Seit vielen Jahren werden Glukokortikoide routinemäßig eingesetzt, um Zeichen der inflammatorischen Reaktion nach kardiochirurgischen Eingriffen unter extrakorporaler Zirkulation (EKZ) zu mildern. Glukokortikoide sind jedoch für ihre immunsuppressiven Wirkungen bekannt, und bisher blieben die möglichen Auswirkungen auf immunologische Funktionen weitgehend hypothetisch. Ziel der vorliegenden Studie war es daher, den Einfluss von Methylprednisolon (MP) und Tirilazad Mesylat (TM), einer antiinflammatorischen Substanz aus der Klasse der Aminosteroide auf immunologische Funktionen nach koronarchirurgischen Eingriffen mit EKZ zu untersuchen. 38 Patienten wurden randomisiert den Behandlungsgruppen Placebo (NaCl 0,9 %, n=13), MP (15 mg/ kg KG, n=12) und TM (10 mg/kg KG, n=13) zugeteilt. Die Verläufe der Plasmakonzentrationen von IL-6 und IL-10, der monozytären HLA-DR Expression und der ex vivo LPS-stimulierten TNF-alpha, IL-1RA, IFN-gamma und IL-12 Sekretion wurden bestimmt. Im Vergleich zu Placebo resultierte die Gabe von MP in geringeren postoperativen Plasmakonzentrationen von IL-6, aber einer deutlichen Erhöhung von IL-10. Die monozytäre HLA-DR Expression nahm postoperativ in allen Gruppen ab mit einer deutlichen Verstärkung durch MP. Die ex vivo stimulierte TNF-alpha Sekretion nahm postoperativ in allen Gruppen deutlich ab, ebenfalls mit einer deutlichen Verstärkung durch MP. Die IL-1RA Sekretion hingegen war zu keinem Zeitpunkt eingeschränkt. Die ex vivo stimulierte IFN-gamma und IL-12 war postoperativ in allen Gruppen stark vermindert ohne Einfluss einer medikamentösen Behandlung. Die Gabe von TM zeigte keinerlei Beeinflussung aller gemessenen Parameter im Vergleich zu Placebo. Nach koronarchirurgischen Eingriffen sind insbesondere monozytäre Funktionen stark eingeschränkt. Diese Suppression wird durch die Gabe von MP verstärkt, während die Gabe von TM nicht in einer zusätzlichen Immunsuppression resultiert. IL-10 scheint eine Schlüsselrolle bei der beobachteten monozytären Funktionseinschränkung einzunehmen. / Glucocorticoids have been routinely applied in cardiac surgery involving cardiopulmonary bypass (CPB) for many years in order to diminish inflammatory stress reactions. On the other hand glucocorticoids are well known for their immunosuppressive effects, and data on the consequences on immune function are scarce. Thus it was the aim of this trial to determine the influence of methylprednisolone (MP) and tirilazad mesylate (TM), an antiinflammatory drug of the class of aminosteroids, on immunological parameters after coronary surgery involving CPB. 38 patients were randomised to receive either placebo (NaCl 0.9 %, n=13), MP (15 mg/kg, n=12) or TM (10 mg/kg, n=13) treatment. Plasma concentrations of IL-6 and IL-10, monocyte surface expression of HLA-DR and the ex vivo endotoxin-stimulated secretion of TNF-alpha, IL-1RA, IFN-gamma und IL-12 were measured. Compared to placebo IL-6 concentrations were lower after MP treatment, whereas IL-10 levels were much higher. The rate of HLA-DR+-monocytes decreased in all groups with a significant aggravation by MP treatment. The ex vivo stimulated TNF-alpha secretion was postoperatively diminished in all groups, with again significantly lower values after MP treatment. IL-1RA secretion was not suppressed at any point. The ex vivo stimulated IFN-gamma and IL-12 secretion was strongly suppressed postoperatively regardless of the treatment. TM treatment resulted in no alterations of any parameter measured. It was demonstrated that especially monocyte functions are depressed after coronary surgery, and that MP treatment results in marked aggravation of this immunosuppression, whereas TM treatment shows no additional immunosuppressive effect. IL-10 seems to play a key role in the observed monocyte functional depression.

The quality of marine bathing water is currently assessed by monitoring the levels of faecal indicator bacteria. Among other drawbacks, results are retrospective using the traditional culture based methods. A rapid method is thus needed as an early warning to bathers for bacterial contamination in marine bathing waters. Total lipopolysaccharide (LPS) was chosen here as a potential general biomarker for bacterial contamination. Levels of total LPS, measured using a Kinetic QCL™ Limulus Amebocyte Lysate (LAL) assay, highly correlated with enumerated Escherichia coli and Bacteroides species. Levels of LPS in excess of 50 EU mL-1 were found to equate with water that was unsuitable for bathing under the current European Union regulations. Results showed that monitoring the levels of total LPS has a potential applicability as a rapid method for screening the quality of marine bathing water. More importantly, the LAL assay overcome the retrospective results when using culture based assessment since the LAL assay takes less than 30 minutes. Although false positive events were not detected, the occurrence of a false positive has been hypothesised, hence a more specific faecal biomarker was also investigated. LPS of five Bacteroides species (B. fragilis, B. caccae, B. ovatus, B. xylanisolvens and B. finegoldii) isolated from marine bathing waters samples were successfully profiled and showed high similarity between isolates in LPS gel electrophoresis banding pattern. Similar results were shown when investigating the endotoxic activity of Bacteroides species with the Kinetic QCL™ LAL assay. The potential biological relevance of Bacteroides LPS was also investigated in cell culture models indicating that Bacteroides showed similar induction of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and generally the biological activity was approximately 100 fold less than E. coli LPS. In addition, an ELISA assay was designed for the detection of Bacteroides LPS. Results showed that the Bacteroides LPS has a high potential to be used as a faecal biomarker, however, further work is required to develop a fully functional assay. The potential biological relevance of LPS present in contaminated bathing waters was also investigated in cell culture models. Results showed that there is a significant difference in the production of proinflammatory cytokines in comparison to “clean” bathing waters. Thus, results suggest that the European Directive regulations should be extended to cover the levels of total LPS in bathing waters to assure safety to the users of marine recreational water.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, characterized by progressive and largely irreversible airflow limitation due to alveolar destruction (emphysema), small airway narrowing, and chronic bronchitis. It is one of the leading causes of morbidity and mortality worldwide and in the UK, it may affect approximately 1.5 per cent of the population; and up to one in eight emergency admissions may be due to COPD,corresponding to over one million bed days, with some 24160 people in the UK dying as a result of COPD in 2005 (Burden of Lung Disease 2nd Edition,British Thoracic Society 2006). Most cases of COPD are triggered by chronic inhalation of cigarette smoke.However, some people do not suffer from COPD even if they smoke for many years. COPD cannot be cured, and patients usually live with poor life quality. Treatments include giving up smoking, medication and oxygen therapy. Genetic factors contribute to the development of COPD. In Northern Europe,Z-AT homozygotes (342Glu Lys) develop emphysema in their third or forth decade. One explanation is AT deficiency because they form inactive polymers. However, this cannot explain why bronchoalveolar lavage fluid (BALF) from Z-AT homozygotes with emphysema contains more neutrophils than BALF from individuals with emphysema and normal AT (M-AT). Inhaling pollutants which include smoking (cigarettes, pipes, cigars, etc.) and other fumes such as those found in many industrial work environments probably also plays a role in an individual’s development of COPD. Previously, it has been shown that the polymeric conformer of AT is present in BALF from Z-AT homozygotes and that it is a chemoattractant for neutrophils in vitro (Parmar JS, 2002). These findings have been confirmed by others (Mulgrew AT, 2004). However, it is unknown where the polymers form and if 4 they are chemotactic in vivo. My colleague Dr Carl Atkison† showed that polymers of Z 1-AT are present in the alveolar wall of Z-AT homozygotes with emphysema, which accounts for 20% of the total AT from lung homogenates.These Z-AT individuals also have an excess of neutrophils in the alveolar wall compared with M-AT homozygotes. Furthermore, neutrophils and polymeric AT co-localize in the alveolar wall (Mahadeva R, 2005). To investigate whether there was a direct relationship between polymers of Z-AT and the excess neutrophils, polymers of AT were instilled into the lungs of wild-type mice (Mahadeva R, 2005). This produced a significant increase in neutrophil influx into the lungs compared with instillation of the native protein.Examination of the time course demonstrated that the influx of neutrophils was closely linked to the presence of polymeric AT. The mechanism of neutrophil recruitment in this mouse model was subsequently shown to be a direct chemotactic effect rather than stimulation of IL-8 homologues or other CXC chemokines. Oxidized AT (Ox-AT) promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF- B and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the anti-elastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway † My colleagues’ contributions are acknowledged in future text where appropriate by the following superscripts: (a) Dr Sam Alam, (b) Dr Jichun Wang, (c) Dr Carl Atkinson, (d) Dr Sabina Janciauskiene. 5 interacts directly with epithelial cells to release chemokines IL-8 and MCP-1,which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells oxidizes AT, perpetuating the cycle, potentially contributing to the pathogenesis of COPD. Furthermore, this demonstrates that molecules such as oxidants, anti-proteinases, and chemokines, rather than acting independently, collectively interact to cause emphysema (Li Z, 2009). To investigate the molecular basis for the interaction between Z-AT and Ox-AT associated with cigarette smoking, female mice transgenic for normal (MAT)or Z-AT on CBA background were exposed to cigarette smoke (CS). Transgenic mice for Z-AT developed a significant increase in pulmonary polymers following acute CS exposure. Increased levels of neutrophils in CSZ lungs were tightly correlated with polymer concentrations. Oxidation of human plasma Z-AT by CS or -chlorosuccinimide greatly accelerated polymerization, which could be abrogated by antioxidants. The results showed that cigarette smoke accelerated polymerization of Z-AT by oxidative modification, which in so doing further reduced pulmonary defense and increased neutrophil influx into the lungs. These novel findings provided a molecular explanation for the striking observation of premature emphysema in ZZ homozygote smokers, and raised the prospect of anti-oxidant therapy in ZAT related COPD (Alam S, 2011).

L’inflammation fait partie des processus réactionnels de défense dont dispose l’organisme en réponse aux agressions, assurant l’intégrité de l’hôte. En réponse au dommage tissulaire, plusieurs médiateurs inflammatoires interviennent dans le processus de l’inflammation. Lors de ces dommages, des signaux de dangers provenant de cellules endommagées sont relâchés dans l’environnement tissulaire, pouvant causer des dommages cellulaires et tissulaires. Les macrophages, tout comme d’autres cellules, peuvent être activés par ces signaux de danger, menant à la sécrétion de molécules telles que des cytokines et des chimiokines pouvant modifier le microenvironnement tissulaire. Les insultes au tissu sain peuvent entrainer la mort cellulaire telle que l’apoptose. Les molécules pouvant être relâchées lors de celle-ci contribuent au microenvironnement, notamment de par l’influence de celles-ci sur le macrophage. Parmi ces médiateurs, nous avons identifié le Milk Fat Globule-Epidermal growth factor 8 (MFG-E8), un acteur important dans la résolution de l’inflammation, comme étant relâché spécifiquement par les cellules apoptotiques. Nous avons émis l’hypothèse que le microenvironnement apoptotique tissulaire, via la relâche de MFG-E8, module le phénotype du macrophage, modifiant le microenvironnement, la réponse inflammatoire ainsi que le devenir de l’insulte tissulaire. Nos objectifs sont 1) de caractériser ce microenvironnement apoptotique tissulaire et la cinétique de relâche du MFG-E8 par les cellules apoptotiques, 2) d’en évaluer son rôle dans la modulation du phénotype du macrophage ainsi que 3) d’en étudier, in vivo, son influence sur l’environnement inflammatoire et le devenir tissulaire. Dans le premier article présenté, nous avons démontré que les cellules endothéliales apoptotiques relâchent le MFG-E8 de façon Caspase-3 dépendante. La stimulation des macrophages par l’environnement conditionné par les cellules endothéliales apoptotiques mène à l’adoption d’un profil macrophagien davantage anti-inflammatoire et moindrement pro-inflammatoire. Ce phénotype est réduit par l’inhibition de la Caspase-3 et il dépend de la présence de MFG-E8. De plus, le potentiel du MFG-E8 à la reprogrammation du macrophage pro-inflammatoire a été démontré via un modèle expérimental de péritonite. Ce changement phénotypique médié par MFG-E8 implique une signalisation STAT3. Ayant démontré que les cellules épithéliales apoptotiques, à l’instar des cellules endothéliales apoptotiques, relâchent elles aussi de façon apoptose-dépendante le MFG-E8, nous avons étudié plus exhaustivement un modèle in vivo riche en apoptose épithéliale, l’obstruction urétérale unilatérale. Dans ce deuxième article présenté, nous rapportons l’implication bénéfique de MFG-E8 dans ce modèle de pathologie rénale obstructive. Nous avons constaté que la présence ou l’administration de MFG-E8 réduit le dommage tissulaire et la fibrose. La protection conférée par MFG-E8 est médiée via la modulation de l’activation de l’inflammasome. De plus, nos résultats illustrent l’importance du phénotype anti-inflammatoire du macrophage médié par le MFG-E8 dans la régulation négative de l’activation de l’inflammasome rénal et du dommage tissulaire. Cette thèse présente la première description de la relâche Caspase-3-dépendante de MFG-E8 par les cellules apoptotiques. Elle démontre également l’importance du MFG-E8 dans le microenvironnement apoptotique inflammatoire dans l’atténuation du phénotype pro-inflammatoire du macrophage. De plus, nous avons démontré son rôle protecteur dans des modèles in vivo de transplantation aortique et de réparation tissulaire, de même que dans un modèle de maladie rénale chronique où nous avons montré que cette protection conférée par MFG-E8 est médiée par la régulation négative de l’inflammasome tissulaire. Nos résultats suggèrent ainsi que le MFG-E8 pourrait être considéré comme un interrupteur inflammatoire et ainsi comme une cible potentielle dans la modulation de maladies inflammatoires. / Inflammation is an important component of the « response to injury » process, allowing host integrity. In response to injury, released inflammatory mediators from damaged cells play a crucial role in the modification of the inflammatory microenvironment, which can lead to more cellular and tissue damages. Macrophages can be activated by those danger signals, leading to a spectrum of cytokines and chemokines secretion and modulating the tissular microenvironment. Tissue injuries can lead to cell death such as apoptosis. Mediators released during apoptosis contribute to the nature of the microenvironment, by their influence on macrophage amongst others. We have identified that Milk Fat Globule-Epidermal growth factor 8 (MFG-E8), an important actor in inflammation resolution, is specifically released by apoptotic cells. We hypothesized that tissular apoptotic microenvironment, through MFG-E8 release, modulates macrophage phenotype, resulting in the modification of microenvironment, inflammatory response and tissu injury outcome. Thus, our objectives were to 1) characterize this tissular apoptotic microenvironment by studying MFG-E8 release kinetic by apoptotic cells, to 2) evaluate its role in macrophage phenotype modulation and to 3) study, in vivo, its influence on inflammatory environment and tissu damage outcome. In the first study, we demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if apoptosis is inhibited or if MFG-E8 is absent from the media. Furthermore, MFG-E8 potential to anti-inflammatory macrophage reprogramming has been demonstrated in the experimental peritonitis model. This MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of STAT-3. As apoptotic endothelial cells, apoptotic epithelial cells also release MFG-E8 in an apoptotic-dependent manner. Thus, we investiguated more exhaustively an in vivo epithelial apoptosis rich model, the unilateral ureteral obstruction. In this second study, we report the positive impact of MFG-E8 in this renal obstructive model. MFG-E8 administration reduced kidney damage and fibrosis compared to the control, whereas its absence in MFG-E8 KO mice was associated with more severe disease. Moreover, we demonstrated that the protective role of MFG-E8 is mediated through inflammasome activation modulation in the kidney. Furthermore, our results showed the importance of the anti-inflammatory macrophage phenotype that results in decreased inflammasome activation, preventing severe tissue damage. This thesis presents the first description of apoptosis-dependent release of MFG-E8 by apoptotic cells. It also demonstrate the importance of MFG-E8 in inflammatory apoptotic microenvironment, leading to pro-inflammatory macrophage phenotype attenuation. Moreover, we demonstrated MFG-E8 protective role in an aortic transplantation and a tissue repair models, as well as in a chronic kidney disease model where we showed that this MFG-E8 confered protection is mediated by negative regulation of tissular inflammasome activation. These data provide valuable insight for identifying MFG-E8 as a novel target in the modulation of inflammatory diseases and could be considerate as an inflammatory switch.

Introdução: A infecção pelo HIV causa um profundo comprometimento da resposta imune do hospedeiro, podendo levar à aids. Várias estratégias terapêuticas têm sido testadas ao longo dos anos, entre elas a imunoterapia com células dendríticas diferenciadas a partir de monócitos (MoDCs), pulsadas com HIV-1 inativado. Neste caso, a produção de vírus para o pulso das MoDCs consiste inicialmente no isolamento do vírus a partir de amostras de sangue do paciente e, em seguida, sua expansão em culturas de células CD4. Também deve ser considerado que quantidade excessiva de vírus pode ser tóxica para as MoDCs a serem pulsadas e do mesmo modo, quantidade insuficiente de vírus pode não ser efetiva para ativar uma resposta imune especifica. Neste contexto, a investigação do efeito de diferentes concentrações de vírus sobre o perfil fenotípico e funcional de MoDCs poderia auxiliar na determinação de uma quantidade ótima de vírus para o pulso das MoDCs e contribuir para o aperfeiçoamento da vacina terapêutica. Objetivo: Avaliar o efeito de diferentes quantidades de partículas virais, sobre o perfil fenotípico e funcional das MoDCs. Metodologia: Monócitos obtidos de indivíduos HIV+ foram diferenciados em MoDCs e pulsadas com HIV quimicamente inativado (3 partículas/MoDC, 30 partículas/MoDC, 300 partículas/MoDC). As células foram analisadas com relação ao perfil fenotípico, capacidade de internalizar p24, expressão de CD38, HLA-DR e CD69 e a produção de IFN-y por linfócitos T CD4+ e CD8+ autólogos. Resultados: O pulso com concentrações crescentes de vírus parece não interferir no perfil fenotípico e funcional das MoDCs. Conclusão: As diferentes quantidades de partículas virais utilizadas para o pulso parecem não ser tóxicas para as MoDCs estudadas, não tendo sido observadas diferenças com relação ao perfil fenotípico ou funcional das MoDCs / Introduction: The infection from HIV causes a profound impairment of the host immune response, which can lead to aids. Several therapeutic strategies have been tested over the years, including immunotherapy with monocyte - derived dendritic cells (MoDCs), pulsed with inactivated HIV-1. In this case, the production of virus for the pulse of the MoDCs initially consists of isolating the vírus from the patient\'s blood samples and then it into CD4+ cell cultures. It should also be considered that excessive amount of virus can be toxic to the MoDCs to be pulsed and likewise, insufficiently amount may not be effective for properly activate a specific immune response. In this context, the investigation of the effect of different virus concentrations on the phenotypic and functional profile of MoDCs could assist in the determination of an optimal amount of virus for the pulse of the MoDCs and contribute to the improvement of the therapeutic vaccine. Objectives: To evaluate the effect of different amounts of viral particles on the phenotypic and functional profile of MoDCs. Methods: MoDCs generated from HIV+ individuals were differentiated into MoDCs and pulsed with chemically inactivated HIV (3 particles /MoDC, 30 particles /MoDC, 300 particles /MoDC). Cells were analyzed for phenotypic profile, ability to internalize p24, expression of CD38, HLA-DR and CD69, and the production of IFN-y by autologous CD4 + and CD8 + T lymphocytes. Results: The pulse with increasing concentrations of virus does not seem to interfere in the phenotypic and functional profile of the MoDCs. Conclusion: The different amounts of viral particles used for the pulse appear to be non-toxic to the MoDCs studied, and no differences were observed regarding the phenotypic or functional profile of the MoDCs

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

Le sepsis constitue une pathologie fréquente, grevée d’une morbi-mortalité encore élevée. Sa physiopathologie fait notamment intervenir des dysrégulations du système immunitaire inné et adaptatif et des voies de l’apoptose. Ce travail aborde l’expression leucocytaire de deux familles de protéines potentiellement impliquées dans sa physiopathologie :les phosphodiestérases (PDE) et les apolipoprotéines L (apoL). L’étude de l’expression des PDE sous-tend le fait que ces enzymes, qui dégradent les nucléotides cycliques (AMPc et GMPc), sont impliquées dans la modulation de nombreux processus inflammatoires, tant d’origine infectieuse que non-infectieuse. L’expression des PDE après administration de LPS chez l’Homme est cependant mal caractérisée, de même qu’au cours du sepsis. Le présent travail teste l’hypothèse selon laquelle le sepsis, caractérisé par un état de dysrégulation immune complexe, s’accompagne d’une répression de l’expression des PDE au sein des leucocytes circulants, contrairement à ce qui est observé dans des modèles standardisés d’inflammation aiguë (LPS) ;il met également en perspective, dans une démarche observationnelle, l’expression des PDE avec l’expression du complexe HLA-DR, un ensemble protéique permettant la présentation de l’antigène et dont l’expression est partiellement dépendante de l’AMPc. Trois études ont ainsi été menées :étude de l’expression des PDE au cours d’une endotoxinémie chez le volontaire sain (1), et au cours du sepsis au sein de leucocytes totaux circulants (2) ou de sous-populations leucocytaires de l’immunité innée (monocytes CD14+ou granulocytes CD15+) (3). Alors que l’administration intraveineuse de LPS chez le volontaire sain mène à l’induction précoce et transitoire de certaines PDE, de façon similaire aux observations in vitro, les patients septiques présentent au contraire dès leur admission, et jusqu’au 5ème jour, une réduction de l’expression de plusieurs PDE en comparaison aux volontaires sains, tant dans les leucocytes totaux que dans les populations CD14+ et CD15+. L’expression de plusieurs de ces PDE est corrélée aux ratios TNF-α/IL-10 qui sont suggestifs d’un état d’immunodépression, attesté par une réduction significative de l’expression du complexe HLA-DR. L’étude des apoL au cours du sepsis sous-tend quant à elle le fait que cette famille de protéines, qui partage des homologies avec des membres du groupe Bcl-2 impliqué dans l’apoptose, a été associée notamment à l’induction de phénomènes pro-apoptotiques ;or, le sepsis est associé à une apoptose retardée des polynucléaires neutrophiles, un phénomène potentiellement délétère au niveau tissulaire. L’hypothèse d’une répression de l’expression des apoL leucocytaires au cours du sepsis est ainsi posée. Dans le présent travail, une diminution de l’expression des apoL-1, 2, 3 et -6 est observée chez les patients de soins intensifs présentant ou non un sepsis en comparaison à des volontaires sains ;cette réduction, corrélée aux taux de protéine C-réactive, concerne tant les populations leucocytaires totales que les granulocytes CD15+, et intéresse tant les ARNm que l’expression protéique (apoL-6 excepté). Le pourcentage de cellules CD15+ apoptotiques est par ailleurs fortement corrélé aux taux d’ARNm des apoL-1 et -2. Ces observations sont reproduites in vitro en incubant des granulocytes CD15+ de volontaire sain avec du sérum de patients septiques ou non septiques. Ces résultats préliminaires suggèrent ainsi une implication des apoL dans la régulation de l’apoptose des neutrophiles au cours du sepsis. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

Le système nerveux central est un organe complexe et privilégié, protégé, entre autres, par la barrière hémato-encéphalique (BHE), sur laquelle se trouvent des transporteurs d'efflux telle que la P-glycoprotéine (Pgp).<br />La BHE est impliquée dans la neuro-inflammation et les maladies cérébrales associées, et la littérature laisse penser que l'endothéline 1 (ET-1) y joue un rôle important. En utilisant un modèle in vitro autologue de BHE humaine adulte et un modèle in vitro de BHE humaine fœtale (co-culture de cellules gliales et de cellules endothéliales microvasculaires cérébrales), nous avons étudié l'effet de l'ET-1 sur la Pgp.<br />Une différence, en termes de modulation de l'activité de la Pgp, a été observée entre la BHE fœtale et la BHE adulte suite à l'exposition à l'ET-1. Nous avons également évalué la modulation induite par l'ET-1 sur la sécrétion de la MCP-1 (Monocyte chemoattractant protein 1) et de l'IL-8. Les résultats montrent que l'ET-1 diminue l'activité de la Pgp et induit la sécrétion de MCP-1 et d'IL-8.

Toxoplasma gondii ist ein obligat intrazellulär lebender einzelliger Parasit, der sich durch ein breites Wirtsspektrum sowie lebenslang persistierende Infektionen bei Menschen und Tieren auszeichnet. T. gondii hat für ein langfristiges Überleben unterschiedliche Mechanismen entwickelt, die ein Gleichgewicht zwischen der Pathogenität des Erregers und der intakten Immunabwehr des Wirtes gewährleisten. In diesem Kontext stellt die Modulation von Signalwegen der Wirtszelle eine wichtige Überlebensstrategie von Toxoplasmen dar. Frühere Arbeiten mit murinen Monozyten haben gezeigt, dass T. gondii die Expression von MHC-Klasse-II-Molekülen auf der Oberfläche infizierter Wirtszellen hemmt und dadurch eine effektive Antigenpräsentation an T-Helfer-Lymphozyten verhindert. Das Ziel der vorliegenden Dissertation war es herauszufinden, inwieweit Toxoplasma gondii auch die Interferon-γ-induzierte MHC-Expression von Monozyten des Menschen vermindert. Analysen mittels Immunfluoreszenzfärbung und Durchflusszytometrie zeigten, dass sowohl primäre, aus PBMC des Menschen isolierte Monozyten als auch permanente humane Monozyten (THP-1) durch eine Infektion mit T. gondii in der Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ signifikant gehemmt werden. Das Ausmaß der Inhibition ist dabei von der Infektionsdosis des Parasiten abhängig und betrifft sowohl die HLA-Expression auf der Zelloberfläche als auch den intrazellulären HLA-Pool. Interessanterweise kann dieser Effekt auch durch hohe Konzentrationen des stimulierenden Zytokins Interferon-γ nicht aufgehoben werden. Es zeigt sich außerdem eine signifikant reduzierte Expression von HLA-DR, -DP, -DQ bei Parasit-negativen Zellen einer T. gondii-infizierten Kultur, was möglicherweise durch sezernierte Proteine des Parasiten oder durch die Produktion hemmender Zytokine durch infizierte Wirtszellen begründet sein kann. Die HLA-Expression in in vitro-infizierten und nicht-infizierten primären Monozyten wurde darüberhinaus zwischen T. gondii-negativen Individuen und Spendern mit chronischer Toxoplasmose verglichen. Chronisch mit T. gondii infizierte Blutspender wurden serologisch anhand von spezifischen IgG-Antikörpern identifiziert. Durchflusszytometrische Analysen zeigten, dass Monozyten aus chronisch mit T. gondii infizierten Blut-Spendern signifikant mehr HLA-A, -B, -C und HLA-DR, -DP, -DQ exprimieren als Monozyten aus Toxoplasma-negativen Spendern. Eine Erklärung für diese gesteigerte MHC-Expression könnte eine Dominanz bestimmter Monozyten-Subpopulationen in Abhängigkeit vom Infektionsstatus ihres Spenders sein. Die Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ wird jedoch sowohl bei Monozyten von T. gondii-positiven als auch nicht-infizierten Individuen durch eine Infektion mit Toxoplasmen in vitro signifikant inhibiert. Analysen mit Hilfe von RT-qPCR zeigten deutlich, dass T. gondii mit der HLA-DR-, -DP-, -DQ-Expressions-Kaskade interferiert und die Synthese der Transkripte von IRF-1 und CIITA dosisabhängig inhibiert. Außerdem sind die Transskripte für HLA-A und HLA-DRα in infizierten Monozyten deutlich reduziert. Dies legt die Annahme nahe, dass T. gondii die Aktivierung von STAT1-abhängigen Promotoren effektiv inhibiert und so die Synthese der sich anschließenden HLA-Expressionskaskade supprimiert. Die Ergebnisse dieser Arbeit eröffnen interessante Ansätze für weitere Untersuchungen, insbesondere eine genauere Charakterisierung von Monozyten-Subpopulationen bei T. gondii-positiven Individuen sowie die Erforschung einer möglicherweise gesteigerten Immunreaktivität gegen andere Infektionserreger im Rahmen einer chronischen Toxoplasmose.