Das Induzieren und Aufrechterhalten einer tumor-protektiven Immunität sind wesentliche Ziele in der Immuntherapie des Neuroblastoms. Eine Erhöhung der Anzahl von tumor-infiltrierenden Leukozyten könnte ein Weg sein, um dieses Ziel zu erreichen. Fractalkine ist ein besonderes TH1 CX3C Chemokin, welches sowohl Adhäsion und Migration von Leukozyten vermittelt. Gerichtetes IL-2 (ch14.18-IL-2) wurde durch eine genetische Fusion von anti-GD2 Antikörper mit IL-2 hergestellt, damit IL-2 spezifisch in das Mikromilieu von Neuroblastomen gebracht werden kann. In dieser Arbeit habe ich die Hypothese getestet, dass Gentherapie mit dem Chemokin Fractalkine (FKN) eine wirksame Antineuroblastom-Immunantwort induziert, welche durch gerichtetes IL-2 amplifiziert wird. Zu diesem Zweck wurden NXS2-Zellen genetisch verändert, damit sie murines FKN produzieren (NXS2-FKN). Transkription und Expression des mFKN Gens konnte in NXS2-FKN Zellen und Tumorgewebe gezeigt werden. Die chemotaktische Eigenschaft von FKN wurde sowohl in vitro als auch in vivo gezeigt. FKN zeigte eine Reduktion des Primärtumorwachstums, welches durch gerichtetes IL-2 mit nicht-kurativen Dosen von ch14.18-IL-2 deutlich verbessert wurde. Ferner wurden experimentelle Lebermetastasen nur in den Mäusen komplett eradiziert, welche die Kombinationstherapie erhalten haben. Die Mechanismen, welche an dieser Antitumorantwort beteiligt sind, schließen eine wirksame T-Zell-Aktivierung (Hochregulation von CD69, CD25, und von TNF-alpha und INF-gamma), sowie eine Erhöhung der tumorspezifischen CTL-Aktivität mitein. Die Depletion von CD4+ und CD8+ T-Zellen in vivo hat diesen therapeutischen Effekt aufgehoben, was die essentielle Rolle von T-Zellen in diesem immuntherapeutischen Ansatz unterstreicht. Zusammenfassend konnte ich zum ersten Mal zeigen, dass Chemokin-Gentherapie mit FKN durch gerichtetes IL-2 amplifiziert wird, was eine Kombination dieser beiden Strategien zur adjuvanten Therapie beim Neuroblastom nahe legt. / Induction and maintenance of tumor-protective immunity are the major goals of neuroblastoma immunotherapy. Enhancing the amount of tumor infiltrating leukocytes might be a way to achieve these goals since they may be associated with residual evidence of the ineffective immune response. Fractalkine is a unique TH1 CX3C chemokine known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Targeted IL-2 (ch14.18-IL-2) was constructed by anti-GD2 antibody fused with IL-2 so that IL-2 can be directed into the microenvironment of neuroblastoma tumor. Here, I tested the hypothesis that chemokine gene therapy with fractalkine (FKN) induces an effective anti-neuroblastoma immune response amplified by targeted IL-2. NXS2 cells were engineered to stably produce murine FKN (NXS2-FKN). Transcrip- tion and expression of the mFKN gene in NXS2-FKN cells and tumor tissue were demonstrated. The chemotactic activity of FKN expressed by NXS2 cells was determined both in vitro and in vivo. Importantly, NXS2-FKN exhibited a reduction in primary tumor growth, which was boosted by targeted IL-2 using non-curative doses of ch14.18-IL-2. Furthermore, experimental liver metastases were completely eradicated in mice receiving the combination therapy, demonstrating the induction of a long-lived tumor protective response. The mechanisms involved in antitumor response included effective T cell activation as indicated by the up-regulation of T-cell activation markers (CD69, CD25) and proinflammatory cytokines (TNF-alpha, INF-gamma) as well as the enhancement of tumor specific CTL activity. The depletion of CD4+ and CD8+ T cells in vivo abrogated the therapeutic effect supporting the crucial role of T cells in this immunotherapeutic approach. In summary, I demonstrated for the first time that chemokine gene therapy with FKN is amplified by targeted IL-2 suggesting a combination of both strategies as an adjuvant therapy for neuroblastoma.

Le cancer épithélial de l'ovaire (CEO) est une cause majeure de mortalité parcancer gynécologique. Il est associé à un mauvais pronostic car il est souventdécouvert à un stade tardif. Mieux comprendre les causes et les mécanismesmoléculaires et cellulaires associés à la progression de ce cancer représente unenjeu majeur.Les deux chimiokines CXCL12/SDF-1 et fractalkine (FKN)/CX3CL1 ont étéimpliquées dans diverses tumeurs. La chimiokine SDF-1, a un effetimmunosuppresseur dans le CEO. Elle est aussi impliquée dans l'angiogenèsetumorale. L'effet de SDF-1 médié par CXCR4 est également impliqué dans larégulation de la prolifération, la survie, la migration et l'invasion des cellulescancéreuses. La FKN, a largement été mise en évidence dans les tissusépithéliaux et dans divers cancers où elle peut avoir soit un rôle anti-tumoral soitun rôle pro-tumoral. Jusqu'à présent la FKN n'a pas été étudié dans le CEO.Dans notre étude, nous avons démontré l'expression de SDF-1 et de la FKNdans l'épithélium de surface de l'ovaire sain et dans les tumeurs bénignes etmalignes. Ces résultats montrent que l'expression de SDF-1 et de la FKNpréexiste à la tumorigenèse. Nous avons démontré une expression hétérogènedes deux chimiokines dans les cellules du CEO. Les niveaux d'expression deSDF-1 dans les cellules tumorales sur une cohorte de 183 patientes n'ont aucunevaleur pronostique sur la survie globale et sur la survie sans progressiontumorale des patientes atteintes par le CEO. L'étude de la corrélation del'expression de la FKN avec les deux marqueurs de prolifération, Ki-67 etGILZ, sur une autre cohorte de 54 patientes, complétée par des expériences invitro, a montré que GILZ augmente l'expression de la FKN et d'autre part que laFKN elle-même augmente la prolifération. Cette étude contribue à élucider lerôle de SDF-1 et de la FKN dans le CEO.

A disfunção temporomandibular (DTM) afeta os músculos da mastigação, a articulação temporomandibular e estruturas associadas, induzindo dor por mecanismos pouco compreendidos. A inflamação tem papel relevante nessas disfunções crônicas pela produção de mediadores químicos. A fractalquina (FK) é uma quimiocina expressa na membrana de neurônios, e seu receptor esta presente na microglia, sugerindo que a FK induz a ativação da microglia, promovendo mudanças funcionais e liberação de mediadores inflamatórios, envolvidas na manutenção de processos nociceptivos. As terapias medicamentosas possuem efeitos indesejáveis. O laser de baixa intensidade (LBI) é uma alternativa promissora para o alívio da dor. O efeito do LBI foi avaliado sobre a nocicepção de animais em modelo de dor persistente e foi capaz de reverter a hipersensibilidade mecânica por uma inibição da resposta inflamatória local e inibição central de fractalquina reforçando papel do LBI como uma alternativa terapêutica importante no alívio dor e modulação da resposta inflamatória em nível central. / Temporomandibular dysfunction (TMD) affects the chewing muscles, the temporomandibular joint and associated structures, inducing pain by poorly understood mechanisms. Inflammation has a relevant role in these chronic dysfunctions by the production of chemical mediators. Fractalkine (FK) is a chemokine expressed on the membrane of neurons, and its receptor is present in the microglia, suggesting that FK induces the activation of microglia, promoting functional changes and release of inflammatory mediators involved in the maintenance of nociceptive processes. Drug therapies have undesirable effects. Low-intensity laser (LILT) is a promising alternative for pain relief. The effect of LILT was evaluated on the nociception of animals in a persistent pain model and was able to reverse mechanical hypersensitivity by an inhibition of the local inflammatory response and central inhibition of fractalkine enhancing LIL\'s role as an important therapeutic alternative in pain relief and modulation Inflammatory response at the central level.

The pathophysiology of atherosclerotic cardiovascular disease (CVD) is highlighted by vascular dysfunction and low-grade vascular inflammation. Furthermore, the site-specific distribution of atherosclerosis throughout the arterial vasculature is primarily determined by local hemodynamic force. Therefore, this dissertation outlines three experiments designed to investigate the role of acute mental and physical (i.e., aerobic exercise), and vascular wall shear stress (SS) on the inflammatory aspects of atherosclerosis. Chapter 2 examines the effect of acute laboratory-induced mental stress on intracellular pro-inflammatory signaling pathways in peripheral blood mononuclear cells. Chapter 3 investigates the impact of acute laboratory-induced mental stress and maximal aerobic exercise on the concentration of soluble VCAM-1 (sVCAM-1) and CX3CL1/fractalkine (sCX3CL1) in human serum. Lastly, Chapter 4 examines the role of short- (30 min) and long-term (24 hr) low-to-negative oscillating SS (LOSS) and high laminar SS (HLSS) on the expression and secretion (i.e., cleavage) of cell-membrane VCAM-1 and CX3CL1 by human umbilical vein endothelial cell cultures in vitro. Together, these experiments provide evidence that acute psychological stress, maximal aerobic exercise, and HLSS influence vascular inflammation and adhesive properties of the vessel wall. More specifically, the results from Chapter 2 provide evidence that acute mental stress promotes the immune-cell mediated synthesis of pro-inflammatory cytokines in circulation. In addition, Chapter 3 and Chapter 4 demonstrate that the elevations in blood flow and hemodynamic force associated with maximal aerobic exercise, and unidirectional high SS may have the capacity to alter the expression of endothelial-bound cellular adhesion molecules, in part by eliciting their release from the vessel wall.

Nos últimos anos, os estudos de nosso grupo foram focados na área de dor, avaliando diferentes modelos experimentais de dor aguda, neuropática e muscular. A busca por mecanismos moduladores destes tipos de dores são alvo de grande estudo, uma vez que o fenômeno da dor é peculiar e torna-se de difícil tratamento em muitos casos. Distúrbios (VER) musculoesqueléticos são as principais causas de incapacidade nas pessoas durante seus anos de trabalho. Diversos estudos tem sido realizados lesionando o músculo gastrocnêmio como modelo experimental em diferentes animais para melhor entendimento deste tipo de dor. O nosso objetivo foi observar possíveis alterações histológicas no tecido muscular, avaliar a alteração na sensibilidade nociceptiva e a atividade locomotora dos animais após a indução de dor muscular crônica, bem como observar o envolvimento das células gliais na medula espinal destes animais. Em adição, avaliamos a participação de determinadas citocinas com o intuito de obter um perfil inflamatório em nosso modelo experimental. Nossos resultados demonstraram um quadro de inflamação instalada no tecido muscular de animais com miosite crônica através das analises histológicas realizadas. Os testes comportamentais tanto para hiperalgesia mecânica como térmica e alodinia confirmaram a instalação do quadro álgico uma vez que os animais com miosite apresentaram uma queda em seus limiares nociceptivos em relação aos grupos controle. A atividade locomotora dos animais também se demonstrou comprometida após a indução de miosite. Em relação à participação das células gliais neste modelo, demonstramos que houve um aumento na expressão de GFAP e OX-42, correspondentes à marcação astrócitos e células da microglia na porção lombar da medula espinal dos animais com miosite, quando comparados ao grupo controle. Quanto à participação dos mediadores sistêmicos, observamos um aumento nos níveis de IL-1β e fractalquina (FKN) no sangue dos animais, enquanto o nível de IL-10 permaneceu baixo em relação ao grupo controle. Com nossos achados esperamos colaborar com o aprimoramento de estratégias terapêuticas para tratamento de dores musculares. / In the last years, the studies of our group were focused on the area of pain, evaluating different experimental models of acute, neuropathic and muscular pain. The search for mechanisms modulating types of pain are the subject of great study, since the phenomenon of pain is peculiar and becomes difficult in many cases. Musculoskeletal (VER) disorders are the leading causes of disability in people during their working years. Several studies have been carried out with the model of experimental model in different animals to better understand this type of pain. Our objective was to observe the non-muscular histological changes, to evaluate the nociceptive sensitivity and a locomotor activity of the animals after an induction of chronic muscular pain, as well as to observe the involvement of the glial cells in the spinal cord of the animals. In addition, we evaluated the participation of certain cytokines in order to obtain an inflammatory profile in our experimental model. Our results demonstrated a picture of inflammation installed without muscle tissue of animals with chronic myositis through histological analysis. Behavioral tests for both mechanical and thermal hyperalgesia and allodynia confirmed the onset of pain since animals with myositis showed a decrease in their nociceptive thresholds in relation to the control groups. The locomotor activity of the animals was also shown to be impaired after an induction of myositis. Regarding the participation of glial cells in this model, we demonstrated that there was an increase in the expression of GFAP and OX-42, corresponding to marking astrocytes and microglia cells in the portion of the spinal cord of animals with myositis, when compared to the control group. Regarding the participation of systemic mediators, we observed an increase in the levels of IL-1β and fractalkin (FKN) in the blood of the animals, while the level of IL-10 remained low in relation to the control group. With the findings we hope to collaborate with the improvement of therapeutic strategies for the treatment of muscular pain.

No description available.

Le cancer épithélial de l'ovaire (CEO) est une cause majeure de mortalité parcancer gynécologique. Il est associé à un mauvais pronostic car il est souventdécouvert à un stade tardif. Mieux comprendre les causes et les mécanismesmoléculaires et cellulaires associés à la progression de ce cancer représente unenjeu majeur.Les deux chimiokines CXCL12/SDF-1 et fractalkine (FKN)/CX3CL1 ont étéimpliquées dans diverses tumeurs. La chimiokine SDF-1, a un effetimmunosuppresseur dans le CEO. Elle est aussi impliquée dans l'angiogenèsetumorale. L'effet de SDF-1 médié par CXCR4 est également impliqué dans larégulation de la prolifération, la survie, la migration et l'invasion des cellulescancéreuses. La FKN, a largement été mise en évidence dans les tissusépithéliaux et dans divers cancers où elle peut avoir soit un rôle anti-tumoral soitun rôle pro-tumoral. Jusqu'à présent la FKN n'a pas été étudié dans le CEO.Dans notre étude, nous avons démontré l'expression de SDF-1 et de la FKNdans l'épithélium de surface de l'ovaire sain et dans les tumeurs bénignes etmalignes. Ces résultats montrent que l'expression de SDF-1 et de la FKNpréexiste à la tumorigenèse. Nous avons démontré une expression hétérogènedes deux chimiokines dans les cellules du CEO. Les niveaux d'expression deSDF-1 dans les cellules tumorales sur une cohorte de 183 patientes n'ont aucunevaleur pronostique sur la survie globale et sur la survie sans progressiontumorale des patientes atteintes par le CEO. L'étude de la corrélation del'expression de la FKN avec les deux marqueurs de prolifération, Ki-67 etGILZ, sur une autre cohorte de 54 patientes, complétée par des expériences invitro, a montré que GILZ augmente l'expression de la FKN et d'autre part que laFKN elle-même augmente la prolifération. Cette étude contribue à élucider lerôle de SDF-1 et de la FKN dans le CEO.

A técnica de laserterapia é um método não-invasivo que demonstra clinicamente ser eficaz na redução da sensibilidade à dor. O presente estudo visou examinar os efeitos da aplicação do LASER sobre a sensibilidade dolorosa induzida pela constrição crônica do nervo isquiático (CCI) de ratos. Os animais foram submetidos a ensaios comportamentais e a dez sessões de laserterapia. Observamos melhora para os testes comportamentais o que corrobora com os ensaios de imunoblotting no gânglio da raiz dorsal, onde observamos uma diminuição de Substância P no grupo de animais tratados com LASER. Com relação aos ensaios imunoenzimático de ELISA, observamos diminuição de citocinas pró-inflamatória e uma tendência ao aumento de citocina anti-inflamatória. Não observamos diferença estatística na análise dos receptores opióides MOR , DOR e KOR . Podemos concluir que o LASER é eficaz e age na modulação da dor neuropática. / The technique of laser therapy is a not invasive method that demonstrates clinically to be effective in reducing sensitivity to pain. This study aimed to examine the effects of application of LASER on pain sensitivity induced by chronic constriction of the sciatic nerve (CCI) in rats. The animals were subjected to behavioral tests and the ten sessions of laser therapy. We observed and improvement for behavioral tests which corroborates with immunoblotting assays in the dorsal root ganglion, where we observes a decrease of substance P in the group of animals treated with LASER. Regarding immunoenzymatic ELISA assay, we observed a decrease of pro-inflammatory cytokines and a possible increase in anti-inflammatory cytokine. No statistical difference in the analysis of opioid receptor MOR, DOR and KOR.

A técnica de laserterapia é um método não-invasivo que demonstra clinicamente ser eficaz na redução da sensibilidade à dor. O presente estudo visou examinar os efeitos da aplicação do LASER sobre a sensibilidade dolorosa induzida pela constrição crônica do nervo isquiático (CCI) de ratos. Os animais foram submetidos a ensaios comportamentais e a dez sessões de laserterapia. Observamos melhora para os testes comportamentais o que corrobora com os ensaios de imunoblotting no gânglio da raiz dorsal, onde observamos uma diminuição de Substância P no grupo de animais tratados com LASER. Com relação aos ensaios imunoenzimático de ELISA, observamos diminuição de citocinas pró-inflamatória e uma tendência ao aumento de citocina anti-inflamatória. Não observamos diferença estatística na análise dos receptores opióides MOR , DOR e KOR . Podemos concluir que o LASER é eficaz e age na modulação da dor neuropática. / The technique of laser therapy is a not invasive method that demonstrates clinically to be effective in reducing sensitivity to pain. This study aimed to examine the effects of application of LASER on pain sensitivity induced by chronic constriction of the sciatic nerve (CCI) in rats. The animals were subjected to behavioral tests and the ten sessions of laser therapy. We observed and improvement for behavioral tests which corroborates with immunoblotting assays in the dorsal root ganglion, where we observes a decrease of substance P in the group of animals treated with LASER. Regarding immunoenzymatic ELISA assay, we observed a decrease of pro-inflammatory cytokines and a possible increase in anti-inflammatory cytokine. No statistical difference in the analysis of opioid receptor MOR, DOR and KOR.