisol levels in response in response to the startle than Polo horses. However, there was no difference between the modalities in the behavioral response after the startle, and Polo horses had significantly higher baseline levels of cortisol than the other modalities and did not change their cortisol levels in response to stress. Thus, we can conclude that 1) the exercise in the field of high intensity and short duration promoted adaptive changes characteristic of stress, being able to be used in studies of reactivity to stress in race horses; 2) acupuncture has a modulating effect on the stress-induced autonomic response in athletic horses, and 3) the equestrian modalities of Dressage, Polo and Endurance present different stress reactivity / O cavalo tem uma predisposi??o natural para o esporte, no entanto, o seu uso em competi??es pode resultar em problemas relacionados ao estresse que prejudicam seu desempenho esportivo e principalmente a sua sa?de. Desta forma ? fundamental n?o apenas entender como os diferentes fatores de risco e de resili?ncia a diferentes estressores influenciam a resposta ao estresse, como tamb?m desenvolver estrat?gias que possam prevenir ou minimizar os efeitos delet?rios do estresse. Neste sentido a acupuntura ? uma t?cnica milenar da Medicina Tradicional Chinesa que tem sido utilizada no tratamento e preven??es de doen?as relacionadas ao estresse. O presente estudo prop?s o uso de dois modelos de estresse: um f?sico (exerc?cio f?sico) e outro psicol?gico (modelo de sobressalto) para verificar a reatividade ao estresse de cavalos atletas. Al?m disso, tamb?m foi avaliado se cavalos de diferentes modalidades esportivas apresentam respostas ao estresse de psicol?gico distintas e se acupuntura pode alterar as respostas ao estresse f?sico. No experimento 1, 16 equinos de corrida da ra?a Puro Sangue Ingl?s foram submetidos ao exerc?cio a campo de alta intensidade e curta dura??o (12 m/s, 4min). Os intervalos RR para an?lise da Variabilidade da Frequencia Card?aca foram adquiridos atrav?s do frequenc?metro card?aco Polar Equine? e as amostras de sangue foram coletadas antes e, imediatamente, 2h, 4h, 6h, e 24h ap?s o exerc?cio. O exerc?cio promoveu altera??es auton?micas no balan?o simpato-vagal uma vez que aumentou significativamente o componente de baixa frequ?ncia (LF), a frequ?ncia card?aca e a raz?o LF/HF e diminuiu o componente de alta frequ?ncia (HF) (p < 0.01). Houve aumento do hemat?crito, das prote?nas plasm?ticas, glicose e lactato plasm?tico imediatamente ap?s o exerc?cio (p < 0.001). Houve aumento (p<0.01) nos valores s?ricos de cortisol ap?s 30 minutos, retornando aos valores basais ap?s 60min. No entanto, n?o foi observado diferen?a significativa nas citocinas plasm?ticas IL-1? e IL-6 entre os momentos ap?s exerc?cio e o momento basal. No experimento 2: os equinos do experimento 1 ap?s o exerc?cio foram aleatoriamente divididos em dois grupos: CTL (C2): Controle (sem manipula??o) e ACUP (C2)ACUP (C2): animais submetidos a sess?es semanais de acupuntura nos pontos VG1, C7, VG20 e B52 durante 10 semanas. Ap?s o per?odo de tratamento os animais repetiram o mesmo exerc?cio e foram analisados os mesmos par?metros. A acupuntura reduziu a raz?o LF/HF, promovendo uma recupera??o mais r?pida dos animais n?o apresentando influ?ncia nos demais par?metros analisados. No experimento 3, foram utilizados 24 equinos, pertencentes a tr?s modalidades equetres: P?lo (ra?a PSI) (n=9), Adestramento (ra?a Brasileiro de Hipismo) (n=6) e Enduro (Puro Sangue ?rabe) (n=9) submetidos ao modelo experimental de sobressalto atrav?s da abertura abrupta de um guarda-chuva. Os resultados mostraram que cavalos de enduro apresentaram resposta auton?mica induzida pelo sobressalto significativamente menos intensa que cavalos de Polo e Adestramento (menor raz?o LF/HF no momento do sobressalto), paradoxalmente cavalos de Enduro possuem n?veis de cortisol em resposta ao sobressalto mais altos que cavalos de Polo. N?o houve diferen?a entre as modalidades na resposta comportamental ap?s o sobressalto, no entanto cavalos de P?lo apresentam n?veis basais de cortisol significativamente mais altos que as demais modalidades e n?o variaram seus n?veis de cortisol em resposta ao estresse. Dessa forma, podemos concluir que 1) o exerc?cio a campo de alta intensidade e curta dura??o promoveu altera??es adaptativas caracter?stica de estresse, podendo ser utilizado em estudos de reatividade ao estresse em cavalos de corrida; 2) a acupuntura tem um efeito modulador da resposta auton?mica induzida pelo estresse em cavalos atletas e 3) as modalidades equestres de Adestramento, Polo e enduro apresentam reatividade ao estresse distintas

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt. A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations. The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement. In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

Les neutrophiles sont généralement considérés résistants aux glucocorticoïdes. Cependant, peu d’études comparant l’effet de ces drogues sur les neutrophiles et les autres leucocytes sanguins (monocytes, lymphocytes et éosinophiles) ont été rapportées. Dans notre étude, nous avons évalué la réponse aux glucocorticoïdes de ces deux populations cellulaires chez le cheval et l’homme. Les cellules, préalablement isolées du sang de 6 chevaux et 4 sujets humains sains, ont été incubées pendant 5 h en présence de lipopolysaccharide (LPS; 100 ng/mL) seul ou combiné avec de l’hydrocortisone, de la prednisolone ou de la dexaméthasone (10-8M et 10-6M). L’expression d’ARNm pour l’IL-1β, le TNF-α, l’IL-8, la glutamine synthétase et le récepteur α des glucocorticoïdes (GR-α) a été quantifiée par qPCR. Les neutrophiles équins ont également été incubés pendant 20 h en présence de ces 3 glucocorticoïdes et la survie cellulaire a été évaluée par cytométrie de flux et microscopie optique. Nous avons démontré que les glucocorticoïdes inhibaient l’expression des gènes pro-inflammatoires induite par le LPS pour les deux populations cellulaires chez les deux espèces étudiées. L’expression de la glutamine synthétase était également significativement augmentée par les glucocorticoïdes chez les neutrophiles et les autres leucocytes sanguins équins. De manière générale, l’intensité de la réponse aux glucocorticoïdes s’est avérée similaire dans les 2 populations leucocytaires et chez les deux espèces. Les glucocorticoïdes augmentaient également la survie des neutrophiles équins, phénomène également rapporté dans d’autres espèces. Ainsi, les glucococorticoïdes exercent des effets d’intensité comparable sur les neutrophiles et les autres leucocytes sanguins. Nous spéculons que la faible réponse à la corticothérapie observée lors de maladies inflammatoires chroniques neutrophiliques comme l’asthme sévère ou la Maladie Pulmonaire Obstructive Chronique (MPOC) ne s’explique pas par une corticorésistance intrinsèque des neutrophiles. / Neutrophils are generally considered resistant to glucocorticoids compared to other inflammatory cells. However, there are few studies comparing the effects of glucocorticoids in neutrophils and those of other blood leukocytes (monocytes, lymphocytes and eosinophils). In our study, we assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and in neutrophil-depleted leukocytes. Cells were isolated from 6 healthy horses and 4 human healthy subjects. They were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10-8M and 10-6M). IL-1β, TNF-α, IL-8, glutamine synthetase and Glucocorticoid Receptor α (GR-α) mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy. We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response was generally similar in both cell populations and species. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Based on these results, it appears that glucocorticoids exert effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic human diseases such as severe asthma or Chronic Obstructive Pulmonary Disease (COPD) is not explained by an inherent attenuated response of neutrophils to these drugs.

An association between inflammation and mood deterioration has been proposed as a potential explanatory mechanism underlying many pathologies. Previous research attributes this consistently reoccurring connection between inflammation and psychopathology that is often reported within the literature, to a relationship between the HPA axis, the body’s stress response system and the immune system. There is evidence of a bidirectional feedback loop between end-products of the immune system and the HPA-axis such as cytokines and cortisol. This is supported by research reporting that components of subjective well-being such as positive affect, optimism and life satisfaction can produce beneficial health outcomes by potentially targeting this feedback loop. The present longitudinal study tested if higher positive affect independently corresponds to lower levels of inflammatory markers Interleukin-6 (IL-6) and C-reactive protein (CRP) and HPA axis marker cortisol. The study further tested if higher subjective well-being decreases levels of IL-6 and CRP as well as cortisol. The study employed a subsample of participants from the Midlife in Japan (MIDJA) Biomarker project (n=174) that underwent testing at two separate time points across a period of 4 years. The data included subjective well-being, positive affect, IL-6, CRP, cortisol, perceived stress, neuroticism and demographic variables. Positive affect was not associated with any inflammatory marker or cortisol. Subjective well-being had no effect on CRP but reduced IL-6 and cortisol even when controlling for all control and demographic variables. It is concluded that subjective well-being may be linked to lower inflammation and HPA axis activity. / Ett samband mellan inflammation och sjukdomsbeteende har föreslagits som en förklaringsmekanism bakom förekomsten av många patologier. Den konsekventa anknytningen mellan inflammation och psykopatologi som många tidigare studier demonstrerat innebär ett samband mellan immunsystemet och HPA-axeln som är den struktur som utgör kroppens svar på stressorer. Det finns tecken på en återkopplingsslinga mellan slutprodukter av det immunologiska systemet och HPA-axeln såsom cytokiner och kortisol. Detta har stöd i tidigare forskning som rapporterat att komponenter av subjektivt välbefinnande så som positiv affekt, optimism och livstillfredställelse kan medföra positiva hälsoutfall genom att potentiellt influera denna återkopplingsslinga. Förevarande longitudinella studie testar om högre positiv affekt leder till lägre nivåer av de inflammatoriska markörerna interleukin-6 (IL-6) och C-reaktivt protein (CRP) samt HPA-axel markören kortisol. Studien testar vidare även om högre subjektivt välbefinnande leder till lägre nivåer av IL-6, CRP och kortisol. Deltagarna är ett subsampel från Biomarkerprojektet (n = 174) inom Midlife in Japan (MIDJA) som genomgick testning vid två separata tidpunkter över en period av 4 år. Data består av subjektivt välbefinnande, positiv affekt, IL-6, CRP, kortisol, upplevd stress, neuroticism samt demografiska variabler. Positiv affekt hade ingen signifikant effekt på någon av de inflammatoriska markörerna eller kortisol. Subjektivt välbefinnande hade inte någon signifikant effekt på CRP men reducerade signifikant IL-6 och kortisol och dessa effekter förblev signifikanta efter kontroll för samtliga kontroll och demografiska variabler. Följaktligen dras slutsatsen att subjektivt välbefinnande kan leda till lägre inflammation och HPA-axel aktivitet.

Dans les patients infectés par HBV, les thérapies avec les analogues de nucléos(t)ides (NAs) ou l'interféron α (IFNα) restent inefficaces pour éradiquer l'infection, à cause d'une forme persistante d'HBV, appelée l'ADN circulaire covalent clos (ADNccc), organisé comme un mini-chromosome. Notre but a été de revisiter l'activité anti-HBV d'un panel de cytokines in vitro en utilisant des hépatocytes non transformés, afin d'identifier de nouvelles options immunothérapeutiques. Parmi toutes les molécules testées, l'IFNβ, l'IFNγ, les IFNλ, le TNFα, l'IL-6, l'IL-1β et le ténofovir (ce dernier utilisé comme contrôle positif) ont montré un effet suppresseur sur la réplication d'HBV aussi fort et parfois plus fort que l'IFNα. La cytokine ayant l'effet le plus élevé sur l'ADN total d'HBV (EC50 ≈ 25 pg/mL), sans cytotoxicité, était l'interleukine-1β (IL-1β), qui est naturellement produite par les cellules de Kupffer (KC), les macrophages du foie. De façon importante, les ARNs totaux d'HBV et l'antigène sécrété HBeAg, mais pas HBsAg, ni l'ADNccc, sont fortement diminués par l'IL-1β. Nous avons donc émis l'hypothèse selon laquelle des promoteurs viraux spécifiques su l'ADNccc pourraient être inhibés, même si l'ADNccc n'est pas dégradé. Ensuite, nous avons étudié le mécanisme de l'activité antivirale de l'IL-1β. Nous avons montré que tous les promoteurs d'HBV sembleraient être inhibés par l'IL-1β. En parallèle, nous avons vérifié que l'IL-1β pouvait activer le promoteur de NF-κB, dont la fonction de transcription a été confirmée. Grâce à cette étude, l'IL-1β a été montré comme ayant un effet antiviral très efficace contre HBV in vitro, par l'intermédiaire de la fixation de NF-κB sur l'ADNccc / In HBV-infected patients, therapies with nucleos(t)ide analogues (NAs) or interferon α (IFNα) remain ineffective in eradicating the infection, because of a persistent form of HBV DNA, namely the covalently closed circular DNA (cccDNA), which is organized as a minichromosome. Our aim was to revisit the anti-HBV activity of a panel of IFNs and pro-inflammatory cytokines in vitro using nontransformed cultured hepatocytes of HBV infection, to identify new immunotherapeutic options. Amongst all molecules tested, IFNβ, IFNγ, IFNλ, TNFα, IL-6, IL-1β and tenofovir showed a suppressive effect on HBV replication at least as strong as, but sometimes stronger than IFNα. The cytokine showing the highest effect on intracellular total HBV DNA without any cytotoxicity, was interleukin-1β (IL-1β), which is naturally produced by Kupffer cells (KC), representing the macrophages of the liver. Importantly, total HBV RNAs and secreted HBeAg, but nor HBsAg, neither cccDNA, were strongly decreased. Thus, we hypothesized that even if cccDNA was not degraded, specific viral promoters on cccDNA could be silenced. Then, we investigated the mechanism of IL-1β antiviral activity. We have shown that all HBV promoters were early inhibited by IL-1β. In the meantime, we have verified that IL-1β can induce nuclear Translocation and expression of NF-κB. We also checked NF-κB functionality. Thanks to this study, IL-1β has been found to have very potent antiviral effect against HBV in vitro, through the binding of NF-κB on cccDNA

No description available.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctorate of Philosophy. / Senecio serratuloides is widely used for wound healing in South Africa but minimal information regarding its efficacy is available. Furthermore toxic pyrrolizidine alkaloids may be present. The following investigation sought firstly to evaluate the efficacy and safety of Senecio serratuloides in a porcine wound model; secondly to assess for a potential mechanism and finally isolate and identify fractions in in-vitro assays. Assessment of Efficacy and Safety Materials and Methods: Deep partial thickness and full thickness wounds were created on 9 pigs. Treatment included an occlusive dressing (negative control), activated carbon, or the Senecio preparation. Wounds were monitored using photographic documentation, pH measurement and histological analysis (skin thickness and collagen content). Toxicity was monitored on blood and liver samples. Results and Discussion: Efficacy of Senecio serratuloides was established with a significantly thicker epidermis, maximal at day 7 post-operative, 2 days before the controls. Effects on collagen content was negligible with no toxicity detected. Mechanistic investigation Materials and Methods: Wound fluid was analysed for IL-10, IL-12, IL-1β, IL-6, IL-8, TNF-α using flow cytometry based assays. Tyrosine phosphorylation and cellular proliferation was assessed using dual immunofluorescence staining. Results and Discussion: IL-1β levels were significantly greater in the Senecio treatment. Tyrosine phosphorylation increased to day 9 post-operative where it stabilised in all groups. In the same period, cellular proliferation was sustained in the Senecio treated wounds but not in the controls. Keratinocyte proliferation was identified as the target for in-vitro assays. Extraction, Isolation and Partial Identification using In-vitro Proliferation Assays. Materials and Methods: The plant was fractionated using solid phase extraction cartridges. Keratinocytes were grown under standard conditions in 96-well plates. Cellular proliferation was assessed spectrophotometrically using a resazurin dye technique. Active fractions were analysed using gas chromatography and mass spectrometry. Results and Discussion: Identified fractions increased the rate of proliferation by 300- 400%. Potential lead compounds were identified. Importantly, pyrrolizidine alkaloids could not be detected. Conclusion Senecio serratuloides is efficacious in treating deep partial thickness wounds without inducing liver toxicity. Sustained keratinocyte proliferation linked to tyrosine phosphorylation may be an underlying mechanism. Although successful, in-vitro detection of active fractions requires further characterisation.

La sclérose en plaques (SP) est une maladie inflammatoire du système nerveux central (SNC) affectant plus de 2,5 millions de personnes dans le monde. Bien que son étiologie soit inconnue, de nombreuses données supportent la contribution des réponses immunitaires à la maladie. La présence de leucocytes au sein des lésions de démyélinisation est un marqueur neuropathologique de la SP. Les traitements actuels diminuent les exacerbations de la SP mais échouent à arrêter sa progression. Ainsi, il est essentiel d’identifier des nouvelles voies contribuant à la pathologie de la SP. NKG2D est un récepteur co-activateur de cellules effectrices qui participent à la surveillance immunitaire. Cependant, cette voie peut contribuer à l’inflammation et aux lésions tissulaires. Le blocage ou l’élimination du NKG2D réduit la gravité de la maladie dans des modèles animaux de SP. Dans la pathologie humaine, des lymphocytes T CD4+ et CD8+ NKG2D+ sont présents dans les lésions et élevés dans le sang périphérique des patients atteints de la forme cyclique de SP durant une poussée. Au moins un ligand du NKG2D (NKG2DL) est exprimé par des oligodendrocytes dans des lésions et les lymphocytes T CD8+ causent la mort in vitro des oligodendrocytes humains de façon NKG2D dépendante. On ignore encore si d’autres cellules neurales expriment des NKG2DL et sont susceptibles à la reconnaissance par le NKG2D. Dans cette thèse, nous avons investigué la contribution de la voie NKG2D à la pathologie de la SP. La première partie présente la caractérisation de l’expression des NKG2DL au sein du SNC. Nous avons trouvé des niveaux élevés de ULBP4, mais aucun autre ligand, dans les lésions et la matière blanche d’apparence normale chez des patients SP. Nous avons identifié des déclencheurs potentiels de l’expression de ULBP4 par les astrocytes. ULBP4 soluble est détecté dans le liquide céphalo-rachidien, et des essais fonctionnels ont démontré sa capacité à renforcer la sécrétion de cytokines inflammatoires par les lymphocytes T CD8+. Dans la seconde partie, nous avons caractérisé les lymphocytes T exprimant NKG2D dans le sang de patients atteints des formes cycliques et progressives de la SP ainsi que chez des sujets témoins. Bien que nous ayons trouvé des proportions similaires, les lymphocytes T NKG2D+CD4+ de patients SP avaient un phénotype mémoire activé associé aux profils Th1 et Th1/Th17. Les lymphocytes T NKG2D+CD8+ étaient diminués chez les patients atteints de la forme cyclique. Cette population affichait une expression prédominante du granzyme B et manifestait des capacités de dégranulation envers les astrocytes exprimant ULBP4. Finalement, les analyses d’imagerie en temps réel ont révélé un rôle pour NKG2D et son ligand ULBP4 dans les interactions durables entre les astrocytes et les lymphocytes T CD8+ humains. Nos travaux identifient un nouveau mécanisme dans le dialogue entre ces types cellulaires. Globalement, ce projet de thèse présente une caractérisation en profondeur de la voie NKG2D dans la SP. De plus, il fournit de nouvelles preuves quant à l’implication de ULBP4 dans la pathophysiologie de la SP. De nouvelles investigations contribueront à élucider la validité de ULBP4 en tant que cible thérapeutique. / Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) that affects more than 2.5 million people worldwide. Despite its unknown etiology, numerous evidences point to aberrant immune responses that contribute to the typical tissue damage. Indeed, the presence of infiltrating immune cells within the characteristic focal demyelinating lesions is a pathological hallmark of MS. Current treatments, which target the immune system, generally control disease exacerbations, but have failed to stop progression. Therefore, it is essential to identify common immune pathways that contribute to MS pathology. NKG2D is a co-activating receptor of immune cells that plays a critical role in immune surveillance. Nevertheless, aberrant NKG2D-mediated responses can contribute to inflammation and tissue damage. Various studies have implicated the NKG2D pathway in MS. NKG2D blocking or depletion reduced disease severity in various EAE models, a commonly used animal model of MS. In the human pathology, NKG2D+CD4+ and CD8+ T lymphocytes have been found in MS lesions and are upregulated in the peripheral blood of RRMS patients under relapse. Moreover, at least one NKG2DL has been observed in oligodendrocytes from MS lesions, which were found near CD8+ T lymphocytes. Furthermore, in vitro studies have demonstrated NKG2D-dependent killing of human oligodendrocytes by CD8+ T lymphocytes. Whether other neural cells express NKG2DL and can thus be susceptible to NKG2D-mediated recognition was still unknown. In this thesis, we investigated further the contribution of the NKG2D pathway to the pathobiology of MS. The first part of this project consisted in the evaluation of NKG2DL expression within the CNS. We found upregulated levels of ULBP4, and no other NKG2DL, in MS lesions and normal appearing white matter from MS patients. Moreover, we identified potential triggers observed in MS lesions that could impact on ULBP4 expression. Soluble ULBP4 was also found in the cerebrospinal fluid, and functional assays demonstrated its capacity to boost inflammatory cytokines secretion by CD8+ T lymphocytes. In the second part, we performed a deep characterization of CD4+ and CD8+ T lymphocytes expressing NKG2D in blood samples from relapsing-remitting and progressive forms of MS as well as age and sex matched healthy controls. Despite finding similar proportions, NKG2D+CD4+ T lymphocytes from MS patients exhibited an activated memory phenotype associated with Th1 and Th1/Th17 responses. In contrast, NKG2D+CD8+ T lymphocytes were reduced in RRMS patients. This subset displayed a predominant granzyme B expression irrespective of the donors’ group, and exhibited degranulating capacities toward ULBP4-expressing astrocytes. Finally, live imaging analysis revealed a role for NKG2D and its ligand ULBP4 in the establishment of long-lasting interaction between astrocytes and CD8+ T lymphocytes. This provides a new mechanism involved in the dialogue between these cell types. Overall, this thesis project provides a deep characterization of the NKG2D pathway in relapsing-remitting and progressive MS patients. Moreover, it provides new evidence for the involvement of ULBP4, a specific NKG2DL, in the pathophysiology of MS. Further investigations will contribute to elucidate the validity of ULBP4 as a therapeutic target in MS.

Réalisé en cotutelle avec le Dr James G Martin de l'Université McGill (Meakins-Christie laboratories) / L’asthme chez l’homme et le souffle chez le cheval sont des maladies inflammatoires chroniques des voies respiratoires partageant plusieurs caractéristiques physiopathologiques dont la bronchoconstriction réversible, l’inflammation des voies respiratoires inférieures, l’hyperréactivité bronchique et le remodelage tissulaire. Les phénotypes cliniques d’asthme se caractérisent en partie selon le type d’inflammation affectant les voies respiratoires et la présence ou non d’allergie. Le souffle chez le cheval s’avère être un modèle adapté pour l’étude des mécanismes impliqués dans l’asthme neutrophilique, lesquels demeurent particulièrement mal compris, en contraste avec ceux associés avec l’asthme éosinophilique. La réponse immunologique sous-jacente au souffle implique entre autres l’expression de cytokines de type Th2, suggestives d’une réponse allergique (immunité acquise). La poussière environnementale qui provoque les symptômes du souffle contient également des agents non-spécifiques dérivés de bactéries, champignons et moisissures, susceptibles d’activer des mécanismes immunitaires innés chez les chevaux atteints du souffle. Nous avons étudié le rôle des neutrophiles dans l’inflammation associée à la réponse innée et acquise chez le cheval atteint du souffle. Dans un premier temps, l’effet de produits dérivés de bactéries sur l’activation des neutrophiles sanguins provenant de chevaux normaux et atteints de souffle a été étudié dans le but d’évaluer la contribution de la réponse innée dans la physiopathologie du souffle. Nous avons évalué l’effet de l’IL-4, une cytokine de type Th2, sur les neutrophiles des deux groupes de chevaux afin d’évaluer de quelle manière le neutrophile peut participer à la réponse acquise associée à la réponse allergique. Finalement, nous avons étudié l’expression des isoformes de l'arginase par les neutrophiles équins car cette enzyme métabolise la L-arginine et est potentiellement impliquée dans le bronchospasme et le remodelage tissulaire associés à l’asthme. Nos résultats suggèrent que les neutrophiles et les mononucléaires sanguins isolés des chevaux atteints du souffle possèdent une réponse inflammatoire exagérée en réponse aux lipopolyssacharides et peptides formylés et surexpriment les cytokines pro-inflammatoires IL-1β, TNF et IL-8. Cette réponse innée aberrante est associée à une inflammation systémique caractérisée par des concentrations sériques élevées de TNF chez les chevaux atteints du souffle en période de rémission clinique. De plus, nos résultats montrent que l’IL-4 active le neutrophile équin et favorise son chimiotactisme de manière autocrine. L’IL-4 induit un phénotype d’activation typique dans le neutrophile équin, caractérisé par l’expression accrue des cytokines pro-inflammatoires (IL-8 et TNF) ainsi que de récepteurs potentiellement impliqués dans la réponse allergique (IL-4Rα et CD23). Enfin, nous montrons que que l’arginase 1 n’est pas un marqueur de l’activation des neutrophiles équins par l’IL-4, mais que ces cellules expriment constitutivement l’isoforme 2 fonctionnelle de l’arginase. La surrégulation des deux isoformes au niveau des poumons périphériques semble être associée à la pathologie du souffle, ce qui est en accord avec les modèles d’asthme chez la souris, le rat et le cobaye. L'ensemble de ces travaux suggère que les neutrophiles sont des cellules effectrices importantes de la réponse innée et acquise dans la pathophysiologie du souffle, un modèle naturel d’asthme neutrophilique. / Human asthma and equine heaves are chronic pulmonary diseases sharing several pathophysiological properties including lower airway inflammation, reversible bronchoconstriction, bronchial hyperresponsiveness, and tissue remodeling. Clinical phenotypes of asthma are characterized in part by the inflammatory cell populations infiltrating the airways, and the presence or absence of allergy. Heaves is a suitable animal model for the study of the poorly defined pathophysiological processes leading to airway neutrophilia. The immune response in heaves involves Th2 cytokine expression, which is, among other features, associated to allergic inflammation (acquired immunity). Environmental dust exposure leading to clinical exacerbation of heaves contains non-specific agents derived from bacteria, molds or fungi which could also activate innate immune responses in heaves affected horses. We studied the role of neutrophils in innate and acquired immune responses in heaves affected-horse. First, innate immune responses of neutrophils isolated from normal and heaves-affected horses to bacterial-derived products were studied. We also assessed the effect of IL-4, a Th2 cytokine, on equine neutrophils isolated from both groups of horses. Finally, we evaluated the arginase isoforms expressed by equine neutrophils as this enzyme that takes part to the L-arginine metabolism and is thought to contribute to bronchospasm and tissue remodeling associated with asthma. Our results suggest that both neutrophils and mononuclear cells from heaves-affected horses, when compared to healthy horses, have an excessive inflammatory response to lipopolyssacharides and formylated peptides characterized by increased IL-1β, IL-8 and TNF expression. This altered innate response was associated with systemic inflammation in asymptomatic susceptible horses as high serum TNF concentrations were detected. Furthermore, we found that equine neutrophils are activated by IL-4 and release neutrophil chemotactic factors in response to this cytokine. IL-4 also induces a distinctive activation phenotype in neutrophils that is characterized by increased expression of the pro-inflammatory cytokines (IL-8 and TNF) and receptors (IL-4Rα and CD23) potentially involved in the allergic response. Finally, we showed that arginase 1 is not a marker of IL-4-activated equine neutrophils although they constitutively express a functionally active isoform 2 of the enzyme. The up-regulation of arginase isoforms in the peripheral lungs of horses with heaves suggests a role for arginase in this model, as it is described in the mouse, rat and guinea pig models. Taken together, this work suggest that neutrophils could play an important role in both innate and acquired immune responses associated with heaves pathophysiology, a natural model of neutrophilic asthma.

L’insuffisance hépatique aiguë (IHA) se caractérise par la perte soudaine de la fonction hépatique résultant de la nécrose massive des hépatocytes en l’absence de pathologie hépatique préexistante. L’IHA s’accompagne de perturbations métaboliques et immunologiques qui peuvent entraîner l’apparition de complications périphériques et cérébrales telles qu’un syndrome de réponse inflammatoire systémique (SIRS), une encéphalopathie hépatique (EH), un œdème cérébral, une augmentation de la pression intracrânienne, et la mort par herniation du tronc cérébral. Les infections sont une complication fréquente de l’IHA et elles sont associées à un risque accru de développer un SIRS et une aggravation subséquente de l’EH avec un taux de mortalité augmenté. L’ammoniaque joue un rôle majeur dans les mécanismes physiopathologiques qui mènent au développement de l’EH et de l’œdème cérébral, et des études récentes suggèrent que les cytokines pro-inflammatoires sont également impliquées. Le but de cette thèse est d’étudier le rôle des cytokines pro-inflammatoires circulantes et cérébrales dans le développement de l’EH et de l’œdème cérébral lors d’IHA. Dans l’article 1, nous démontrons que l’inhibition périphérique du facteur de nécrose tumorale-α (TNF-α) par l’etanercept retarde la progression de l’EH en diminuant le dommage hépatocellulaire, réduisant l’inflammation périphérique et centrale ainsi que le stress oxydatif/nitrosatif hépatique et cérébral associé chez la souris avec une IHA induite par l’azoxyméthane (AOM). Ces résultats démontrent un rôle important du TNF-α dans la physiopathologie de l’EH lors d’IHA d’origine toxique et suggèrent que l’etanercept pourrait constituer une approche thérapeutique dans la prise en charge des patients en attente de transplantation hépatique. Dans l’article 2, nous simulons la présence d’une infection chez la souris avec une IHA induite par l’AOM pour mettre en évidence une éventuelle augmentation de la réponse inflammatoire. Nous démontrons que l’endotoxémie induite par le lipopolysaccharide (LPS) précipite la survenue du coma et aggrave la pathologie hépatique. Les cytokines pro-inflammatoires systémiques et cérébrales sont augmentées de façon synergique par le LPS lors d’IHA et résultent en une activation accrue de la métalloprotéinase matricielle-9 cérébrale qui s’accompagne d’une extravasation d’immunoglobulines G (IgG) dans le parenchyme cérébral. Ces résultats démontrent une augmentation majeure de la perméabilité de la barrière hémato-encéphalique (BHE) qui contribue à la pathogenèse de l’EH lors d’IHA en condition infectieuse. Les résultats de l’article 3 démontrent que l’augmentation de la perméabilité de la BHE lors d’IHA induite par l’AOM en condition non infectieuse ne résulte pas de l’altération de l’expression des protéines constitutives de la BHE. Dans l’article 4, nous démontrons que l’exposition d’astrocytes en culture à des concentrations physiopathologiques d’ammoniaque ou d’interleukine-1β résulte en l’altération de gènes astrocytaires impliqués dans la régulation du volume cellulaire et dans le stress oxydatif/nitrosatif. Un effet additif est observé dans le cas d’un traitement combiné au niveau des gènes astrocytaires impliqués dans le stress oxydatif/nitrosatif. L’ensemble des résultats de cette thèse démontre un rôle important de l’inflammation périphérique et cérébrale dans la survenue des complications neurologiques lors d’IHA et une meilleure compréhension des mécanismes physiopathologiques impliqués pourrait contribuer à la mise en place de stratégies thérapeutiques chez les patients atteints d’IHA en attente de transplantation. / Acute liver failure (ALF) is the clinical manifestation of an abrupt loss of hepatic function resuting from a massive hepatocyte necrosis in a patient with no preexisting liver disease. ALF is associated with metabolic and immunological disturbances that may lead to peripheral and cerebral complications such as systemic inflammatory response syndrome (SIRS), hepatic encephalopathy (HE), brain edema, increased intracranial pressure (ICP) and ultimately death by cerebral herniation. ALF is frequently complicated by infections, which are known to increase the risk of developing a SIRS with a subsequent worsening of HE and higher mortality rates. Ammonia plays a pivotal role in the pathophysiological mechanisms leading to HE and brain edema, and recent studies suggest that pro-inflammatory cytokines may also be involved. The aim of this thesis is therefore to investigate the role of circulating and cerebral pro-inflammatory cytokines in the setting of HE and brain edema during ALF. In article No. 1, we demonstrated that peripheral inhibition of tumor necrosis factor-alpha (TNF-α) by etanercept delays the progression of HE by reducing hepatocellular damage, decreasing peripheral and cerebral inflammation as well as associated oxidative/nitrosatif stress in mice with ALF induced by azoxymethane (AOM). These findings demonstrate an important role of TNF-α in the pathophysiology of HE during toxic liver injury and suggest that etanercept may provide a therapeutic approach in the management of patient awaiting liver transplantation. In article No. 2, we mimicked infection in mice with AOM-induced ALF in order to better understand the effects of an increased inflammatory response. We demonstrated that endotoxemia induced by lipopolysaccharide (LPS) precipitates the onset of coma and worsens the liver pathology. Peripheral and brain pro-inflammatory cytokines are synergistically raised by LPS during ALF and result in a large increase in cerebral matrix metalloprotease-9 (MMP-9) activity that was associated with immunoglobulin G (IgG) extravasation in the brain parenchyma. These results demonstrate a major increase of blood-brain barrier (BBB) permeability that contributes to the pathogenesis of HE during ALF with superimposed infection. Results from article No. 3 demonstrate that increase of BBB permeability during AOM-induced ALF without superimposed infection is not due to alteration of BBB constitutive proteins. In article No. 4, we demonstrated that exposure of cultured astrocytes to pathophysiological concentrations of ammonia or interleukin-1β results in an alteration of the expression of astrocytic genes implicated in cell volume regulation and oxidative/nitrosative stress. An additive effect on astrocytic genes implicated in oxidative/nitrosative was made evident in case of co-treatment. Taken together, results of the present thesis demonstrate a major role of peripheral and cerebral inflammation in the onset of neurological complications during ALF and a better understanding of the pathophysiological mechanisms implicated may contribute to new therapeutic strategies for ALF patients awaiting transplantation.