Le mécanisme biologique responsable pour l’augmentation de l’expression de la protéine nestin dans les cellules souches neurales (CSN) du cœur après un infarctus du myocarde (IM) demeure inconnu. Des études antérieures ont démontré que le traitement au dexamethasone, un glucocorticoïde aux propriétés anti-inflammatoires, abolit la régulation positive de nestin après un IM. Ceci suggère un lien avec la réponse inflammatoire. Nous avons vérifié dans cette étude l’hypothèse que la cytokine inflammatoire interleukin-1beta (IL-1beta) peut modifier le phénotype de cellules souches neurales. Le deuxième objectif de l’étude fut d’établir l’impact, suivant un IM, de l’inhibition de la signalisation de IL-1beta sur la fonction et la guérison cardiaque. Suite à une ligature complète de l’artère coronaire du rat mâle, le dysfonctionnement contractile du ventricule gauche fut associé à une régulation positive de la protéine nestin dans le myocarde non-infarci. Le traitement avec Xoma 052 (1 mg/kg), un anticorps anti-IL-1beta, 24h, 7 et 14 jours après un évènement ischémique, eu aucun effet sur la taille de l’infarctus ou la contractilité du ventricule gauche. De plus, le traitement avec Xoma 052 après un IM n’a pu supprimer l’augmentation de l’expression de nestin et Bcl-2 malgré une réduction modeste du niveau de la protéine Bax. Pour déterminer directement le rôle de la réponse inflammatoire en l’absence d’ischémie, nous avons injecté des rats mâles avec du LPS (10mg/kg, 18hrs). Dans le coeur du rat-LPS, nous avons noté une augmentation significative du niveau d’ARNm de IL-1beta et de l’expression de la protéine nestin. Le prétraitement avec 10mg/kg de Xoma 052 a aboli l’augmentation de l’expression de nestin dans le coeur des rats-LPS. Ces observations indiquent que les cellules souches neurales pourraient représenter une cible potentielle de l’IL-1beta. / The underlying biological event(s) implicated in the increased expression of nestin in cardiac resident neural stem cells (NSC) following myocardial infarction (MI) remain unknown. Past studies have shown that dexamethasone treatment, a synthetic glucocorticoid with potent anti-inflammatory properties, abolished the upregulation of nestin in the non-infacted left ventricle (NILV) following MI. This suggests an association with the inflammatory response. The present study tested the hypothesis that the inflammatory cytokine interleukin-1beta (IL-1beta) can influence the neural stem cell phenotype. A secondary goal of the study was to assess impact of IL-1beta signalling inhibition on cardiac function and wound healing following MI. Following complete coronary artery ligation of the adult male rat heart, left ventricular contractile dysfunction was associated with the upregulation of the pro-apoptotic protein bax, the anti-apoptotic protein Bcl-2 and nestin in the non-infarcted myocardium. Xoma 052 (1 mg/kg), an IgG antibody directed against IL-1beta, was administred 24 hrs after ischemic injury and subsequently injected 2x over a period of 21 days. Treatment did not alter infarct size or improve left ventricular contractility. In addition, it failed to abolish the increased expression of nestin and Bcl-2, and modestly reduced bax protein levels. To directly assess the relationship between inflammation and the expression of nestin in the absence of an ischemic insult, adult male rats were injected with lipopolysaccharide (LPS) (10mg/kg;18 hrs). In the heart of LPS- treated rats, IL-1beta mRNA levels were significantly increased and associated with elevated nestin protein expression. The pre-treatment with Xoma 052 (10mg/kg) abolished the increased expression of nestin in the heart of LPS-treated rats. These data indicate that neural stem cells may represent a target for IL-1beta. However, futher investigation is required to elucidate the role of IL-1beta on NSC following MI.

La nature du lien qui unit les deux aspects du système immunitaire, que sont la défense de l’hôte contre les agressions extérieures et la genèse des maladies auto- immunes, n’a pas été élucidée. L’axe IL-23/Th17 joue un rôle dans les deux versants, ce qui le place en bonne position pour être un potentiel chaînon manquant. Objectif : connaître l’implication de cet axe dans un modèle infectieux, Mycoplasma hominis, et un modèle de maladie auto-immune, la sclérodermie systémique (ScS), dans lesquels il n’a pas encore été étudié. Les lipoprotéines membranaires de M. hominis sont capables d’induire une maturation des cellules dendritiques humaines. Elle s’accompagne d’une sécrétion d’IL-23 variable selon l’origine clinique des isolats, via TLR2, et d’une polarisation vers la voie Th17. Nous avons observé une augmentation de la fréquence des cellules Th17 et Th22 dans le sang périphérique des patients ScS, potentialisée par l’iloprost, via entre autres la production monocytaire d’IL-23. Dans la peau des patients ScS, il existe une augmentation des cellules produisant l’IL-17 inversement corrélé au score de fibrose cutanée. In vitro, l’IL-17 est capable d’inhiber partiellement l’expression d’α-SMA induite par le TGF-ß et d’induire la sécrétion de MMP1 par des fibroblastes dermiques humains. L’axe IL-23/IL-17 et les cellules Th17 jouent un rôle dans la défense contre M. hominis et dans la physiopathologie de la ScS. / Relationship between both aspects of the immune system, ie host defense against external aggression and genesis of autoimmune diseases, has not been elucidated. IL-23/Th17 axis plays a role in both sides, which puts him in a good position to be a potential missing link. Objective: To understand the implication of this axis in a model of infection, Mycoplasma hominis, and a model of autoimmune disease, systemic sclerosis (SSc), in which it has not yet been studied.
The membrane lipoproteins of M. hominis are capable of inducing human dendritic cell maturation. It occurs along with an IL-23 secretion changing with the clinical origin of isolates, via TLR2, and a T cell polarization towards Th17. Then we observed an increase in the Th17 and Th22 cell frequency in peripheral blood of SSc patients, further enhanced by iloprost via monocyte production of IL-23 among others. In the skin of SSc patients, we showed an increase in IL-17-producing cells with an inverse correlation to the skin fibrosis score. In vitro, IL-17 is able to partially inhibit the expression of α-SMA induced by TGF-ß and to induce the secretion of MMP1 in human dermal fibroblasts. The IL-23/IL-17 axis and Th17 cells play a role in defense against M. hominis and in the pathogenesis of SSc.

4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...

Introduction. Les mécanismes physiopathologiques du remodelage vasculaire pulmonaire chez le patient BPCO sont encore mal élucidés. L'inflammation pourrait jouer un rôle déterminant.Objectifs. Déterminer le rôle de l'interleukine 6 (IL6) dans la physiopathologie de l'hypertension pulmonaire (HTP) associée à la BPCO.Méthodes. Nous avons étudié le lien entre l'IL6 et l'HTP sur une population de patients atteints de BPCO, l'effet de l'hypoxie sur le développement d'une HTP chez des souris IL6-/- et l'effet in vitro de l'IL6 sur les cellules endothéliales et les cellules musculaires lisses d'artère pulmonaire humaine.Résultats. Les patients BPCO avec HTP présentaient un taux plasmatique d'IL6 circulante plus élevé. Le génotype GG de l'IL6 était corrélé à un risque plus élevé de développer une HTP. L'IL6 est produite par tous les acteurs cellulaires impliqués dans la physiopathologie du remodelage vasculaire pulmonaire et en particulier la cellule musculaire lisse. Sa synthèse, ainsi que celle de ses récepteurs, est très nettement stimulée par l'hypoxie aigue et chronique. L'IL6 participe probablement à l'entretien de la dysfonction endothéliale, à la migration des cellules musculaires lisses et au recrutement des cellules inflammatoires. Les souris IL6-/- sont partiellement protégées de l'hypertension pulmonaire hypoxique et présentent un moindre recrutement pulmonaire de cellules inflammatoires induit par l'hypoxie. Le taux d'IL6 est corrélé à une longueur télomérique plus courte chez les patients BPCO, témoignant d'un processus de vieillissement biologique accéléré. L'HTP associée à la BPCO ou à l'hypoxie chronique pourrait résulter d'une accentuation du processus de sénescence des cellules vasculaires pulmonaires, favorisé par l'inflammation.Conclusion. L'inflammation et plus particulièrement l'IL6 semblent fortement impliquées dans la physiopathologie du remodelage vasculaire pulmonaire chez le patient BPCO. / Introduction. The pathophysiological mechanisms responsible for pulmonary vascular remodeling in COPD remain poorly understood. Inflammation may play a major role.Objectives. To study the role of interleukin-6 (IL6) in the pathophysiology of pulmonary hypertension associated with COPD.Methods. We studied the relationship between IL6 and PH in a population of patients with COPD, the effect of hypoxia on the development of PH in mice IL6-/- and the effect of IL-6 on endothelial cells and smooth muscle cells of human pulmonary artery in vitro.Results. COPD patients with PH were characaterised by a more pronounced hypoxia and higher plasma levels of circulating IL-6. The GG genotype of IL-6 also correlated with a higher risk of developing PH in these patients. Each of the different cellular elements that promote pulmonary vascular remodeling are known to produce IL-6, with smooth muscle cells known to be a particularly important source of this cytokine In addition, synthesis of IL-6 and its associated receptors is increased in response to acute and chronic hypoxia. It is likely that IL-6 contributes to endothelial dysfunction, the migration and, indirectly, proliferation of smooth muscle cells, and recruitment of inflammatory cells. Indeed, IL6-/- mice are partially protected from hypoxia-associated pulmonary hypertension and demonstrate attenuated hypoxia-induced lung recruitment of inflammatory cells. Levels of IL-6 also correlate with shorter telomere length in patients with COPD, indicating a process of accelerated biological aging. This suggests that pulmonary hypertension secondary to COPD or chronic hypoxia may be due to inflammation-associated acceleration of normal pulmonary vascular cell sénescence.Conclusion. Inflammation in general, and IL-6 in particular, appear to be strongly involved in the pathophysiology of pulmonary vascular remodeling in patients with COPD

Les cellules épithéliales constituent la première ligne de défense face à l’infection et jouent un rôle actif dans l'immunité innée. Par la sécrétion locale de cytokines, ces cellules sont capables d'orchestrer la réponse immunitaire contre les pathogènes invasifs. L'activation des récepteurs de reconnaissance de pathogènes, qu’ils soient intracellulaires ou extracellulaires, conduit à une cascade de signalisation complexe. Cette dernière entraîne l'activation du facteur de transcription NF-kB ainsi que la production ultérieure de cytokines pro-inflammatoires. Cependant, les mécanismes moléculaires qui gouvernent ce processus n'ont pas été entièrement élucidés. La bactérie à Gram négatif Shigella flexneri est un pathogène humain majeur à l’origine de la dysenterie bacillaire. Cette maladie se caractérise par une inflammation aiguë du colon qui peut entraîner la destruction du tissu intestinal et même dans les cas les plus graves, la mort. En effet, S. flexneri peut envahir les cellules épithéliales du colon et se répliquer dans leur cytoplasme. Après la détection de bactéries intracellulaires, les cellules infectées et non infectées déclenchent des voies de signalisation inflammatoire, ce qui entraîne une production massive d'interleukine-8. En utilisant S. flexneri comme modèle d'infection, nous avons identifié une nouvelle voie de signalisation qui joue un rôle central dans l'activation de NF-kB et la production d'IL-8 qui en résulte lors des infections bactériennes à Gram négatif. Après la détection cytosolique des bactéries, les protéines TIFA forment des oligomères à travers un processus dépendant de leur thréonine en position 9, ainsi que de leur domaine « Forkhead-associated ». D’une part, ces oligomères interagissent avec TRAF6, ce qui conduit à l’oligomérisation de cette dernière et à l'activation subséquente de NF-kB. D'autre part, nous montrons que l'oligomérisation de TIFA dépend de la kinase ALPK1 et que cette voie est activée en réponse au métabolite bactérien heptose-1, 7-bisphosphate. Ces observations pourraient être étendues au pathogène entéro-invasif Salmonella typhimurium ainsi qu'à la bactérie extracellulaire Neisseria meningitidis. Nos résultats démontrent donc le rôle central de la voie de signalisation ALPK1-TIFA-TRAF6 en réponse aux pathogènes bactériens à Gram négatif intracellulaires et extracellulaires. Ainsi, ces travaux contribuent à une meilleure compréhension des mécanismes moléculaires régissant la réponse immunitaire des cellules épithéliales aux bactéries pathogènes. / Epithelial cells represent the first line of defense against pathogens and play an active role in innate immunity. Via local secretion of cytokines, they are able to orchestrate the immune response against invading pathogens. The activation of both intracellular and extracellular pathogen recognition receptors leads to a complex signaling cascade, resulting in the activation of the transcription factor nuclear factor kB(NF-kB)and the subsequent production of pro-inflammatory cytokines. However, the molecular mechanisms governing this process have not been fully elucidated. The Gram-negative bacterium Shigella flexneriis an important human pathogen and the causative agent of bacillary dysentery. This disease is characterized by acute inflammation of the colon resulting in the destruction of the intestinal tissue and, in severe cases, death. S. flexneri can invade and replicate within colonic epithelial cells. Following detection of the bacteria, both infected and uninfected bystander cells initiate inflammatory signaling pathways, which result in massive interleukin-8 (IL-8) production by the latter. Using S. flexneri as a model of infection, we have identified a novel signaling pathway, which is central to the activation of NF-kB and the subsequent production of IL-8 during Gram-negative bacterial infections. Following the cytosolic detection of bacteria, the protein TRAF-interacting factor with forkhead-associated domain (TIFA) forms oligomers, a process dependent on its threonine at position 9 and theforkhead-associated domain. These oligomers interact withTNF receptor associated factor (TRAF)6, leading to its oligomerization and the subsequent activation of NF-kB. In addition, we show that oligomerization of TIFA is dependent on the kinase alpha-kinase(ALPK)1 and that this pathway is activated in response to the detection of the bacterial metabolite heptose-1, 7-bisphosphate (HBP). These observations could be extended to the enteroinvasive pathogen Salmonella typhimurium as well as the extracellular bacteria Neisseria meningitidis. Our results therefore demonstrate the central role of the ALPK1-TIFA-TRAF6 signaling pathway in response to HBP of both intracellular and extracellular Gram-negative bacterial pathogens, and offer a better understanding of the molecular mechanisms governing the epithelial cell immune response to pathogenic bacteria.

A leishmaniose tegumentar americana (LTA) apresenta um amplo espectro de manifestações clínicas e imunopatológicas resultante da interação entre as diferentes espécies de Leishmania e os mecanismos de resposta imune do hospedeiro. Leishmania (Viannia) braziliensis e Leishmania (Leishmania) amazonensis são as espécies de maior potencial patogênico para o homem e de importância médica no Brasil. As células TCD4, quando ativadas por antígenos via MHC II podem se diferenciar em linhagens de células efetoras como Th1, Th2, Th17 e células T reguladoras (Treg). IL-23 é indispensável para as funções efetoras e manutenção de células Th17. O objetivo deste trabalho foi avaliar a expressão de Foxp3, IL-17 e IL-23 em lesões cutâneas de pacientes com diferentes formas clínicas da LTA. Biópsias parafinadas de 44 pacientes foram submetidas à imunoistoquímica, sendo 6 casos de leishmaniose cutânea anérgica difusa (LCADIDRM-) e leishmaniose cutânea disseminada borderline (LCDBIDRM-), ambas causadas por L.(L) amazonensis e 16 casos de leishmaniose cutânea localizada (LCLIDRM+) também causada por L.(L.) amazonensis; 9 casos de LCLIDRM+, 2 casos de LCDBIDRM- e 5 casos de leishmaniose cutâneo-mucosa (LCMIDRM+), todos causados por L.(V.) braziliensis. A densidade de células Tregs Foxp3+ no espectro clínico da LTA mostrou um aumento progressivo partindo das formas centrais LCL causadas por L.(V.) braziliensis (170mm2) e L.(L) amazonensis (140mm2) para as formas polares, LCADIDRM- (289mm2) e LCDBIDRM- (183mm2) causada por L.(L) amazonensis, LCDBIDRM- (189mm2) e LCMIDRM+, causadas por L.(V.) braziliensis (158mm2). A comparação entre as densidades de células IL-17+ nas diferentes formas clínicas da LTA mostrou um perfil semelhante também com um aumento progressivo da expressão de IL-17 partindo das formas centrais LCLIDRM+ causadas por L.(V.) braziliensis (232mm2) e L.(L) amazonensis (197mm2) em direção as formas polares, LCADIDRM- (470mm2) e LCDBIDRM- (340mm2) causada por L.(L.) amazonensis, LCDBIDRM- (431mm2) e LCMIDRM+ (372mm2) causada por L.(V.) braziliensis. A densidade de células IL-23+ mostrou perfil similar ao de IL-17 como no espectro de doença causada por L. (V.) braziliensis ou L. (L.) amazonensis: LCADIDRM- (687mm2), LCDBIDRM- (518mm2) e LCLIDRM+ (348mm2) por L.(L.) amazonensis, LCLIDRM+ (457mm2), LCDBIDRM- (609mm2) e LCMIDRM+ (568mm2) L. (V.) braziliensis. Diante dos nossos achados, observa-se que as células Foxp3+, IL-17+ e IL-23+ desempenham um papel importante na imunopatogênese das diferentes formas clínicas da LTA causadas por L. (V.) braziliensis ou L. (L.) amazonensis, caracterizada por uma resposta imune polarizada de diferente expressão patológica / The American cutaneous leishmaniasis (ACL) presents a wide spectrum of clinical and immunopathological manifestations resulting from the interaction between the different species of Leishmania and the mechanisms of the host immune response. Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis are the species with the largest pathogenic potential for humans and medical importance in Brazil. The CD4+ T cells can be differentiated into effector cell lines as Th1, Th2, Th17 and regulatory T cells (Treg). IL-23 is essential for effector functions and maintenance of Th17 cells, that produces IL-17. The aim of this study was to evaluate the expression of Foxp3, IL-17 and IL-23 in cutaneous lesions of patients with different clinical forms of ACL. Paraffin embedded biopsies from 44 patients were submitted to immunohistochemistry, there were 6 cases of anergic diffuse cutaneous leishmaniasis (ADCLDTH-) and borderline disseminated cutaneous leishmaniasis (BDCLDTH-) both caused by L. (L.) amazonensis 16 cases of cutaneous leishmaniasis (LCLDTH+) caused by L. (L.) amazonensis, 9 cases of LCLDTH+, 2 cases of BDCLDTH- and 5 cases of mucocutaneous leishmaniasis (MCLDTH+) all caused by L. (V.) braziliensis. The density of Treg Foxp3+ cells in the clinical spectrum of ACL showed a progressive increase starting from the central forms LCLDTH+ caused by L. (V.) braziliensis (170mm2) and L. (L) amazonensis (140mm2) towards the polar forms ADCLDTH- (289mm2). The intermediate clinical forms BDCLDTH- (183mm2) caused by L. (L) amazonensis and BDCLDTH-(189mm2) by L. (V.) braziliensis as well as, MCLDTH+(158mm2) did not present any significant differences. The comparison between the densities of IL-17+ cells in different clinical forms of ACL showed progressive increasing starting from the central forms LCLDTH+ caused by L. (V.) braziliensis (232mm2) and L. (L) amazonensis (197mm2) towards the polar forms, ADCLDTH-(470mm2) and BDCLDTH-(340mm2) caused by L. (L.) amazonensis BDCLDTH- (431mm2) and MCLDTH+ (372mm2) caused by L. (V.) braziliensis. The density of IL-23+ cells showed a similar profile to that of IL-17 at the disease spectrum caused by L. (V.) braziliensis and L. (L.) amazonensis: ADCLDTH-(687mm2) BDCLDTH-(518mm2) and LCLDTH+ (348mm2) by L. (L.) amazonensis; LCLDTH+ (457mm2) LCDBDTH-(609mm2) and MCLDTH+ (568mm2) L. (V.) braziliensis. In view of our findings, we notice that the Foxp3+, IL-17+ and IL-23+ cells play an important role in the immunopathogenesis of different clinical forms of ACL caused by L. (V.) braziliensis and L. (L.) amazonensis, characterized by an immune polarized response with different pathological expression

Diabetes Mellitus tipo 1A (DM1A), doença autoimune clássica, decorrente da quebra de tolerância imune por fatores ambientais em indivíduos geneticamente predispostos, é caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas. As células T auxiliadoras 17 (Th17) são células potentes, altamente inflamatórias, que produzem a interleucina 17A (IL-17A), citocina mediadora de várias desordens imunológicas como, artrite reumatóide, esclerose múltipla, encefalite experimental autoimune, psoríase e asma, e em animais, o diabetes autoimune. No entanto, seu papel na patogênese do DM1A em humanos não está definido O objetivo de nosso estudo foi avaliar a influência da IL-17A na predisposição ao DM1A através da identificação de variantes alélicas no gene da IL-17A (por sequenciamento automático) e da determinação dos níveis séricos de IL-17A (por ELISA) e da expressão do seu receptor em linfócitos T periféricos (por citometria de fluxo). Foram analisados 103 pacientes com DM1A (idade 15,15 ± 10,38) e 102 controles normais (idade 18,29 ± 10,83). O estudo da expressão do receptor da IL-17A em linfócitos T periféricos bem como o da proteína sérica foram conduzidos em 24 pacientes com DM1A recente (duração inferior a 6 meses) e 23 controles normais. Resultados: Nos 3 exons da IL-17 A analisados, a freqüência das 14 variantes alélicas já descritas em bancos de dados e de três novas variantes alélicas na região não codificadora do exon 3 (3UTR) não diferiu entre diabéticos e controles. Detectamos, pela primeira vez, diminuição estatisticamente significativa da expressão proporcional do receptor de IL-17A em células TCD3+ (p = 0,041) e TCD4+ (p = 0,0019) periféricas de pacientes com DM1A de início recente quando comparados com controles normais. As concentrações séricas de IL-17A foram menores nos diabéticos. Não observamos correlação entre a expressão dos receptores com a resposta humoral (níveis de autoanticorpos pancreáticos anti-GAD65 e anti-IA2) ou com variáveis metabólicas (glicemia e HbA1c). Nossos resultados sugerem que mutações ou polimorfismos no gene da IL-17A não estão implicadas na predisposição ao DM1A em humanos. A reduzida expressão dos receptores de IL-17A em linfócitos T CD3+ e CD4+ periféricos e das concentrações séricas de IL-17A nos pacientes diabéticos não indicam a participação ativa da via Th17 na periferia na patogênese do DM1A em humanos. No entanto, não descartamos a possibilidade de que, ao estudarmos variáveis na periferia e não do local de agressão imune (as ilhotas pancreáticas), tenhamos obtido valores que não expressem o processo adequadamente. Um eventual mecanismo de regulação negativa da via Th17, na tentativa de proteção do organismo contra o processo inflamatório autoimune, poderia explicar a diminuição de expressão de IL-17RA nos linfócitos periféricos / Type 1A diabetes mellitus (T1AD), a classical autoimmune disease related to the loss of immune tolerance is determined by environmental factors in genetically predisposed individuals. Pancreatic infiltration of T and B lymphocytes, macrophages and dentric cells characterize the process. T helper 17 (Th17) cells are potent, highly inflammatory cells, which initiate tissue inflammation and induce infiltration of other inflammatory cells in target organs. They produce the Interleukin 17A (IL-17A), considered a mediator of various immune disorders such as rheumatoid arthritis, multiple sclerosis, experimental autoimmune encephalitis, psoriasis and asthma, and in animals, autoimmune diabetes. However, its role in T1AD pathogenesis in humans is not defined. The aim of our study was to evaluate the influence of IL-17A in T1AD predisposition in humans. The allelic variants of IL-17A gene (by automatic sequencing), the expression of IL-17A receptors in peripheral lymphocytes (by flow cytometry assay) and the serum levels of IL-17A (by ELISA) were analyzed. Our casuistic was composed of 103 patients with T1D (15,15 ± 10,38 years) and 102 normal controls (18,29 ± 10,83 years). The expression of IL-17A receptor in peripheral lymphocytes and the serum concentration of IL-17A were determined in a subgroup of 24 recent-onset T1D (less than 6 months) and 23 normal controls. Results: The frequency of the 14 allelic variants on the 3 exons of IL- 17A gene already described on data bases did not differ between patients with diabetes and controls. We detected three new allelic variants at the final non-coding region of exon 3. Their frequency was also similar between patients and controls. We detected for the first time a statistically significant decrease in the proportional expression of the receptor of IL-17 on CD3+ (p=0,041) and CD4+ (p=0,0019) T lymphocytes in patients with recent-onset type 1A diabetes. IL- 17A serum concentrations were also lower in patients. There was no correlation between the expression of IL-17A receptor and titles of pancreatic autoantibodies (anti-GAD65 or anti-IA2) or metabolic variables (glucose and HbA1c levels). Our results suggest that mutations or polymorphisms of IL-17A gene are not implicated in the pathogenesis of T1AD in humans. The reduced expression of IL-17A receptors in peripheral T lymphocytes and of IL-17A serum concentrations in patients with diabetes did not indicate a role of Th17 via at the periphery in the autoimmune process. There is however the possibility that by studying the peripheral and not the local immune aggression (pancreatic islets) we have obtained values that do not adequately express the process. A possible mechanism of negative regulation of receptors in an attempt to protect the organism against autoimmune inflammatory process could explain the decrease of IL-17A levels and of IL-17RA expression in peripheral lymphocytes

Objetivo: determinar as concentrações séricas e peritoneais das interleucinas 1ß (IL-1ß) e 6 (IL-6) e da proteína amilóide A (SAA) em pacientes com endometriose pélvica. Métodos: foram avaliadas 97 pacientes submetidas à videolaparoscopia, 57 com endometriose (A), 27 com sintomas sugestivos porém sem endometriose (B) e 13 sem sintomas e doenças (C). Foram coletados no ato cirúrgico líquido peritoneal e sangue. As concentrações dos mediadores foram determinadas em pg/ml (IL-1 e 6) e ng/ml (SAA) por método imunoenzimático e leitura óptica. Resultados: (líquido peritoneal e sangue medianas) IL-1ß: A- 11,22 and 1,83; B- 15,62 and 1,16; C- 1,92 and 0,80; IL-6: A- 6,80 and 3,70; B- 8,60 and 3,90; C- 3,40 and 2,0; SAA- A- 310,30 and 14,01; B- 306,20 and 10,39; C- 53,4 e 9,5. Conclusão: as concentrações dos mediadores de inflamação avaliados estão elevadas no líquido peritoneal e no soro das mulheres com endometriose. Estas concentrações foram semelhantes às das pacientes com sintomas sugestivos porém sem a doença. A fase do ciclo menstrual, o tipo histológico envolvido e o local de acometimento da doença não influíram de forma significante nas concentrações séricas ou peritoneais de IL-1ß, IL-6 e SAA. / Objective: to assess peritoneal and seric interleukin-1ß (IL-1ß), 6 (IL-6), and protein amyloid A (SAA) concentrations in patients with pelvic endometriosis. Methods: 97 patients were submitted to video laparoscopic surgery, 57 with endometriosis (A), 27 with suggestive symptoms but no endometriosis (B), and 13 without symptoms and diseases (C). Peritoneal fluid and blood were collected during the procedure. Mediator s concentration was determined in pg/ml (IL-1ß and IL-6) and ng/ml (SAA) through immunoenzimatic test and optic measure. Results: (peritoneal fluid and blood -medians) IL-1ß: A- 11,22 and 1,83; B- 15,62 and 1,16; C- 1,92 and 0,80; IL-6: A- 6,80 and 3,70; B- 8,60 and 3,90; C- 3,40 and 2,0; SAA- A- 310,30 and 14,01; B- 306,20 and 10,39; C- 53,4 e 9,5. Conclusion: the inflammation mediators are increased in the peritoneal fluid and blood of women with pelvic endometriosis. The concentrations are similar to those found in patients with suggestive symptoms but no endometriosis. The place of the disease, the histological type and the day of the menstrual cycle didn t alter peritoneal or seric concentration of these cytokines.

Alternatives mRNA-Splicing ist ein häufig beobachtetes Phänomen, das es der Zelle ermöglicht, unterschiedliche Proteine aus einem Gen zu generieren. In den letzten Jahren wurden immer mehr alternativ gespleißte Transkripte entdeckt, und einigen der daraus resultierenden Protein-Isoformen konnten geänderte biologische Funktionen zugeordnet werden. In dieser Arbeit ist erstmals ein alternativ gespleißtes Transkript der Interleukin-4 Rezeptor alpha (IL-4R-alpha) Kette beschrieben. Dieser mRNA Splice-Variante, genannt IL-4R-alpha-IT, fehlt im membranproximalen Bereich der zytoplasmatischen Domäne ein komplettes Exon. Dies führt zur Verschiebung des Leserasters und so zur Entstehung eines vorzeiten Stop-Codons. Der resultierenden Protein-Isoform fehlt der größte Teil der intrazellulären Kette mit den dort enthaltenen, für die Signaltransduktion essentiellen Domänen. Die Untersuchung der biologischen Funktion der Rezeptor-Varianten in einem geeigneten Zellsystem der Maus zeigte, daß die Splice-Variante IL-4R-alpha-IT keine Proliferation der Zellen vermitteln und auch den Übergang der Zellen in die Apoptose nicht verhindern kann. Bei der Quantifizierung der Expression von IL-4R-alpha-IT-mRNA in Relation zum IL-4R-alpha voller Länge mit einer kompetitiven RT-PCR an Knochenmark und peripheren Blutlymphozyten von Kindern mit ALL zeigte sich zunächst ein irreführender Unterschied zwischen Proben von Kindern mit ALL-Ersterkrankung und Rezidiv. Weitere Untersuchungen ergaben jedoch, daß der Zeitraum zwischen Abnahme und Aufarbeitung des Untersuchungsmaterials für diesen scheinbaren Zusammenhang verantwortlich war. Während direkt nach Abnahme aufgearbeitetes Untersuchungsmaterial eine nur niedrige relative Expression der Splice-Variante zeigte, nahm diese bei verzögerter Aufarbeitung drastisch zu. Diese Beobachtung wurde experimentell an Proben gesunder Probanden wiederholt bestätigt. Interessanterweise konnte derselbe Effekt in unterschiedlicher Ausprägung auch bei Splice-Variante anderer Zytokine und -Rezeptoren wie IL-7, IL-7R und beta-C beobachtet werden. mRNA-Stabilitäts-Assays und die Bestimmung der einzelnen Transkripte mit einer semiquantitativen RT-PCR zeigten, daß es tatsächlich zu einer absoluten Hochregulation der IL-4R-alpha-IT-mRNA in den verzögerte aufgearbeiteten Proben kommt. Wurden die Zellen wieder in Kultur genommen, war dies innerhalb weniger Stunden reversibel. Desweiteren scheinen auch unterschiedlichen mRNA-Stabilitäten eine Rolle zu spielen. / Alternative pre-mRNA splicing is a widespread mechanism contributing to the diversity of gene expression. The number of newly detected alternatively spliced transcripts has continuously risen, and distinct biological functions have been attributed to some protein isoforms resulting from these mRNA variants. We report on the detection of a novel alternatively spliced transcript of the human interleukin-4 receptor alpha (IL-4R-alpha) chain, which has been called IL-4R-alpha-IT mRNA. A premature stop codon due to omission of one exon in the membrane-proximal region of the cytoplasmic domain leads to an mRNA variant, which encodes an intracellular truncated receptor protein lacking domains which are essential for signal transduction. The investigation of the biological function of the IL-4Ra splice variants in a suitable mouse cell system showed, that the truncated receptor variant is not able to mediate cell proliferation or prevention of apoptosis. Bone marrow and peripheral blood samples from children with acute lymphoblastic leukemia were analyzed for the expression of IL-4R-alpha-IT mRNA relative to the full-length receptor transcript by competitive RT-PCR. Initially, there was found a difference of IL-4R-alpha-IT mRNA expression in patients with initial ALL versus relapsed ALL. However, this difference turned out to be due to the time interval between collection and preparation of samples. While freshly isolated material was associated with low levels of IL-4R-alpha-IT mRNA, samples with a longer period until cell preparation exhibited a drastic increase of IL-4R-alpha-IT mRNA levels. The same results were obtained for peripheral blood samples from healthy donors by imitating a prolonged time of transport until cell preparation. Interestingly, a similar effect could be demonstrated for splice variants of other cytokine receptors and cytokines (beta-C, IL-7R, and IL-7), although to different extents. mRNA stability assays and semiquantitative RT-PCR specific for IL-4Ra or IL-4R-alpha-IT, respectively, indicated that the expression of IL-4R-alpha-IT mRNA increases absolutely in these samples, although mRNA degradation may be of importance as well.

Das Knochenmarkmikromilieu produziert eine Reihe von unterschiedlichen Wachstumsfaktoren, die für das maligne Wachstum und die Medikamentenresistenz von Myelomzellen von grosser Bedeutung sind. Einer der wichtigsten Faktoren, der in manchen experimentellen Systemen sogar als essentiell für das Wachstum und Überleben von Myelomzellen beschrieben wurde, ist Interleukin-6. Aus diesem Grund könnte die Entwicklung von Substanzen, die die Wirkung von IL-6 oder dem IL-6 Rezeptor inhibieren von Bedeutung für die Therapie des Myeloms sein. In dieser Arbeit wurde die Wirkung des IL-6 Rezeptorantagonisten SANT7 auf das Überleben der IL-6 abhängigen Myelomzellinie INA-6 sowie primären Myelomzellen in Gegenwart oder Abwesenheit von primären humanen Knochenmarkstromazellen (KMSZ) untersucht. Von besonderem Interesse war hierbei die Frage ob SANT7 die wachstumsinhibitorische Wirkung von Dexamethson (Dex) und All-Trans-Retinolsäure (ATRA) verstärken kann. Keine der drei Substanzen, SANT7 eingeschlossen, konnte bei alleiniger Applikation in Gegenwart von primären humanen KMSZ eine nennenswerte Wachstumsinhibition induzieren. Wenn jedoch Dex und ATRA mit SANT7 kombiniert wurden konnte sowohl in INA-6 als auch primären Myelomzellen eine starke Wachstumsinhibition erzielt werden. Dieser Effekt beruht sowohl auf Apoptose als auch eines Zellzyklusarrests. / The bone marrow microenvironment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is interleukin-6 (IL-6). Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have a potential therapeutic value for the treatment of multiple myeloma. In this work the effect of the IL-6 receptor antagonist SANT7 on growth and survival of the IL-6 dependent MM cell lines INA-6 as well as primary MM cells in the presence or absence of bone marrow stromal cells (BMSC) was analyzed. Of particular interest was the question whether SANT7 might enhance the growth inhibitory effects of dexamethasone (Dex) and all-trans retinoic acid (ATRA). None of the drugs, when tested as a single substance, including SANT7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, if Dex and ATRA were given in combination with SANT7 a strong growth inhibition was achieved in INA-6 and primary MM cells. This effect is due to cell cycle arrest and induction of apoptosis.