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Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatmentDallas, Shannon, Block, Michelle, Thompson, Deborah, Bonini, Marcelo, Ronaldson, Patrick, Bendayan, Reina, Miller, David January 2013 (has links)
BACKGROUND:Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide.METHODS:To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS).RESULTS:3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters.CONCLUSIONS:The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-kappabeta and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.
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Localisation, mécanisme d’induction et rôle physiopathologique du récepteur B1 des kinines dans de modèles expérimentaux de douleur chez le ratTalbot, Sébastien 06 1900 (has links)
Les kinines sont des peptides neuro- et vaso- actifs impliqués dans les processus hémodynamiques, inflammatoires et douloureux. Leurs effets biologiques sont produits par l’entremise de deux types de récepteurs couplés aux protéines G, soit B1 (B1R) et B2 (B2R). Le B1R est inductible, son expression est augmentée à la suite d’un dommage tissulaire ou de l’exposition à des endotoxines bactériennes (lipopolysaccharide bactérien (LPS)), à des cytokines pro-inflammatoires (interleukine-1β (IL-1β), facteur de nécrose tumorale-α (TNF-α)) ou à des espèces réactives oxygénées (ROS). Les travaux présentés dans cette thèse avaient pour objectif d’élucider et/ou de raffiner les connaissances sur 1) la localisation, 2) le mécanisme d’induction et 3) le rôle physiopathologique du B1R dans des modèles expérimentaux de douleur chez le rat. Nos données ont permis de démontrer pour la première fois que le B1R est augmenté de façon significative dans la moelle épinière du rat diabétique de type 1 où il est localisé sur les fibres sensorielles de type C, les astrocytes et les cellules de la microglie (1er article). Également, l’inhibition de l’activation des cellules de la microglie supprime les neuropathies diabétiques, l’expression de médiateurs pro-inflammatoires ainsi que l’activité pro-nociceptive du B1R (2e et 3e articles). Finalement, nous avons démontré que la stimulation systémique du TRPV1 par la capsaïcine induit une surexpression du B1R au niveau microgliale, via un mécanisme impliquant l’augmentation de la production de ROS et possiblement de cytokines (4e article). Ces données nous permettent de mieux comprendre les mécanismes impliqués dans l’expression et l’activité du B1R. Aussi, elles nous permettent d’imaginer de nouvelles stratégies pour prévenir l’induction du B1R (inhibition du TRPV1) ou son activité délétère (inhibition de l’activation des cellules de la microglie) dans la douleur inflammatoire et neuropathique. / Kinins are vaso- and neuro-active peptides involved in hemodynamic, inflammatory and pain processes. Their biological effects are mediated by two G Protein Coupled Receptors (GPCR), termed B2R (constitutive) and B1R (inducible). B1R is expressed following tissue damage or exposure to bacterial endotoxin (LPS), pro-inflammatory cytokines (IL-1β, TNF-α) and increased reactive oxygen species (ROS) levels. The objectives of this doctoral thesis were to define 1) the localisation, 2) the mechanism of induction and 3) the pathophysiological role of B1R in experimental models of pain in rat. Our data showed that B1R is significantly upregulated on sensory C fibers, astrocytes and microglia in spinal cord of type 1 diabetic rat (paper #1). Moreover, pharmacological inhibition of microglia reversed diabetic pain neuropathy, reduced levels of pro-inflammatory mediators and prevented B1R pro-nociceptive activity (papers #2 and 3). Finally, our data showed that systemic stimulation of TRPV1 with capsaicin upregulated B1R expression, mainly on microglia, through the increase of ROS and possibly cytokines (paper #4). Altogether, these data increased our knowledge related to B1R mechanism of induction and B1R activity. Also, these data shed light on new strategies to prevent B1R expression (TRPV1 blockade) and B1R deleterious activity (inhibition of microglia activation) in inflammatory and neuropathic pain.
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La rigidité artérielle, induite par une calcification des carotides, altère l’homéostasie cérébrale chez la sourisSadekova, Nataliya 04 1900 (has links)
La rigidité artérielle est considérée comme un facteur de risque important pour le développement du déclin cognitif. Toutefois, les effets précis de la rigidité artérielle sur le cerveau sont peu connus et, à ce jour, aucun modèle animal ne permet d’étudier l’effet isolé de ce facteur sur l’homéostasie cérébrale. Dans cette étude, nous avons développé un nouveau modèle de rigidité artérielle qui se base sur la calcification de l’artère carotide chez la souris. Au niveau artériel, ce modèle présente une fragmentation de l’élastine, une augmentation de la distribution du collagène et de l’épaisseur intima-média ainsi qu’une diminution de la compliance et de la distensibilité artérielles démontrant la rigidité artérielle. De plus, le modèle ne présente pas d’augmentation de pression artérielle ni de changement de rayon du lumen indiquant une absence d’hypoperfusion globale et d’anévrisme. Au niveau cérébral, les résultats montrent que la rigidité artérielle induit une augmentation de la pulsatilité du flux sanguin cérébral menant ainsi à une augmentation du stress oxydatif. Ce dernier induit une inflammation cérébrale, détectée par l’activation de la microglie et des astrocytes, induisant ultimement une neurodégénérescence. Ces effets sont surtout observés au niveau de l’hippocampe, la région cruciale pour la mémoire et la cognition. Ainsi, cette étude montre que la rigidité artérielle altère l’homéostasie cérébrale et mérite d’être considérée comme une cible potentielle dans la prévention et le traitement des dysfonctions cognitives chez les personnes âgées. / Arterial stiffness is considered as an important risk factor for the development of cognitive decline in the elderly population. However, its precise effects on the brain are unknown and, to date, no animal model allows to study the precise outcome of arterial stiffness on the brain homeostasis. In this study, we developed a new animal model of arterial stiffness based on the calcification of the carotid artery in mice. On the arterial level, this model shows a fragmentation of elastin, increased collagen distribution and intima-media thickness as well as decreased arterial compliance and distensibility, thus fulfilling the major arterial stiffness properties. In addition, this model does not a show an increase in blood pressure or change in arterial lumen radius indicating a lack of global hypoperfusion and aneurysm. Regarding the brain, the results show that arterial stiffness induces an increase in cerebral blood flow pulsatility leading to increased oxidative stress. Oxidative stress induces brain inflammation, detected by the activation of microglia and astrocytes, ultimately leading to neurodegeneration. These effects are particularly observed in the hippocampus, a crucial area for memory and cognition. Thus, this study shows that arterial stiffness alters brain homeostasis and therefore should be considered as a potential therapeutical target for the prevention and treatment of cognitive dysfunction in the elderly.
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Resolution of Inflammation Rescues Axon Initial Segment DisruptionGeorge, Nicholas M 01 January 2016 (has links)
Axonal domains are required for proper neuron function. These domains are unstable and degenerate concurrent with the inflammation in multiple sclerosis (MS) and the inflammatory disease models experimental autoimmune encephalomyelitis (EAE) and lipopolysaccharide (LPS) induced inflammation. Previous studies from our laboratory have shown that the axon initial segment (AIS) is maintained independently of the presence of myelin, but that AIS disruption is seen in MS as well as EAE and LPS-mediated inflammation. AIS loss can be interrupted in the early stage of EAE using the anti-inflammatory drug Didox. However, the potential for Didox directed repair of the AIS in later stages of disease has not been investigated. Here, we utilize two models of CNS inflammation to assess the possibility of reversing AIS pathology. Based on our findings, we present the first evidence that AIS degeneration, an axonal pathology observed in MS and in chronic inflammation, is reversible.
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Rôle et expression de l'interleukine-27 dans le contexte de la sclérose en plaquesSénécal, Vincent 03 1900 (has links)
Des études antérieures ont indiqué que l’IL-27 supprime le développement de l’encéphalomyélite auto-immune expérimentale (EAE), un modèle murin de la sclérose en plaques (SEP). L’expression en ARNm d’IL-27 est maximale au pic du développement de l’EAE. Cependant, sa contribution dans la pathogenèse de la SEP demeure irrésolue. Nous avons investigué si l’IL-27 contribue à moduler les réponses immunes dans le système nerveux central (SNC) de patients SEP. Nos résultats d’immunohistochimie sur échantillons post-mortem de cerveaux humains ont révélé que la production des deux sous-unités d’IL-27 (EBI-3 et p28) est plus élevée chez des patients comparés à des contrôles. De plus, les astrocytes (GFAP) et les microglies/macrophages (Iba1) représentent des sources biologiques importantes de l’IL-27 dans les lésions. Les lymphocytes T CD4 et CD8 qui infiltrent le SNC des patients expriment d’ailleurs le récepteur de l’IL-27 composé des chaînes gp130 et TCCR, supportant le concept que ces cellules pourraient répondre aux sources locales d’IL-27. Nous avons également démontré que des combinaisons de cytokines pro-inflammatoires (IFNγ, IL-1β et TNF) augmentent l’expression in vitro d’IL-27 par les astrocytes et macrophages humains, et que les microglies/macrophages de phénotype M1 produisent l’IL-27. Enfin, nous avons démontré que les astrocytes humains expriment aussi le récepteur à l’IL-27 et répondent à l’IL-27 par la phosphorylation de STAT1, mais pas de STAT3. Une telle signalisation dans ces cellules mène à l’augmentation d’expression de la molécule de co-inhibition PD-L1 et de la sécrétion de la chimiokine CXCL10. / Multiple lines of evidence support the notion that IL-27 can dampen the severity of experimental autoimmune encephalomyelitis (EAE). Whether this cytokine contributes to the pathogenesis of the human disease multiple sclerosis (MS) is still unresolved. We investigated whether IL-27 locally contributes to modulate the immune responses in the central nervous system (CNS) of MS patients. We performed immunohistochemistry on human post-mortem brain tissue samples and observed that both IL-27 subunits (EBI-3 and p28) are elevated in MS brains compared to controls. Moreover, we identified that astrocytes (GFAP+ cells) as well as microglia/macrophages (Iba1+ cells) are important sources of IL-27 in MS brains. Infiltrating CD8 and CD4 T cells present in the CNS of MS patients express the receptor for IL-27, composed of gp130 and TCCR chains, supporting the notion that these cells can respond to the local sources of IL-27. We demonstrated that combinations of inflammatory cytokines (IFNγ, IL-1β and TNF) up-regulate in vitro IL-27 expression by human astrocytes and macrophages, and that M1 microglia/macrophages produce IL-27. Lastly, we demonstrated that human astrocytes also express IL-27R and respond to the cognate cytokine by triggering the phosphorylation of STAT1, but not STAT3. Such signaling leads to the upregulation of PD-L1 expression and CXCL10 production by astrocytes. Our data demonstrate that IL-27 and its receptor are elevated in the CNS of MS patients and that inflammatory mediators present in MS lesions contribute to up-regulate such expression. Finally, astrocytes are key mediators of the production of IL-27 but also respond to this cytokine.
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Role gliových buněk v imunitní odpovědi myší infikovaných neurotropní motolicí Trichobilharzia regenti / Role of glial cells in the immune response of mice infected by neurotropic fluke Trichobilharzia regentiMacháček, Tomáš January 2015 (has links)
A central nervous system (CNS) can be invaded by plenty of parasites, including parasitic helminths. Host's immune response during such infections includes not only participation of peripheral lymphocytes, but also astrocytes and microglia, resident glial cells present in the CNS. Activation of astrocytes and microglia has been recently demonstrated also in mice infected by neurotropic avian trematode Trichobilharzia regenti (Digenea: Schistosomatidae) for which mammals represent accidental hosts. The parasite does not mature in them and elicits development of inflammatory reaction in the CNS which may take part in parasite's destruction. Employing in vitro experiments, this thesis aimed at evaluation of the possible role of astrocytes and microglia in murine immune response to T. regenti. For this purpose, primary astrocyte and microglia culture preparations were established and the cells were then stimulated by antigens of T. regenti (homogenate of transformed cercariae, recombinant cathepsins B1.1 and B2). After that, production of nitric oxide and proinflammatory cytokines (IL-1 beta, IL-6, TNF-alpha) was measured. The results revealed that in vitro stimulated astrocytes and microglia increase production of nitric oxide, IL-6 and TNF-alpha. Such response to parasite's antigens could influence...
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Perfil de expressão gênica da micróglia humana e suas alterações relacionadas ao glioma / Human microglia expression profile and its alterations related to gliomaGalatro, Thais Fernanda de Almeida 12 September 2016 (has links)
A micróglia é essencial para a homeostase do Sistema Nervoso Central (SNC), função neuro-imune inata, e exerce papel importante na neurodegeneração, envelhecimento cerebral e tumorigênese. Gliomas difusos são tumores cerebrais primários caracterizados por crescimento infiltrativo e altas taxas de heterogeneidade, o que torna a doença praticamente incurável. Avanços em análises genéticas caracterizaram alterações moleculares relacionadas ao tempo de sobrevida e à resposta clínica desses pacientes, especialmente em glioblastomas (GBM). No entanto, a tumorigenicidade dos gliomas não é controlada unicamente por suas alterações genéticas. As interações entre as células tumorais, a micróglia residente e os macrófagos/monócitos infiltrados desempenham um papel crucial na modulação do crescimento e agressividade do glioma. Neste estudo, analisamos o fenótipo de ativação da micróglia/macrófagos em gliomas, incluindo astrocitomas e oligodendroglimas de diferentes graus de malignidade, apresentamos o perfil de expressão gênica da população pura de micróglia cortical e do tecido cerebral total correspondente. Usando sequenciamento de DNA de alta performance, classificamos as amostras de GBM em Proneural, Clássico e Mesenquimal. Em seguida, avaliamos os status de ativação da micróglia/macrófagos dessas amostras. Apesar do alto grau de heterogeneidade, pudemos observar níveis mais altos dos marcadores mielóides (IBA1, CD11b and CD68) em tumores astrocíticos comparados a tumores de origem oligodendrocítica e ao tecido não-neoplásico. Marcadores de anti-inflamação, como CD163, foram mais abundantes em astrocitomas, bem como em GBMs do subtipo Mesenquimal e Clássico; enquanto que marcadores de pró-inflamação, como IL1-beta, mostraram uma expressão mais heterogênea entre as amostras. Em seguida, micróglia foi isolada de 25 amostras de córtex parietal provenientes de autópsia de indivíduos cognitivamente preservados e foi feito o RNA-seq. Os resultados foram comparados à micróglia de camundongo e a outras células mielóides. Boa parte dos genes expressos pela micróglia humana foram similares àqueles expressos pela micróglia murina, como CX3CR1, P2YR12 e ITGAM. Porém, foram identificados genes de característica imune, abundantemente expressos na micróglia humana e não identificados na micróglia de camundongos, como TLR, Fcy, receptores do tipo SIGLEC, e fatores de transcrição NLRC5 e CIITA. A comparação dos dados de expressão gênica da micróglia com monócitos e macrófagos identificou novos marcadores que distinguem a micróglia humana de outras células mielóides. Nossos dados sobre a micróglia em gliomas sugerem características de imunossupressão e de pró-crescimento em tumores de pior prognóstico, ligado a um fenótipo específico de ativação das células mielóides. Este é o primeiro estudo a identificar o transcriptoma da micróglia humana pura, demonstrando que ela é claramente diferente da micróglia murina e de outras células mielóides. Esses resultados abrem portas para estudos de populações específicas de células mielóides em gliomas / Microglia are essential for central nervous system (CNS) homeostasis and innate neuroimmune function, and play important roles in neurodegeneration, brain aging and tumorigenesis. Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular alterations leading to impact on patients\' overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells, resident microglia and infiltrating monocytes/macrophages plays a crucial role in modulating glioma growth and aggressiveness. Here, we assess the activation status of microglia/macrophages in gliomas,including astrocytomas and oligodendrogliomas of different grades of malignancy, and present the gene expression profile of pure cortical human microglia and corresponding unsorted brain tissue. Using high-throughput DNA sequencing, we have classified GBM samples in Proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Next, microglia were isolated from the parietal cortex of 25 autopsy samples of cognitively preserved humans and RNA sequenced. Overall, genes expressed by human microglia are similar to mouse microglia, such as CX3CR1, P2YR12, and ITGAM. Interestingly, a number of immune genes, not identified as mouse microglia signature genes, were abundantly expressed in human microglia, such as TLR, Fcy and SIGLEC receptors and NLRC5 and CIITA transcription factors. Comparison of microglia to monocyte and macrophage expression data underscored the CNS-specific functions of microglia and new markers were identified that distinguish human microglia from other myeloid cells. Our glioma-related data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. This data is the first comprehensive pure human microglia gene expression profile; human microglia clearly differ from mouse microglia and other myeloid cells. These results will help further studies focusing on pure myeloid cells populations in glioma
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Einwanderung und Differenzierung von hämatogenen Zellen zu Mikroglia im adulten ZentralnervensystemWehner, Tim 26 January 2004 (has links)
Zur langfristigen Markierung von hämatogenen Zellen wurde Knochenmark mit dem Gen für das grüne fluoreszierende Protein (GFP) transduziert und in bestrahlte Empfängermäuse transplantiert. Die GFP-Expression im peripheren Blut dieser Tiere war über den untersuchten Zeitraum von vier Monaten stabil. Die Hirne der Empfängertiere wurden zu den Zeitpunkten zwei, vier, acht und fünfzehn Wochen nach Knochenmarktransplantation auf die Präsenz von GFP-exprimierenden Zellen untersucht. Es fand sich eine im Zeitverlauf zunehmende Einwanderung und Differenzierung von GFP-exprimierenden hämatogenen Zellen zu ramifizierten Mikrogliazellen in der grauen und weißen Substanz. Nach vier Monaten stammten bis zu ein Viertel aller regionalen Mikrogliazellen aus dem transplantierten Knochenmark. Nach fokaler cerebraler Ischämie wanderten deutlich mehr GFP-positive Zellen aus dem Blut in das ischämische Areal ein und differenzierten zu ramifizierten Mikrogliazellen. Diese Ergebnisse implizieren einen Weg für den Transfer des humanen Immunodefizienzvirus in das Zentralnervensystem und offerieren einen nichtinvasiven Weg, genetisch manipulierte Zellen in das adulte Hirnparenchym einzuschleusen. / In order to stably label hematogenous cells, bone marrow was transduced with the gene for the green fluorescent protein (GFP) and transplanted into irradiated recipient mice. The GFP- expression in peripheral blood cells of these animals was stable within the examined time frame of four months. Brains of recipient animals were examined for the presence of GFP- expressing cells at two, four, eight and fifteen weeks after bone marrow transplantation. An increasing migration and differentiation of hematogenous GFP-expressing cells into ramified parenchymal microglia within the white and grey matter was found. After four months, up to quarter of regional microglia were bone-marrow derived. Following focal cerebral ischemia, an increased influx of GFP-positive blood-borne cells differentiating into ramified microglia was observed. These results imply a route for the human immunodeficiency virus into the central nervous system, and they offer a noninvasive approach for the transfer of genetically manipulated cells into the adult brain parenchyma.
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T-Zell-vermittelte AutoimmunitätGimsa, Ulrike 26 February 2004 (has links)
Die vorliegende Arbeit befaßt sich mit T-Helferzellen und ihren Interaktionen mit Gewebszellen, wie sie im gesunden Organismus und in Autoimmunerkrankungen auftreten. Es werden Fragen der Toleranzinduktion durch orale Gabe von Antigenen, speziell der oralen Verabreichung von Collagen II bei Patienten mit rheumatoider Arthritis diskutiert. Eine Immundeviation als Mittel, inflammatorische Th1-Zellantworten in anti-inflammatorische Th2-Zellantworten zu verwandeln, kann durch Eingriffe in die T-Zell-Signaltransduktion erreicht werden. Es werden neue Ansätze zu Mechanismen diskutiert, die das Immunprivileg des Zentralnervensystems gewährleisten. Die hirnresidenten Immunzellen, zu denen Mikrogliazellen und Astrozyten zählen, besitzen Eigenschaften, die eine Entzündung unwahrscheinlich machen. Sie müssen aktiviert werden, um Antigene präsentieren zu können. In organtypischen entorhinal-hippocampalen Schnittkulturen konnte gezeigt werden, dass Mikrogliazellen durch Th1-Zellen aktiviert, von Th2-Zellen hingegen deaktiviert werden. Die Möglichkeit, dass die Costimulation über CD80 oder CD86 differentielle Effekte auf den Charakter der Immunantwort hat, wird diskutiert. Der Einfluß von pro-inflammatorischen Zytokinen auf Mikrogliaaktivierung und den Erhalt von Nervenfasern wurde ebenfalls in Hirnschnittkulturen untersucht. Astrozyten sind wesentlicher Bestandteil der Blut-Hirn-Schranke. Diese kann jedoch von aktivierten T-Zellen überwunden werden. In dieser Arbeit wird gezeigt, dass Astrozyten über eine Expression von CD95L in aktivierten T-Zellen Apoptose induzieren können. Davon sind jedoch nicht alle T-Zellen betroffen. Andererseits wird eine T-Zellproliferation unterdrückt, indem T-Zellen unter Astrozyteneinfluß verstärkt CTLA-4 exprimieren, was einen Zellzyklusarrest zur Folge hat. Darüber hinaus ist eine verstärkte Produktion von Nervenwachstumfaktor (NGF; nerve growth factor) nach antigenspezifischer Interaktion von Astrozyten mit Th1- und Th2-Zellen als zusätzliches Mittel, eine Neuroinflammation einzudämmen, anzusehen. Die Arbeit stellt diese Ergebnisse in fünf Kapiteln dar, welche gleichzeitig eine Einführung in die als Anlagen enthaltenen zehn Publikationen geben. / This thesis deals with T helper cells and their interactions with tissue cells as they occur in the healthy organism and in autoimmune diseases. Questions of tolerance induction by oral application of antigens are discussed especially oral treatment with type II collagen in patients with rheumatoid arthritis. In order to transform inflammatory Th1 responses into anti-inflammatory Th2 responses, immune deviation can be reached by interference with T-cell signal transduction. New approaches towards the different ways that the immune privilege of the central nervous system is maintained are discussed. The resident immune cells, i.e. microglia and astrocytes possess properties that make inflammation unlikely. They have to be activated in order to present antigens. It has been shown in organotypic entorhinal-hippocampal slice cultures that Th1 cells activate whereas Th2 cells deactivate microglial cells. The possibility is discussed as to whether costimulation via CD80 or CD86 differentially influences the character of the immune response. The influence of pro-inflammatory cytokines on microglial activation and preservation of nerve fibers has also been studied in brain slice cultures. Astrocytes are an essential part of the blood-brain barrier, which can be crossed by activated T cells. The thesis shows that astrocytes can induce apoptosis in activated T cells via expression of CD95L. However, not all T cells are affected. T cell proliferation is suppressed by increased CTLA-4 expression in T cells under the influence of astrocytes, resulting in a cell cycle arrest. An additional mechanism of confining neuroinflammation is increased production of the nerve growth factor (NGF) following antigen-specific interaction of astrocytes and Th1 and Th2 cells, respectively. These results are presented in five chapters that also introduce the ten attached publications.
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Untersuchungen zur strukturellen und funktionellen Plastizität des 20S-Proteasoms der Maus und seiner Modulierung durch den Proteasomaktivator PA28Stohwasser, Ralf 17 November 2000 (has links)
Die Studie beinhaltet eine biochemisch-molekularbiologische Analyse des 20S-Proteasoms und seiner Aktivierung durch Proteine der PA28-Familie. Das 20S-Proteasom ist die zentrale Epitop-prozessierende cytosolisch-nukleäre Protease des MHC-Klasse-I-Antigenpräsentationsweges. In Mikroglia, wie auch in anderen Zellen, unterliegt das Proteasom einer Interferon-g-(IFN-g)-vermittelten strukturellen Plastizität, d.h. einer Substitution der Untereinheiten der Aktiven Zentren. Durch diesen Austauschmechanismus werden proteolytische Schnittpräferenzen modifiziert, was für die Hierarchie von cytotoxischen T-Zellantworten von Bedeutung ist. Lipopolysaccharide (LPS) bewirken in Mikroglia ebenfalls Veränderungen der proteasomalen Zusammensetzung des 20S-Komplexes und seines PA700-Aktivators. Dies ist ein Hinweis auf die Rolle des Proteasoms auch bei der Prozessierung von Antigenen des endolysosomalen Antigenpräsentationsweges. Die Modulation der Zusammensetzung des 20S-Proteasoms in Mikroglia durch IFN-g und LPS ist ein weiterer Beleg für die Rolle der Mikroglia bei der zellulären Immunantwort im Zentralnervensystem. Funktionen des Proteasoms in der MHC-Klasse-I-Antigenpräsentation werden durch den Proteasomaktivator PA28 optimiert. Die PA28-Proteinfamilie besteht aus den Proteinen PA28a, PA28b und PA28g. Diese Studie trägt - basierend auf kinetischen Modellierungen, Mutagenese- und Protein-Protein-Interaktionsstudien und Untersuchungen zur MHC-Klasse-I-Antigen-präsentation in PA28-Transfektanten - zur funktionellen Neubewertung dieser drei Proteine bei. Die drei PA28-Proteine sind autonome Aktivatoren des Proteasoms. Heteromere PA28ab-Komplexe verursachen eine stärkere Aktivierung des Proteasoms als die homomeren PA28a-oder PA28b-Komplexe. Das PA28g-Protein ist in vitro ein schwacher Aktivator verschiedener proteasomaler Peptidaseaktivitäten, der dennoch in unserem in vivo-Modell eine verbesserte MHC-Klasse-I-Antigenpräsentation bewirkt. Kinetische Argumente sprechen für Funktionen der PA28a und PA28b-Proteine als Translokasen für Peptidsubstate und -produkte des Proteasoms. Anhand des HBx-Proteins des Hepatitis B-Virus wird die Möglichkeit illustriert, das vorgestellte Modell zur Analyse viraler Faktoren anzuwenden, die mit der Aktivierung des Proteasoms durch PA28 interferieren. / The 20S proteasome is the proteolytic core complex of the main cytoplasmic and nuclear protein degradation system. One of the numerous functions assigned to the 20S proteasome is the generation of antigenic peptides from intracellular proteins. MHC class I surface presentation of antigenic peptides is one key event of the cellular immune response. The presented study was aimed on the biochemical and molecular-biological analysis of the 20S proteasome and its activation by regulatory proteins of the PA28 family. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. Cultured mouse microglia was used as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. In response to interferon-g , constitutive active site subunits were replaced by inducible subunits as described for other cellular systems. These replacements result in altered proteolytic cleavage preferences, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Since lipopolysaccharide (LPS), a glycolipid of bacterial pathogens, also alters proteasome subunit composition of the 20S proteasome and its activator PA700, a function of microglial proteasome in processing of antigens of the endolysosomal pathway has been postulated. The modulation of the 20S proteasome subunit composition indicates that microglia is part of the cellular immune response in the central nervous system. The proteasome activator PA28 has been reported to optimize MHC class I antigen presentation. The PA28 protein family is composed of three proteins: PA28a, PA28b and PA28b. Based on kinetic modelling approaches, mutagenesis of activator proteins, protein-protein interaction studies and investigation of MHC class I antigen presentation in PA28-transfected cell lines a evaluation of functions of these three proteins has been performed. In vitro investigations revealed that the recombinant PA28 proteins are activators of peptidase activities of the proteasome. Heteromeric PA28ab complexes are stronger activators than homomeric PA28a o PA28b complexes. Recombinant PA28g is a weak activator of several peptidase activities. Nevertheless, in PA28g transfected B8-fibroblasts preliminary results indicate an improved MHC class I presentation. Based on kinetic evidence, a model has been presented indicating that PA28a and PA28b proteins act as peptide translocases, performing the import of substrates or the export of products into the catalytic chamber of the 20S proteasome. Finally, as examplified with the HBx protein of Hepatitis B virus, the opportunity is presented to use an in vitro reconstitution system of proteasomal activation to examine viral proteins interfering with proteasomal activation.
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