INTRODUÇÃO: Estudos recentes em modelos experimentais de sepse demonstraram as propriedades antioxidante e anti-inflamatória do etilpiruvato. Diferentes modelos experimentais também demonstraram que pequenos volumes de solução salina hipertônica (7,5%) melhoram a hemodinâmica, a microcirculação e modulam o sistema imunológico. Este estudo teve como objetivo investigar os efeitos do etil-piruvato, da solução salina hipertônica e da solução de Ringer lactato sobre a microcirculação mesentérica em modelo de sepse induzida por Escherichia coli viva em ratos. MÉTODOS: Ratos Wistar machos receberam por via endovenosa uma suspensão de E. coli ou foram submetidos ao procedimento cirúrgico do grupo falso-operado. Após três horas da infusão bacteriana os animais foram randomizados em: grupo controle não tratado, grupo tratado com solução de Ringer lactato (4mL/kg i.v.); grupo tratado com solução de Ringer lactato (4 mL/kg i.v.) associado a etil-piruvato (50mg/kg) e grupo tratado com solução salina hipertônica (7,5%, 4 mL/kg i.v.). Após 24 horas da bacteremia, as interações leucócito-endotélio foram investigadas por microscopia intravital, e a expressão de P-selectina e da molécula de adesão intercelular (ICAM)-1 determinada por imuno-histoquímica. Leucograma e contagem de plaquetas foram realizadas no início do estudo, 3 horas e 24 horas após a inoculação de E. coli. RESULTADOS: Os grupos não tratado e tratado com solução de Ringer lactato exibiram um aumento no número de leucócitos rollers (~ 2,5 vezes), leucócitos aderidos (~ 3,0 vezes), e de leucócitos migrados (~ 3,5 vezes) comparados ao grupo falso operado. O tratamento com etil-piruvato reduziu o número de leucócitos rollers, aderidos e migrados aos níveis obtidos no grupo falso operado (p > 0,05). Efeitos semelhantes foram observados nos animais tratados com a solução salina hipertônica (p > 0,05). A expressão de P-selectina e de ICAM-1 aumentou significativamente na microcirculação mesentérica no grupo não tratado, comparado ao grupo falso operado (p < 0,001). Todos os tratamentos reduziram a expressão de ambas moléculas de adesão, sendo os grupos tratados com etil-piruvato e solução salina hipertônica mais eficazes. A infusão de bactérias provocou leucopenia significante, seguida por leucocitose com granulocitose, e concomitante redução progressiva no número de plaquetas. CONCLUSÕES: Os dados apresentados sugerem que o etil-piruvato e a solução salina hipertônica (7,5%) eficientemente reduziram a resposta inflamatória na microcirculação mesentérica no presente modelo experimental de sepse induzida por E. coli viva; associada, pelo menos em parte, a menor expressão de moléculas de P-selectina e ICAM-1 / BACKGROUND: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. It has been shown that small volumes of hypertonic saline solution (7.5%) improve hemodynamics, the microcirculation, and modulate the immune system. This study aimed to investigate the effects of ethyl pyruvate, hypertonic saline and lactated Ringer\'s solution on mesenteric microcirculation in a sepsis model induced by live Escherichia coli in rats. METHODS: Male Wistar rats were underwent an intravenous suspension of E. coli bacteria or submitted to the sham procedure. After 3h of bacteria infusion rats were randomized into: control, without treatment; treated with lactated Ringer\'s solution (4 mL/kg, i.v.); treated with lactated Ringer\'s solution (4mL/kg, i.v.) plus ethyl pyruvate (50mg/kg), and treated with hypertonic saline solution (7.5%, 4 mL/kg i.v.). At 24h after bacteria infusion leukocyte-endothelial interactions were investigated by intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule (ICAM)- 1 evaluated by immunohistochemistry. White blood cell and platelet counts were determined at baseline, 3h and 24h after E. coli inoculation. RESULTS: Both non-treated and lactated Ringer\'s-treated groups exhibited an increase in the number of rolling leukocytes (~2.5-fold), adherent (~3.0-fold), and migrated cells (~3.5-fold) compared to sham. Treatment with Ringer\'s ethyl pyruvate solution reduced the number of rolling, adherent and migrated leukocytes to the levels attained in the sham group (p > 0.05). Similar effects were observed when animals were treated with hypertonic saline (p > 0.05). The expression of P-selectin and ICAM-1 significantly increased on mesenteric microvessels in non-treated group compared with sham (p < 0.001). All treatments reduced the expression of both adhesion molecules being ethyl pyruvate and hypertonic saline solution more effective than lactated Ringer\'s solution. Infusion of bacteria caused a significant leucopenia (3h), followed by a leucocytosis with granulocytosis (24h). There was an intense and progressive reduction in the number of platelets. No differences were observed after treatment with the different solutions. CONCLUSIONS: Data presented suggest that ethyl pyruvate and hypertonic saline solution (7.5%) efficiently reduce the inflammatory response at mesenteric microcirculation in an experimental model of sepsis induced by live E. coli associated, at least in part, with a down-regulation of P-selectin and ICAM-1

INTRODUÇÃO: Estudos recentes em modelos experimentais de sepse demonstraram as propriedades antioxidante e anti-inflamatória do etilpiruvato. Diferentes modelos experimentais também demonstraram que pequenos volumes de solução salina hipertônica (7,5%) melhoram a hemodinâmica, a microcirculação e modulam o sistema imunológico. Este estudo teve como objetivo investigar os efeitos do etil-piruvato, da solução salina hipertônica e da solução de Ringer lactato sobre a microcirculação mesentérica em modelo de sepse induzida por Escherichia coli viva em ratos. MÉTODOS: Ratos Wistar machos receberam por via endovenosa uma suspensão de E. coli ou foram submetidos ao procedimento cirúrgico do grupo falso-operado. Após três horas da infusão bacteriana os animais foram randomizados em: grupo controle não tratado, grupo tratado com solução de Ringer lactato (4mL/kg i.v.); grupo tratado com solução de Ringer lactato (4 mL/kg i.v.) associado a etil-piruvato (50mg/kg) e grupo tratado com solução salina hipertônica (7,5%, 4 mL/kg i.v.). Após 24 horas da bacteremia, as interações leucócito-endotélio foram investigadas por microscopia intravital, e a expressão de P-selectina e da molécula de adesão intercelular (ICAM)-1 determinada por imuno-histoquímica. Leucograma e contagem de plaquetas foram realizadas no início do estudo, 3 horas e 24 horas após a inoculação de E. coli. RESULTADOS: Os grupos não tratado e tratado com solução de Ringer lactato exibiram um aumento no número de leucócitos rollers (~ 2,5 vezes), leucócitos aderidos (~ 3,0 vezes), e de leucócitos migrados (~ 3,5 vezes) comparados ao grupo falso operado. O tratamento com etil-piruvato reduziu o número de leucócitos rollers, aderidos e migrados aos níveis obtidos no grupo falso operado (p > 0,05). Efeitos semelhantes foram observados nos animais tratados com a solução salina hipertônica (p > 0,05). A expressão de P-selectina e de ICAM-1 aumentou significativamente na microcirculação mesentérica no grupo não tratado, comparado ao grupo falso operado (p < 0,001). Todos os tratamentos reduziram a expressão de ambas moléculas de adesão, sendo os grupos tratados com etil-piruvato e solução salina hipertônica mais eficazes. A infusão de bactérias provocou leucopenia significante, seguida por leucocitose com granulocitose, e concomitante redução progressiva no número de plaquetas. CONCLUSÕES: Os dados apresentados sugerem que o etil-piruvato e a solução salina hipertônica (7,5%) eficientemente reduziram a resposta inflamatória na microcirculação mesentérica no presente modelo experimental de sepse induzida por E. coli viva; associada, pelo menos em parte, a menor expressão de moléculas de P-selectina e ICAM-1 / BACKGROUND: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. It has been shown that small volumes of hypertonic saline solution (7.5%) improve hemodynamics, the microcirculation, and modulate the immune system. This study aimed to investigate the effects of ethyl pyruvate, hypertonic saline and lactated Ringer\'s solution on mesenteric microcirculation in a sepsis model induced by live Escherichia coli in rats. METHODS: Male Wistar rats were underwent an intravenous suspension of E. coli bacteria or submitted to the sham procedure. After 3h of bacteria infusion rats were randomized into: control, without treatment; treated with lactated Ringer\'s solution (4 mL/kg, i.v.); treated with lactated Ringer\'s solution (4mL/kg, i.v.) plus ethyl pyruvate (50mg/kg), and treated with hypertonic saline solution (7.5%, 4 mL/kg i.v.). At 24h after bacteria infusion leukocyte-endothelial interactions were investigated by intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule (ICAM)- 1 evaluated by immunohistochemistry. White blood cell and platelet counts were determined at baseline, 3h and 24h after E. coli inoculation. RESULTS: Both non-treated and lactated Ringer\'s-treated groups exhibited an increase in the number of rolling leukocytes (~2.5-fold), adherent (~3.0-fold), and migrated cells (~3.5-fold) compared to sham. Treatment with Ringer\'s ethyl pyruvate solution reduced the number of rolling, adherent and migrated leukocytes to the levels attained in the sham group (p > 0.05). Similar effects were observed when animals were treated with hypertonic saline (p > 0.05). The expression of P-selectin and ICAM-1 significantly increased on mesenteric microvessels in non-treated group compared with sham (p < 0.001). All treatments reduced the expression of both adhesion molecules being ethyl pyruvate and hypertonic saline solution more effective than lactated Ringer\'s solution. Infusion of bacteria caused a significant leucopenia (3h), followed by a leucocytosis with granulocytosis (24h). There was an intense and progressive reduction in the number of platelets. No differences were observed after treatment with the different solutions. CONCLUSIONS: Data presented suggest that ethyl pyruvate and hypertonic saline solution (7.5%) efficiently reduce the inflammatory response at mesenteric microcirculation in an experimental model of sepsis induced by live E. coli associated, at least in part, with a down-regulation of P-selectin and ICAM-1

Certains virus respiratoires comme rhinovirus semblent favoriser la colonisation par staphylococcus aureus. Cependant, les détails des mécanismes impliqués dans cette synergie n’ont pas été suffisamment élucidés. Le but de cette thèse a été de développer et valider un modèle in vitro mimant la colonisation du vestibule nasal par s. aureus en utilisant les kératinocytes humains hacat. Ce modèle a permis d’étudier (i) les pouvoirs d’adhésion et d’internalisation d’une collection de souche clinique de s. aureus, (ii) l’efficacité intracellulaire des molécules antimicrobiennes utilisées dans le cadre de la décolonisation nasale de s. aureus, (iii) l’effet de la clarithromycine sur l’infection par rhinovirus et (iv) l’impact de l’infection par rhinovirus ou de l’inflammation non spécifique sur la colonisation par s. aureus. ce travail a principalement permis d’identifier un nouveau mécanisme alternatif de l’internalisation de s. aureus à travers la liaison entre la protéine bactérienne eap (extracellular adherence protein) et le récepteur cellulaire icam-1 (intracellular adhesion molecule 1). Cette voie alternative est favorisée en cas d’infection par rhinovirus ou d’inflammation, ce qui pourrait expliquer les observations cliniques de l’augmentation de la charge de s. aureus ou du risque d’infection par cette bactérie lors des infections virales respiratoires ou d’inflammation post-traumatique. Les résultats de cette thèse illustrent la complexité des interactions entre les cellules épithéliales de la muqueuse, s. aureus et les pathogènes viraux et ouvrent les perspectives sur d’autres études nécessaires afin de proposer des stratégies préventives ou thérapeutiques adaptées. / Some respiratory viruses such as rhinoviruses seem to promote staphylococcus aureus colonization. However, the details of the bacterial and cellular mechanisms involved in this synergy have not been sufficiently elucidated. The aim of this thesis was to develop and validate an in vitro model mimicking s. aureus colonization of the nasal vestibule by using hacat human keratinocytes. This model allowed to study (i) the adhesion and internalization capacities of various clinical s. aureus strains, (ii) the intracellular efficiency of the antimicrobial molecules used for s. aureus nasal decolonization, (iii) the effect of clarithromycin on rhinovirus infection, and (iv) the impact of rhinovirus infection and non-specific inflammation on s. aureus colonization. This work has mainly identified a new alternative mechanism for the internalization of s. aureus through the binding between the bacterial protein eap (extracellular adherence protein) and the cell receptor icam-1 (intracellular adhesion molecule 1). This alternative pathway is favored in case of rhinovirus infection or inflammation; which could explain the clinical observations of the increase of the load of s. aureus or the risk of infection by this bacterium during respiratory viral infections or post-traumatic inflammations. The results of this thesis illustrate the complexity of the interactions between the mucosal epithelial cells, s. aureus and viral pathogens and suggest that other studies are needed to propose appropriate preventive or therapeutic strategies.

Le paludisme à P. falciparum est l’une des causes majeures de mortalité et de morbidité dans le monde. Ce parasite est responsable de plusieurs manifestations cliniques allant du portage asymptomatique et infections non compliquées aigüe au paludisme grave et compliqué, tel que le neuropaludisme. Nous avons émis l’hypothèse que l’expression différentielle des gènes contribue à la variation phénotypique de parasites, entraînant des interactions spécifiques avec l’hôte, qui à son tour déterminent le type de manifestations cliniques du paludisme. L’objectif principal de cette étude était d’identifier les facteurs génétiques de P. falciparum impliqués dans la pathogenèse du neuropaludisme. Ceci a été réalisé par l’analyse complète du transcriptome d’isolats provenant d’enfants camerounais porteurs asymptomatiques (PA) ou atteints d’accès simple (AS) ou de neuropaludisme (NP). Le transcriptome du clone non sélectionnée (3D7) et la lignée sélectionnée (3D7-Lib) a été également analysé. Les résultats ont montré la surexpression de plusieurs gènes chez des isolats provenant d’enfants atteints de neuropaludisme et chez la lignée 3D7-Lib, par rapport à ceux provenant d’enfants asymptomatiques et 3D7, respectivement. L’analyse de l’ontologie de gène indique que les gènes potentiellement impliqués dans la pathogenèse, la cytoadhérence et l’agrégation des érythrocytes sont surreprésentés parmi les gènes surexprimés chez les isolats de CM et 3D7-Lib. Les résultats les plus marquants étaient la surexpression des gènes var (groups A et B) portant les domaines cassettes DC4, DC5, DC8 et DC13 et les gènes avoisinants rif chez les isolats de NP et la lignée 3D7-Lib, par rapport aux isolats de PA et au clone non sélectionné 3D7, respectivement. Le rôle joué par ces gènes dans la virulence parasitaire est lié à la cytoadhérence, c’est-à-dire la capacité de leurs protéines exprimées à interagir entre les érythrocytes parasités et les récepteurs endothéliaux post capillaires. Parmi ces récepteurs, le CD36 et inter cellular adhesion molecule 1 (ICAM-1) ont été les plus couramment utilisés par les isolats. L’étude sur l’implication de ces deux récepteurs, ainsi que celle des ligands PfEMP-1, dans la pathogenèse du neuropaludisme devrait être approfondie poursuivie. Nous avons analysé le phénotype de cytoadhérence et les profils de transcription des variantes de Pfemp-1 des isolats frais provenant des enfants béninois atteints de NP ou AS à l’aide du test d’adhérence statique aux récepteurs CD36, ICAM-1 et CSPG et au moyen de RT-PCR quantitative pour les groupes A, B, var2, var3, DC8 et DC13. Nos résultats montrent que le niveau de cytoadhérence des parasites associés au neuropaludisme au CD36 est significativement plus important que celui des parasites associés à l’accès simple. En outre, nous n’avons pas trouvé de différence significative entre la cytoadhérence des isolats de deux groupes cliniques à ICAM-1 et au CSPG. En outre, les niveaux d’expression des groupes var A, B, var2, var3 et du DC8 et DC13 sont plus élevés chez les isolats associés au neuropaludisme que chez les isolats associés à l’accès simple. Nos résultats montrent également que, chez les parasites provenant de NP le haut niveau de cytoadhérence des parasites au CD36 est corrélé au niveau de l’expression de groupe B de gènes var. En revanche, les profils d’expression des groupes spécifiques du gène var et le phénotype de cytoadhérence aux récepteurs ICAM-1 et CSPG n’étaient pas corrélés. Nos résultats suggèrent un rôle important du récepteur CD36 et des protéines codées par les variantes de PfEMP-1 codées par le groupe B dans la pathogenèse du neuropaludisme. / Plasmodium falciparum infection is a major cause of mortality and morbidity worldwide. This parasite is involved in several clinical manifestations, ranging from asymptomatic carriage and acute uncomplicated to severe and complicated malaria, including cerebral malaria. We hypothesized that differential gene expression contributes to phenotypic variation of parasites leading to specific interaction with the host which induces several clinical categories of malaria. The principal aim of this study was to identify parasite genetic factors implicated in the pathogenesis of cerebral malaria. We investigated the whole transcriptome of parasites isolated from Cameroonian children with asymptomatic (AM), uncomplicated (UM) and cerebral malaria (CM). We also investigated the transcriptome of 3D7 clone and the selected 3D7-Lib line. Our results revealed the up-regulation of several genes in CM isolates and 3D7-Lib line compared to AM isolates and 3D7 clone respectively. Gene ontology analysis indicates an over-representation of genes implicated in pathogenesis, cytoadherence, and erythrocyte aggregation among up-regulated genes in CM and 3D7-Lib. The most remarkable outcomes were the up-regulation of UPS A and B var genes containing architectural Domains Cassettes DC4, DC5, DC8, and DC13 and their neighboring rif genes in isolates from CM and 3D7-Lib line, compared with isolates from AM and the unselected 3D7 line, respectively. The involvement of these genes in parasite virulence rises from the ability of their encoded proteins to mediate cytoadherence of infected erythrocytes to post-capillary endothelial receptors. Of these receptors, CD36 and Inter Cellular Adhesion Molecule-1 (ICAM-1) were found as the most commonly used by the isolates. The implication of these two receptors, as well as that of PfEMP-1 ligands in the pathogenesis of CM needs to be more elucidated. We examined the adhesive phenotype and the transcription patterns of Pfemp-1 variants of fresh isolates from Beninese children with CM or UM malaria by static binding assay to CD36, ICAM-1 and CSPG and RT-qPCR for groups A, B, var2, var3, DC8, and DC13. Our findings showed that isolates from CM patients bind more to CD36 than those from UM cases. No differences were observed in binding levels to ICAM-1 or CSPG between these two groups. Furthermore, CM isolates transcribed groups A, B, var2, var3, DC8 and DC13 of var genes at higher levels than UM isolates. Interestingly, the high transcription levels of group B in CM parasites correlated with their higher level of binding to CD36. In contrary, the expression profiles of a specific var group and the binding phenotype of isolates to ICAM-1 and to CSPG were not correlated. Our findings support the implication of CD36 along with PfEMP-1 variants encoded by group B in cerebral malaria pathogenesis.

Neben aktivierten Astrozyten weisen zahlreiche Zytokine und Wachstumsfaktoren, die in der Nähe amyloidogener Ablagerungen im Hirn des Alzheimer-Patienten auftreten, auf eine Alzheimer-assoziierte Neuroinflammation hin, die an der Ausprägung der Neurodegeneration ursächlich beteiligt sein kann. Bisher ist die Bedeutung aktivierter Astrozyten für die Alzheimer-Pathogenese wenig untersucht worden. Von besonderem Interesse war in diesem Zusammenhang das interzelluläre Adhäsionsmolekül ICAM-1, da es mit amyloidogenen Ablagerungen und Astrozyten assoziiert vorliegt. Um einen ersten Hinweis auf eine mögliche pathophysiologische Rolle des ICAM-1 in der Alzheimer-assoziierten Neuroinflammation zu erhalten, wurde im ersten Teil der vorliegenden Arbeit die astrozytäre ICAM-1-Expression in Abhängigkeit unterschiedlicher Mitogene untersucht, die für die Alzheimer-Pathogenese von Bedeutung sind. Aus diesen Untersuchungen ging hervor, daß Astrozyten, die mit den Zytokinen IL1[beta], TNF[alpha] und TNF[alpha] mit IFN[gamma] behandelt wurden, ihre ICAM-1-Expression verstärkten. Es konnte erstmals gezeigt werden, daß eine Erhöhung der intrazellulären cAMP-Konzentration durch Forskolin, Rolipram und Prostglandine die Zytokin-verstärkte ICAM-1-Expression verminderte. Allerdings konnte eine schon bestehende astrozytäre Aktivität durch cAMP-erhöhende Substanzen nicht beeinflußt werden. Im Rahmen weiterer Untersuchungen wurde in dieser Arbeit gezeigt, daß die astrozytäre ICAM-1-Expression nicht durch das Amyloid[beta]-Protein ausgelöst oder verstärkt wird, dagegen konditionierte Medienüberstände A[beta]-aktivierter Mikroglia einen starken Einfluß auf die astrozytäre Aktivität ausüben. Im zweiten Teil dieser Arbeit ist die Untersuchung aktivierter Astrozyten in der Alzheimer-assoziierten Amyloidogenese beschrieben, die im Zusammenhang mit der amyloidogenen Prozessierung des Amyloid-Vorläuferproteins APP steht. Bisher standen Neurone als Hauptproduzenten des Amyloid [beta]-Proteins im Vordergrund der Alzheimer-Forschung. Da jedoch verschiedene Veröffentlichungen darauf hinweisen, daß Astrozyten ebenfalls eine direkte Rolle in der Entstehung der amyloidogenen Ablagerungen spielen können, wurde die amyloidogene APP-Prozessierung aktivierter Astrozyten untersucht. Neben vergleichenden Analysen der astrozytären und neuronalen APP-Prozessierung konnte im Rahmen dieser Arbeit erstmals gezeigt werden, daß sich die APP-Prozessierung aktivierter Astrozyten zugunsten des nicht-amyloidogenen Prozessierungsweges verlagert, d.h. aktivierte Astrozyten verringerten die Sekretion des A[beta]-Proteins. Ein charakteristisches Merkmal aktivierter Astrozyten ist eine verstärkte [alpha]-Sekretaseaktivität bei der APP-Prozessierung. Astrozyten, die mit den Zytokinen IL1[beta], TNF[alpha] und TNF[alpha] mit IFN[gamma] aktiviert wurden, akkumulierten nicht-amyloidogene C-terminale Fragmente, sekretierten verstärkt sekretorisches APP nach [alpha]-Sekretaseaktivität und zeigten eine verringerte A[beta]-Sekretion bei gleichzeitig verstärkter Sekretion des nicht-amyloidogenen p3-Fragmentes. Abschließend wurde die pathophysiologische Bedeutung der Astrozyten in der Alzheimer-Pathogenese im Zusammenhang mit den erzielten Ergebnissen aus dieser Arbeit und mit den Befunden anderer Forschungsgruppen diskutiert und ein Modell über die zellulären, neuroinflammatorischen Vorgänge in der Alzheimer-Pathogenese entwickelt. / Activated astrocytes, number of cytokines and growth factors are associated with amyloidogenic deposits in the brain of Alzheimer-patients, pointed to an Alzheimer-associated neuroinflammation, that could be involved in the progression of the neurodegeneration. So far the implication of activated astrocytes in the pathogenesis of Alzheimer's disease is not clear. In this connection the adhesion molecule ICAM-1 was of particularly interest, because it is associated with astrocytes and amyloidogenic deposits. To get insights in a possible pathophysiological role of ICAM-1 in the Alzheimer-associated neuroinflammation, in the first part of this thesis the astrocytic expression of ICAM-1 was investigated dependent on different mitogens, that are relevant to the pathogenesis of Alzheimer's disease. It was shown that the expression of ICAM-1 was enhanced in astrocytes treated with the cytokines IL1[beta], TNF[alpha] and the combination TNF[alpha] with IFN[gamma]. It was shown that an elevation of intracellular concentration of cAMP by forskolin, rolipram and prostaglandines results in an suppressed cytokine-stimulated ICAM-1 expression. But it was impossible to reduce an existing astrocytic activity by these cAMP-elevating agents. Within the scope of additional investigations it was shown that the expression of ICAM-1 was not induce by the amyloid [beta]-peptide, whereas the ICAM-1 expression was strongly enhanced by conditioned media of A[beta]-activated microglia. In the second part of this thesis the investigation of activated astrocytes in the Alzheimer-associated amyloidogenesis is described, the astrocytic processing of the amyloid precursor protein (APP). So far it is believed that the neurons are the main producers of the amyloid [beta]-peptide. But different publications pointed to the possibility that astrocytes could play a direct role in the generation of the amyloidogenic deposits. From this reason the amyloidogenic processing of APP was investigated in activated astrocytes. Besides comparative study of astrocytic and neuronal APP processing it was first shown that activated astrocytes practise more the non-amyloidogenic pathway of APP processing. A characteristic hallmark of activated astrocytes is an increased activity of [alpha]-secretase. Astrocytes that are activated by the cytokines IL1[beta], TNF[alpha] or TNF[alpha] with IFN[gamma] showed an accumulation of non-amyloidogenic C-terminal fragments, increased APP-secretion followed by [alpha]-secretase activity, decreased A[beta]-secretion and enhanced p3-secretion. In conclusion the pathophysiological role of astrocytes in the pathogenesis of Alzheimer's disease was discussed leading to a model of cellular neuroinflammatory events in the pathogenesis of Alzheimer's disease.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Les métastases sont responsables de 90 % des décès causés par le cancer. Les métastases sont des foyers cancéreux secondaires qui se forment à distance de la tumeur d’origine. Des cellules cancéreuses quittent la tumeur primaire, rejoignent la circulation sanguine puis colonisent des organes voisins par migration à travers l’endothélium vasculaire. Ce phénomène d’adhésion à l’endothélium et de migration à travers l’endothélium appelé l’extravasation est une étape clé du processus métastatique. L’identification des molécules impliquées constitue une priorité dans le but d’élaborer de nouvelles drogues anticancéreuses. Nous avons précédemment montré que la molécule d’adhésion cellulaire InterCellular Adhesion Molecule-1 (ICAM-1) exprimée par les cellules endothéliales, est impliquée dans l’interaction des cellules de cancer de la vessie (BCs) avec l’endothélium. Cependant les ligands d’ICAM-1 n’ont pas été étudiés. Dans cette étude, nous utilisons des tests d'adhésion cellulaire et la microscopie à force atomique (AFM) afin d’identifier les ligands d’ICAM-1 et de mesurer les forces impliquées dans l’interaction ligand-ICAM-1. Nous avons identifié que les protéines MUC1 et CD43 exprimées par les BCs les plus invasives se lient à ICAM-1 en développant des forces d’intensité différente selon le couple considéré. Une analyse détaillée des événements de rupture suggère que CD43 est fortement lié au cytosquelette et que son interaction avec ICAM-1 correspond principalement à des sauts brusques. Au contraire, MUC1 semble être lié faiblement au cytosquelette et ses interactions avec ICAM-1 sont principalement associées à la formation de filaments membranaires ou « tethers ». Les forces mises en jeu lors de la migration des cellules cancéreuses à travers l'endothélium ont été étudiées par microscopie de forces de traction (TFM). Les résultats préliminaires montrent que les tractions exercées par les cellules cancéreuses lors de l’extravasation sont mesurables par TFM. / Cancer metastasis is associated with 90% cancer-associated deaths, when cancer cells escape from the primary tumor and form metastatic colonies in secondary sites. Extravasation is an important step in cancer metastasis, where cancer cells carried in blood, adhere and transmigrate through the endothelium. Therefore identifying the key molecules involved during the adhesion process could enable to develop new anticancer cancer drugs able to inhibit the adhesion of cancer cells to the endothelium. We have previously shown that InterCellular Adhesion Molecule-1 (ICAM-1) expressed by endothelial cells is involved in the interactions of bladder cancer cells (BCs) with the endothelium. However the ICAM-1 ligands have never been investigated. In this study, we combined adhesion assays and Atomic Force Microscopy (AFM) to identify the ligands involved and to quantify the forces relevant in such interactions. We report the expression of MUC1 and CD43 on BCs and demonstrate that these ligands interact with ICAM-1 to mediate cancer cell-endothelial cell adhesion in the case of the more invasive BCs. AFM experiments were performed to quantify the force ranges involved by MUC1 and CD43 during their interaction with ICAM-1. AFM measurements combined with a Gaussian Mixture Model showed distinct force ranges for the interaction of ICAM-1 with MUC1 and ICAM-1 with CD43. Furthermore, a detailed analysis of the rupture events suggests that CD43 is strongly connected to the cytoskeleton and that its interaction with ICAM-1 mainly corresponds to force ramps followed by sudden jumps. On the contrary, MUC1 seems to be weakly connected to the cytoskeleton as its interactions with ICAM-1 are mainly associated with the formation of tethers. The forces involved during the transmigration of cancer cells through the endothelium was investigated using Traction Force Microscopy (TFM). Preliminary results showed that tractions exerted by cancer cells during transmigration can be studied and quantified using TFM.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

L'adhésion cellulaire est un processus biologique fondamental contrôlé par des liaisons moléculaires spécifiques entre ligands et récepteurs attachés à des surfaces. La formation et la rupture de ces liens dépendent de facteurs cinétiques, mécaniques et structurels. Le but de ce travail était d'observer comment l'interaction ICAM-1 (Inter- Cellular Adhesion Molecule 1) - anti ICAM-1 pouvait être modifiée en jouant i) sur la multivalence de molécules impliquées dans la liaison ii) sur la topographie de surface et iii) sur la mobilité des ligands. A cette fin, on a principalement utilisé une chambre à flux laminaire, complété par une détection de molécule unique par fluorescence. L'étude sur les effets de multivalence, utilisant des monomères et dimères d'ICAM-1, a été réalisée en absence ou en présence d'une force mécanique, montrant la plus grande stabilité des liaisons divalentes. En outre, un renforcement avec la force et le temps a été trouvé et décrit avec une fonction à deux paramètres, montrant, pour les liaisons divalentes, un comportement intermédiaire entre rupture parallèles et successives des liaisons monovalentes. La fréquence d'adhésion des liaisons monovalentes et divalentes présente différentes valeurs causées par la différence de longueur de ces molécules. Les expériences d'adhésion ont été effectuées en variant la topographie du substrat à l'échelle nanométrique pour les molécules étudiées. Une comparaison des cinétiques de liaisons sur ces surfaces ne montrent pas de différences soit dans la formation ou dans la rupture. Dans l'écoulement, le temps de contact entre les molécules est contrôlé par la convection de microsphères. Des résultats récents montrent qu'un temps minimum est requis pour former la liaison (Robert et al. 2011). Pour tester cette prédiction, les ligands sont ancrés à une bicouche lipidique (SLB) pour étudier comment la diffusion peut modifier l'adhésion. Expérimentalement, les fréquences d'adhésion des liaisons ont montré un comportement similaire pour les SLB fixes et fluides. Toutefois, une simulation numérique 2D prédit un effet sur la formation de la liaison, même lorsque la diffusion des ligands est faible. Il semblerait que la diffusion joue un rôle dans la dissociation de la liaison, limitant fortement la dissociation de la bicouche fluide. Cet effet peut être expliqué par la présence éventuelle de liaisons multiples dues à l'accumulation des ligands sur la surface de contact. La chambre à flux laminaire et le suivi de particule individuelle a permis de mieux comprendre les mécanismes d'adhésion et le comportement de l'interaction des molécules d'ICAM-1 au niveau de molécule individuelle, lorsque l'environnement moléculaire a été modifiée. Des travaux similaires peuvent être effectuées sur d'autres molécules d'adhésion afin d'atteindre une connaissance beaucoup plus large des mécanismes d'adhésion, ou sur les liaisons entre TCR et pMHC qui sont extrêmement importantes dans la réponse immunitaire.

O presente estudo teve como objetivo avaliar o perfil da molécula relacionada à inflamação (ICAM-1), citocinas e da atividade das NTPDases como potencial influência sobre a genotoxicicdade em trabalhadores expostos ocupacionalmente a HPA. Este estudo incluiu 45 taxistas e 40 indivíduos com atividades administrativas (não-expostos ocupacionalmente), ambos nãi fumantes. O monitoramento biológico foi realizado pela quantificação do 1-hidroxipireno (1-pireno OH) urinário. A expressão de ICAM-1 (CD54) em neutrófilos foi realizada por citometria de fluxo. O perfil de hidrólise das NTPDases em plaquetas foi determinada pelo método colorimétrico. Além disso, os níveis de malondialdeído no plasma (MDA), citoquinas inflamatórias (IL-1β, IL-6, IL-10, TNF-α e IFN-γ) e o dano ao DNA (ensaio cometa e do micronúcleo) foram também avaliados. Os resultados demonstraram que os níveis de 1-OH pireno foram significativamente aumentados nos motoristas de táxi em comparação com o grupo não exposto ocupacionamente (p <0.0001); também foi positivamente correlacionada com neutrófilos ICAM-1, níveis de MDA e biomarcadores de danos no DNA. A expressão de ICAM-1 em neutrófilos foi significativamente elevado em motoristas de táxi (p <0.05), bem como os níveis de MDA (p <0.01), sendo a última positivamente correlacionada com a % de DNA na Cauda e frequência de MN. Aumento da hidrólise de ATP e ADP forma encontrados nos taxistas. Concentrações dos marcadores pró-inflamatórios foram aumentadas e anti-inflamatórias (IL-10) diminuída no grupo exposto. Para o teste de ensaio de micronúcleos e cometa, houve aumento significativo em motoristas de táxi, inclusive depois da adição de enzimas de reparo. Correlações positivas foram encontradas entre IL-1β, IL-6, TNF-α, IFN-γ e preditores de danos no DNA (% de DNA na cauda e frequência de MN), enquanto que a IL-10 está negativamente correlacionada com os biomarcadores de lesão ao DNA. Em resumo, a exposição ocupacional à poluição do ar pode levar a anormalidade homeostática como potencial contribuição para o processo aterosclerótico. Este estudo mostrou também que a exposição crônica à poluição do ar pode causar danos no DNA relacionado com a peroxidação lipídica e processo inflamatório. / The present study aimed to evaluate the profile of inflammatory molecule (ICAM-1), cytokines and the NTPDases activity as potential influence on genotoxicity process in workers exposed occupationally to PAH. This study included 45 taxi drivers and 40 non-occupationally exposed subjects, both non smorkers. Biological monitoring was performed by quantification of urinary 1-hydroxypyrene (1-OH pyrene). The expression of ICAM-1 (CD54) in neutrophil was performed and the hydrolysis profile of the NTPDases in platelets was determined. Plasma malondialdehyde (MDA) levels, inflammatory cytokines and DNA damage (comet and micronucleus assays) were also evaluated. The results demonstrated that the 1-OH pyrene levels were significantly increased in taxi drivers (p<0.0001); were also positively correlated to neutrophil ICAM-1 expression, MDA levels and biomarkers of DNA damage. ICAM-1 expression in neutrophil was significantly elevated in taxi drivers (p<0.05), as well as MDA levels (p<0.01), being the last positively correlated with % Tail DNA and MN frequency. ATP and ADP hydrolysis was increased in taxi drivers. Pro-inflammatory markers concentrations were increased and anti-inflammatory (IL-10) was decreased in exposed group. For the comet assay and micronucleus test, increase was significant in taxi drivers, inclusive after repair enzymes. Positively correlations were found between IL-1β, IL-6, TNF-α, IFN-γ and predictors of DNA damage (%Tail DNA and MN frequency), while IL-10 is negatively correlated with the biomarkers of DNA lesion. In summary, occupational exposure to air pollution, especially to PAHs, may be related with homeostatic abnormality as potential contribute to atherosclerosis process. This study showed also that the chronic exposure to outdoor air pollution may cause DNA damage related with lipid peroxidation and inflammatory process.