La goutte est une arthrite particulièrement douloureuse due à une réponse immunitaire contre des cristaux d’urate monosodique (MSU). Le neutrophile est un leucocyte qui contribue grandement à la perpétuation de l’inflammation lors d’une crise de goutte. Avant d’être recrutés au site d’inflammation, les neutrophiles rencontrent plusieurs médiateurs pro-inflammatoires, dont la protéine S100A9. Nous montrons ici que S100A9 accentue plusieurs réponses effectrices des neutrophiles humains aux MSU, notamment la production de dérivés réactifs de l’oxygène (ROS), la sécrétion de CXCL8/IL-8 et d’IL-1β et la glycolyse. S100A9 augmente également la mobilisation intracellulaire de calcium du neutrophile en plus d’augmenter la phosphorylation des tyrosines, la phosphorylation des sérines des substrats de la PKC, d’AKT et de la p38. Nous avons identifié un des mécanismes par lesquels S100A9 contribue à la pathogenèse de la goutte ainsi que les voies de signalisation impliquées dans ce phénomène qui sont des cibles thérapeutiques potentielles pour cette maladie. / Gout is the common and painful type of inflammatory arthritis. It is caused by an immune response against monosodium urate crystals (MSU) that form in the affected joint. Neutrophils are the most abundant leukocytes in the gout joint and play a key role in perpetuating inflammation during a gout flare. During their recruitment to the site of inflammation, neutrophils are exposed to several pro-inflammatory mediators such as S100A9. Although blocking S100A9 dampens MSU-induced inflammation, the role of this protein in the pathogenesis of gout remains incompletely characterized. We identified a novel role for S100A9 in MSU-induced inflammation, the priming of neutrophils towards MSU activation. We provide evidence for the ability of S100A9 to enhance several effector functions of human neutrophils triggered by MSU including the production of reactive oxygen species (ROS), the secretion of CXCL8/IL-8 and IL-1β as well as glycolysis. As for intracellular signaling, S100A9 increases the mobilisation of calcium, induces tyrosine phosphorylation of intracellular proteins as well as serine phosphorylation of PKC, AKT and p38 kinase. In summary, we report for the first time that S100A9 acts as a priming agent during MSU-induced inflammation and identify the underlying signaling pathways that could be targeted to treat gout.

Thèse (Ph. D.)--Université Laval, 2008. / Titre de l'écran-titre (visionné le 2 sept. 2008). Bibliogr.

Ce mémoire de maîtrise se focalise sur l'influence du cholestérol sur l'interaction de la protéine S100A10 avec les membranes cellulaires, dans le contexte de la macropinocytose. Ce processus, essentiel à la survie cellulaire, permet l'ingestion de liquides extracellulaires et joue un rôle clé dans la régulation de fonctions cellulaires critiques telles que l'immunité, la migration cellulaire et la signalisation. Il est caractérisé par sa capacité à permettre aux cellules de s'adapter rapidement aux changements environnementaux en ingérant des nutriments et en répondant à des stimuli variés. Ce mémoire s'est concentré à décortiquer le rôle spécifique du cholestérol sur la liaison membranaire de la protéine S100A10. L'accent a, dans un premier temps, été mis sur l'optimisation de la surexpression et la purification de cette protéine, par le biais d'approches méthodologiques rigoureuses. Ces efforts ont permis de sonder en profondeur les interactions entre cette protéine et différents constituants des membranes cellulaires. La technique de la tensiométrie de surface a été particulièrement révélatrice, dévoilant l'influence significative de la présence et de la concentration du cholestérol sur la liaison de la S100A10 avec divers phospholipides membranaires. En complément, les isothermes de compression ont permis de dévoiler comment le cholestérol modifie la compressibilité et la stabilité des monocouches lipidiques. Ces données ont mis en évidence l'importance de l'architecture membranaire dans les mécanismes cellulaires impliquant la protéine S100A10. En somme, ce mémoire révèle que la modulation des interactions entre la S100A10 et les phospholipides par le cholestérol est un processus complexe, pouvant avoir des répercussions directes sur la macropinocytose. Les résultats de cette étude apportent une contribution significative à la compréhension des mécanismes régissant le comportement des membranes biologiques. / This master's thesis is centered on the influence of cholesterol on the interaction of the S100A10 protein with cellular membranes, in the context of macropinocytosis. This process, essential for cellular survival, allows the ingestion of extracellular fluids and plays a key role in regulating critical cellular functions such as immunity, cell migration, and signaling. It is characterized by its ability to allow cells to quickly adapt to environmental changes by ingesting nutrients and responding to various stimuli. This master's thesis focused on dissecting the specific role of the cholesterol in the membrane binding of the S100A10 protein. Initially, the priority was placed on optimizing the overexpression and purification of this protein, using rigorous methodological approaches. These efforts enabled an in-depth probing of the interactions between this protein and different constituents of cellular membranes. The technique of surface tensiometry was particularly revealing, uncovering the significant influence of the presence and concentration of cholesterol on the binding of S100A10 to various membrane phospholipids. Additionally, compression isotherms revealed how cholesterol modifies the compressibility and stability of lipid monolayers. These findings highlighted the importance of membrane architecture in cellular mechanisms involving the S100A10 protein. In summary, this thesis reveals that the modulation of interactions between S100A10 and phospholipids by cholesterol is a complex process, which can have direct repercussions on macropinocytosis. The results of this study make a significant contribution to understanding the mechanisms governing the behavior of biological membranes.

Les protéines appartenant à la famille S100 et les annexines interviennent lors de différents mécanismes membranaires vitaux. En effet, le complexe protéique S100A10-annexine A2 permettrait le recrutement d'une partie du C-terminal de la protéine AHNAK à la membrane en présence de calcium, avant de former une plateforme qui initierait la réparation membranaire. Cependant, aucune donnée moléculaire n'est à ce jour disponible sur la liaison membranaire de la S100A10 et du segment C-terminal d'AHNAK de ce complexe. Leur liaison membranaire doit donc être étudiée afin de mieux comprendre leurs rôles lors du processus de réparation membranaire. Afin de combler le manque de données sur le rendement et la quantité de S100A10 solubilisée et purifiée parmi la littérature existante, le protocole de surexpression et purification de la S100A10 a été optimisé dans un premier temps. La protéine a été identifiée par spectrométrie de masse et la stabilité de sa structure secondaire a été analysée par dichroïsme circulaire à différentes températures au cours du temps. La S100A10 obtenue est stable pendant au moins 60 jours à plusieurs températures, dont 20 °C, permettant d'effectuer des expériences biophysiques à température ambiante avec la protéine non-dénaturée. En parallèle, le segment peptidique C-terminal d'AHNAK (pAHNAK) a été synthétisé commercialement. L'étude par spectroscopie infrarouge de réflexion totale atténuée (ATR) montre que sa structure secondaire ne subit pas de changement majeur en présence et en absence de lipides. Deux modèles membranaires ont été choisis dans nos études. D'abord, celui des monocouches de Langmuir a été utilisé pour mimer les membranes cellulaires afin de caractériser l'interaction des protéines avec différents phospholipides composant les membranes. Les études de la liaison membranaire de ces protéines ont été effectuées avec ce modèle en le couplant avec la tensiométrie de surface. Ces travaux ont démontré que le pAHNAK interagissait plus fortement avec les lipides insaturés, en particulier les lipides monoinsaturés, qu'avec les lipides saturés. De plus, le pAHNAK interagit préférentiellement avec la tête polaire des phospholipides de type phosphatidylsérine (PS), qui sont chargées négativement, puis avec ceux de type phosphatidyléthanolamine (PE) et enfin avec ceux de type phosphatidylcholine (PC). Avec le même modèle, la profondeur d'insertion des protéines dans des monocouches lipidiques a été déterminée par ellipsométrie. Les expériences avec des phospholipides monoinsaturés démontrent que la profondeur d'insertion du pAHNAK suit la même tendance (PS˃PE˃PC). Le deuxième modèle employé est celui des bicouches lipidiques avec lequel l'affinité de ces protéines pour les têtes polaires des phospholipides a spécifiquement été évaluée par résonance magnétique nucléaire (RMN) du ³¹P à l'état solide. Ces études de RMN confirment la tendance de préférence du pAHNAK pour les différents types de phospholipide à 37 °C observée précédemment. La liaison membranaire de la S100A10 a ensuite été étudiée selon la même stratégie que pour le pAHNAK. Les résultats de la tensiométrie montrent que la S100A10 préfère interagir avec les phospholipides insaturés avec des têtes polaires PE ou PS, et surtout les phospholipides polyinsaturés contenant de longues chaînes acyles. Les expériences d'ellipsométrie suggèrent que la S100A10 suit l'ordre d'insertion PC > PE > PS, ce qui pourrait être expliqué par un changement d'orientation de la protéine. De plus, les résultats de RMN suggèrent qu'à 20 °C et à 37 °C, la S100A10 pourrait s'insérer partiellement dans la membrane près des têtes polaires PS. L'ensemble de nos travaux de recherche démontrent que, dans un environnement physiologique à 37 °C, le pAHNAK et la S100A10 peuvent probablement interagir avec des phospholipides monoinsaturés ayant une tête polaire chargée négativement. Cependant, le pAHNAK aura tendance à s'insérer dans les chaînes acyles alors que la S100A10 s'insérerait partiellement près des têtes polaires. Ce projet a ainsi permis de développer les connaissances sur la liaison membranaire de la protéine S100A10 et du pAHNAK et d'émettre de nouvelles hypothèses quant au rôle des PS dans la réparation membranaire. De plus, la poursuite de ces travaux mènera à l'identification potentielle des conditions conduisant à une modification de leur liaison membranaire, et éventuellement à une perte de fonction. Ainsi, les connaissances développées dans cette thèse permettent de mieux comprendre la liaison membranaire de la S100A10 et du C-terminal d'AHNAK afin de mieux déterminer leurs rôles dans la réparation membranaire, ainsi que dans les autres mécanismes physiologiques auxquels ces protéines participent. / Proteins belonging to S100 family proteins and annexins are involved in various vital membrane mechanisms. The S100A10-annexin A2 protein complex was postulated to recruit part of the C-terminal segment of the AHNAK protein to the membrane in the presence of calcium, before forming a platform which can initiate membrane repair. However, no molecular data is currently available on the membrane binding of S100A10 and of the C-terminal segment of AHNAK of this complex. Their membrane binding should therefore be studied in order to better understand their roles during the membrane repair process. In order to fill the lack of data on the yield and the quantity of purified and solubilized S100A10 in the existing literature, the protocol for the overexpression and purification of S100A10 was first optimized. The protein was identified by mass spectrometry and its secondary structure stability was analyzed by circular dichroism at different temperatures over time. The S100A10 obtained is stable for at least 60 days at several temperatures, including 20 °C, allowing to perform biophysical experiments at room temperature with a non-denatured protein. In parallel, the C-terminal peptide segment of AHNAK (pAHNAK) was commercially synthesized. Attenuated total reflection infrared spectroscopy (ATR) study shows that its secondary structure does not undergo major changes in the presence and absence of lipids. Two membrane models were chosen in our studies. First, the Langmuir monolayers model was used to mimic cell membranes in order to characterize the interaction of proteins with the different phospholipids found in membranes. Membrane binding studies of these proteins were carried out with this model by coupling it with surface tensiometry. This work demonstrated that pAHNAK interact more strongly with unsaturated lipids, in particular monounsaturated lipids, than with saturated lipids. In addition, pAHNAK preferentially interacts with the phosphatidylserine (PS) polar head group which is negatively charged, then with phosphatidylethanolamine (PE) and finally with phosphatidylcholine (PC). With the same model, the insertion depth of pAHNAK into lipid monolayers was determined by ellipsometry. Experiments with monounsaturated phospholipids demonstrate that the insertion depth of pAHNAK follows the same trend (PS˃PE˃PC). The second model membrane used is lipid bilayers with which the affinity of pAHNAK for phospholipid polar head groups has specifically been evaluated by solid state ³¹P nuclear magnetic resonance (NMR). These NMR studies confirm the previously observed preference trend of pAHNAK at 37 °C for the different polar head groups. The membrane binding of S100A10 was then studied according to the same strategy as pAHNAK. The results of the tensiometry show that S100A10 prefers to interact with unsaturated phospholipids bearing PE or PS polar head groups, and especially polyunsaturated ones containing long fatty acyl chains. Ellipsometric experiments suggest that S100A10 follows the order of insertion PC> PE> PS, which could be explained by a change in orientation of the protein. Moreover, NMR results suggest that, at 20 and 37 °C, S100A10 could partially insert into the membrane near the PS polar head group. All of our research demonstrates that, in a physiological environment of 37 °C, pAHNAK and S100A10 can probably interact with monounsaturated phospholipids containing a negatively charged polar head group. However, pAHNAK will tend to insert into the acyl chains while S100A10 would insert partially near the polar head group of phospholipids. This project has thus made it possible to develop knowledge on the membrane binding of the S100A10 protein and of pAHNAK as well as to provide new hypotheses regarding the role of PS in membrane repair. In addition, the continuation of this work will lead to potentially identify the conditions leading to a modification of the membrane binding of these proteins, and possibly to a loss of function. Thus, the knowledge developed in this thesis allows to improve our understanding of the membrane binding of S100A10 as well as of the C-terminal segment of AHNAK in order to better determine their roles in membrane repair, as well as in other physiological mechanisms in which these proteins are involved.

La réaction inflammatoire est un mécanisme de défense de l’hôte, toutefois, dans certaines circonstances, il arrive que cette réaction se retourne contre l’organisme. Il est donc important de comprendre l’origine de ce problème afin d’y trouver des solutions. Une étape majeure de la réaction inflammatoire est la migration des leucocytes du sang vers la région affectée. De ces cellules, le neutrophile est le premier à se rendre au site inflammatoire. Outre son rôle de défense de première ligne, le neutrophile a également la capacité de libérer divers médiateurs qui lui permettent d’orchestrer la réponse immunitaire. Le neutrophile est un réservoir majeur de protéines S100A8, S100A9 et S100A12. Depuis une vingtaine d’années, ces protéines ne cessent d’être associées à diverses pathologies infectieuses ou inflammatoires. Elles sont en effet détectées à de fortes concentrations dans le sérum de patients atteints d’arthrites, de maladies inflammatoires de l’intestin, de tuberculose, etc. Elles sont au demeurant des marqueurs sensibles de l’activité de ces maladies. Des études indiquent que ces protéines sont sécrétées dans des conditions inflammatoires et qu’elles exercent dans le milieu extracellulaire des fonctions pro-inflammatoires. Plus particulièrement, elles sont associées à la migration des leucocytes. Toutefois, les mécanismes d’action précis de ces protéines restent à élucider. Dans ces travaux de thèse, nous nous sommes intéressés aux rôles des protéines S100 dans la migration transendothéliale et tissulaire des neutrophiles. Nous avons observé que l’ajout de la protéine S100A9, dans des modèles in vitro, favorise l’adhésion des neutrophiles d’une part à l’endothélium et d’autre part à la fibronectine par l’activation des intégrines β2 et favorise ainsi la migration de ces cellules au site inflammatoire. S100A9 joue donc un rôle d’amplificateur de la réponse inflammatoire. Ces travaux nous aident à mieux comprendre le rôle des protéines S100 dans l’inflammation et plus particulièrement dans la migration des neutrophiles. Ces protéines constituant des cibles thérapeutiques potentielles, il est donc important de détailler leurs rôles dans le contexte inflammatoire. / Inflammation is one of the body’s defence mechanisms. In some cases, this reaction can be detrimental to the host it is supposed to protect. It is thus important to understand the origin of such reactions in order to find solutions. A key step of inflammatory reactions is leukocyte migration from blood to the injured area. Among leukocytes, neutrophils are the first to reach the inflammatory site. In addition to their role as the body’s first line of defence, neutrophils also help orchestrate the immune response through the release of inflammatory mediators. The neutrophils are a major reservoir of S100A8, S100A9, and S100A12 proteins. In the past 20 years, these proteins have been increasingly associated with various infectious and inflammatory diseases as they were detected at high concentrations in the serum of patients suffering from arthritis, inflammatory bowel diseases and tuberculosis to name a few. Moreover, they were shown to be sensitive markers of these diseases’ activity. Some studies have indicated that these proteins are secreted in inflammatory conditions and that they have pro-inflammatory activities in the extracellular space. Specifically, they were associated with leukocyte migration. However, the precise mechanisms through which these proteins affect the inflammatory response remain to be elucidated. In this work, we investigated the roles of S100 proteins in neutrophil transendothelial and tissue migration. We observed that S100A9 protein increased neutrophil adhesion to the endothelium and fibronectin by activating β2 integrins which led to the stimulation of cell migration to the inflammatory site in in vitro models. This work provided insight on the roles of S100 proteins in inflammation and more specifically in neutrophil migration. As these proteins represent potential therapeutic targets, it remains important to further understand their roles in inflammatory conditions.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2016-2017 / Les leucémies myéloïdes aiguës (LMA) sont des hémopathies rares, mais très agressives. Elles résultent d’un dérèglement du processus d’hématopoïèse qui se caractérise par une prolifération incontrôlée de cellules sanguines immatures engagées dans la lignée myéloïde. En dépit des traitements actuels qui reposent sur l’utilisation d’agents chimiothérapeutiques ciblant les cellules en prolifération, le pronostic des patients souffrants de LMA est très sombre. En effet, seuls 30% des patients souffrants de LMA survivent au-delà de 5 ans suivant la prise en charge thérapeutique. L’identification des acteurs participant au développement et au maintien des LMA est donc cruciale pour l’élaboration d’une stratégie thérapeutique efficace et ciblée. S100A8 et S100A9 sont des protéines fixatrices de calcium exprimées par les neutrophiles et les monocytes. Ce sont des alarmines jouant des rôles clés dans l’inflammation et dans des pathologies causées par une inflammation excessive. Les protéines S100A8 et S100A9 exercent également de multiples fonctions dans divers tumeurs solides. Elles favorisent la formation de niche pré-métastasique et inhibe la réponse immunitaire antitumorale. Une analyse du génome par séquençage a mis en évidence que S100A8 et S100A9 sont fortement exprimées chez les patients atteints de LMA. De plus, l’expression de la protéine S100A8 chez les patients souffrants de LMA serait corrélée avec un faible taux de survie. Principalement étudiées dans les tumeurs solides, les fonctions des protéines S100A8 et S100A9 dans les néoplasies hématologiques telles que les leucémies sont très peu documentées. Dans ces travaux de thèse, nous nous sommes donc intéressés aux rôles exercés par les protéines S100A8 et S100A9 dans les leucémies myéloïdes aiguës. À l’aide d’un modèle murin de LMA induit par la surexpression des facteurs HOXA9 et MEIS1 dans des cellules souches/progénitrices hématopoïétiques, nous avons démontré l’existence d’une fraction de cellules exprimant les protéines S100A8 et S100A9. Celle-ci est également retrouvée chez les patients atteints de leucémies aiguës myélomonocytaires et monocytaires (M4-M5 d’après la classification FAB). Les études menées in vivo et in vitro révèlent que la protéine S100A9 induit la différenciation des cellules leucémiques, tandis que la protéine S100A8, préviens l’effet de S100A9 permettant de maintenir ainsi le phénotype immature des cellules LMA. Le traitement par la protéine recombinante S100A9 permet d’accroitre la maturation des cellules LMA, diminue leur prolifération et prolonge la survie des souris LMA. De la même façon le traitement par les anticorps anti-S100A8 provoque un effet similaire au traitement par la protéine S100A9. Nos résultats suggèrent que de forts ratios de S100A9 sur S100A8 sont requis pour induire la différenciation des cellules LMA. Le mécanisme intracellulaire par lequel S100A9 induit la différenciation des cellules leucémiques a également été étudié dans le cadre de cette thèse. Nous avons identifié que S100A9 via la liaison au récepteur TLR (Toll-like receptor) active les voies de signalisations Mitogen Activated Protein Kinase p38, Jun N-terminal Kinase et extracellular signal-regulated kinases 1 et 2 et provoque la différenciation des cellules leucémiques. Les essais menés sur des cellules primaires de patients malades ont permis de confirmer la capacité de S100A9 et de S100A8 à réguler la différenciation des cellules leucémiques. En somme, les données présentées dans cette thèse contribuent à une meilleure compréhension des rôles des protéines S100A8 et S100A9 dans la différenciation des cellules myéloïdes. Par ailleurs, nos données permettent également d’entrevoir les bénéfices thérapeutiques liés au blocage de S100A8 ou à l’augmentation de S100A9 dans les LMA. / Acute myeloid leukemias (AMLs) are rare but still aggressive hematological diseases. They are the result of a perturbed hematopoietic process characterized by an uncontrolled proliferation of hematopoietic cells committed to the myeloid lineage. Despite current therapy based on chemotherapeutic agents, aimed at killing proliferating cells, prognosis of AML patients is dismal and only 30 % of patients survived beyond 5 years. Identification of actors involved in the initiation and sustaining LMA is crucial to the development of efficient and targeted therapy strategy. S100A8 and S100A9 are calcium-binding proteins predominantly expressed by neutrophils and monocytes, and play key roles in both normal and pathological inflammation. Recently, both proteins were found to promote tumor progression through the establishment of pre-metastatic niches and to inhibit antitumor immune responses. Although S100A8 and S100A9 have been studied in solid cancers, their functions in hematological malignancies remain poorly understood. However, S100A8 and S100A9 are highly expressed in acute myeloid leukemia (AML), and S100A8 expression has been linked to a poor prognosis in AML. Although the roles of these proteins were studies in solid tumor, little is known in their functions in hematological malignancies. We studied in this thesis the role of S100A8 and S100A9 in acute myeloid leukemia. Using AML mouse model of AML surexpressing HOXA9 and MEIS1 in hematopoietic stem and progenitor cells, we identified a small subpopulation of cells expressing S100A8 and S100A9. This subpopulation was consistently found in AML samples from patients with myelomonocytic and monocytic leukemias (M4 and M5 according FAB classification). In vitro and in vivo analyses revealed that S100A9 induces AML cell differentiation, whereas S100A8 prevents differentiation induced by S100A9 activity and maintains AML immature phenotype. Treatment with recombinant S100A9 proteins increased AML cell maturation, induced growth arrest, and prolonged survival in an AML mouse model. Interestingly, anti-S100A8 antibody treatment had effects similar to S100A9 therapy in vivo, suggesting that high ratios of S100A9 over S100A8 are required to induce differentiation. In this thesis, the mechanism of S100A9 leading to differentiation of leukemic cells was also study. Our in vitro studies on the mechanisms/pathways involved in leukemic cell differentiation revealed that binding of S100A9 to toll-like receptor 4 (TLR4) promotes activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2, and Jun N-terminal kinase signaling pathways, leading to myelomonocytic and monocytic AML cell differentiation. Overall, our findings indicate that S100A8 and S100A9 are regulators of myeloid differentiation in leukemia and have therapeutic potential in myelomonocytic and monocytic AMLs.

Compreender a função das diferentes proteínas e citocinas durante a vigência de alterações inflamatórias pode contribuir para um melhor conhecimento do sistema imunológico dos mamíferos marinhos e sua relação com os processos patológicos. A toninha, Pontoporia blainvillei, é um cetáceo costeiro e o único golfinho brasileiro considerado ameaçado de extinção devido às atividades antrópicas. O objetivo desse trabalho foi avaliar as imunoexpressões das citocinas TGF-β, TNF-α e IL-12 e proteínas S-100 e lisozima em amostras pulmonares e esplênicas de 34 espécimes de P. blainvillei. Com base nas alterações histopatológicas, os animais foram divididos em dois grupos: G1 (n = 8) - animais sem doença inflamatória pulmonar; G2 (n = 26) - animais com doenças inflamatórias pulmonares. Os fragmentos de pulmão e baço foram processados de acordo com os protocolos de rotina. A padronização das reações de imuno-histoquímica e análise morfométrica foram realizadas conforme procedimentos descritos na literatura. Os nossos dados demonstram que as P. blainvillei pertencentes ao G2 apresentam imunoexpressão pulmonar das citocinas TGF-β, TNF-α e IL-12 e proteínas S-100 e lisozima significativamente maior que os animais do G1. Tais resultados corroboram os dados disponíveis na literatura, indicando que a P. blainvillei apresenta citocinas com funções semelhantes às encontradas em outros mamíferos.Evidenciamos também, que as P. blainvillei pertencentes ao G2 não apresentam imunoexpressão esplênica das citocinas TGF-β, TNF-α e IL-12 e da proteína lisozima significativamente maior que os animais do G1. Estudos mais detalhados, incluindo aqueles relacionados com os métodos utilizados, são necessários para a adequada compreensão destes resultados. E finalmente, apesar de tratar-se de dados ainda preliminares, observou-se uma possível associação entre elevadas concentrações de organoclorados no tecido adiposo subcutâneo de parcela dos exemplares de P. blainvillei estudados na presente investigação e a ocorrência de depleção linfóide esplênica nestes animais. Tais dados ratificam a importância do desenvolvimento de análise mais detalhada sobre estes tópicos em futuras investigações científicas. / The understanding of the role of different proteins and cytokines during the inflammatory diseases could lead to better knowledge of the marine mammal immune system and its relation to pathological process. Franciscana dolphin, Pontoporia blainvillei, is a coastal cetacean and the only Brazilian dolphin considered threatened with extinction, due to the human activities. The aim of the current study was to investigate the immunoexpression of TGF-β, TNF-α and IL-12 cytokines, and S-100 and Lysozyme proteins in pulmonary and splenic samples of 34 Pontoporia blainvillei. Based on histopathological examination, the animals were divided in two groups: G1 (n = 8) animals without pulmonary inflammatory disease; G2 (n = 26) animals with pulmonary inflammatory diseases. Fragments of lung and spleen were processed according to routine protocols. The immunohistochemistry and morphometric procedures were tested and optimized according to proceedings described in literature. Our results showed that P. blainvillei from G2 presented significant increase in the pulmonary immunoexpression of TGF-β, TNF-α and IL-12 cytokines, and S-100 and Lysozyme proteins in comparison with animals from G1. These results are according to the literature and suggest that P. blainvillei presents same cytokines observed in other mammals. We evidenced also that the P. blainvillei belonging to the G2 did not show splenic immunoexpression to TGF-β, TNF-α and IL- 12 cytokines and Lysozyme protein significantly higher than animals from G1. Further studies, including those related to the methods used, are necessary for the proper understanding of these results. And finally, although these are still preliminary data, we observed a possible association between high concentrations of organochlorines in subcutaneous adipose tissue of a portion of P. blainvillei studied in this research and the occurrence of splenic lymphoid depletion in these animals. These data confirm the importance of developing more detailed analysis on these topics in future scientific investigations.

Compreender a função das diferentes proteínas e citocinas durante a vigência de alterações inflamatórias pode contribuir para um melhor conhecimento do sistema imunológico dos mamíferos marinhos e sua relação com os processos patológicos. A toninha, Pontoporia blainvillei, é um cetáceo costeiro e o único golfinho brasileiro considerado ameaçado de extinção devido às atividades antrópicas. O objetivo desse trabalho foi avaliar as imunoexpressões das citocinas TGF-β, TNF-α e IL-12 e proteínas S-100 e lisozima em amostras pulmonares e esplênicas de 34 espécimes de P. blainvillei. Com base nas alterações histopatológicas, os animais foram divididos em dois grupos: G1 (n = 8) - animais sem doença inflamatória pulmonar; G2 (n = 26) - animais com doenças inflamatórias pulmonares. Os fragmentos de pulmão e baço foram processados de acordo com os protocolos de rotina. A padronização das reações de imuno-histoquímica e análise morfométrica foram realizadas conforme procedimentos descritos na literatura. Os nossos dados demonstram que as P. blainvillei pertencentes ao G2 apresentam imunoexpressão pulmonar das citocinas TGF-β, TNF-α e IL-12 e proteínas S-100 e lisozima significativamente maior que os animais do G1. Tais resultados corroboram os dados disponíveis na literatura, indicando que a P. blainvillei apresenta citocinas com funções semelhantes às encontradas em outros mamíferos.Evidenciamos também, que as P. blainvillei pertencentes ao G2 não apresentam imunoexpressão esplênica das citocinas TGF-β, TNF-α e IL-12 e da proteína lisozima significativamente maior que os animais do G1. Estudos mais detalhados, incluindo aqueles relacionados com os métodos utilizados, são necessários para a adequada compreensão destes resultados. E finalmente, apesar de tratar-se de dados ainda preliminares, observou-se uma possível associação entre elevadas concentrações de organoclorados no tecido adiposo subcutâneo de parcela dos exemplares de P. blainvillei estudados na presente investigação e a ocorrência de depleção linfóide esplênica nestes animais. Tais dados ratificam a importância do desenvolvimento de análise mais detalhada sobre estes tópicos em futuras investigações científicas. / The understanding of the role of different proteins and cytokines during the inflammatory diseases could lead to better knowledge of the marine mammal immune system and its relation to pathological process. Franciscana dolphin, Pontoporia blainvillei, is a coastal cetacean and the only Brazilian dolphin considered threatened with extinction, due to the human activities. The aim of the current study was to investigate the immunoexpression of TGF-β, TNF-α and IL-12 cytokines, and S-100 and Lysozyme proteins in pulmonary and splenic samples of 34 Pontoporia blainvillei. Based on histopathological examination, the animals were divided in two groups: G1 (n = 8) animals without pulmonary inflammatory disease; G2 (n = 26) animals with pulmonary inflammatory diseases. Fragments of lung and spleen were processed according to routine protocols. The immunohistochemistry and morphometric procedures were tested and optimized according to proceedings described in literature. Our results showed that P. blainvillei from G2 presented significant increase in the pulmonary immunoexpression of TGF-β, TNF-α and IL-12 cytokines, and S-100 and Lysozyme proteins in comparison with animals from G1. These results are according to the literature and suggest that P. blainvillei presents same cytokines observed in other mammals. We evidenced also that the P. blainvillei belonging to the G2 did not show splenic immunoexpression to TGF-β, TNF-α and IL- 12 cytokines and Lysozyme protein significantly higher than animals from G1. Further studies, including those related to the methods used, are necessary for the proper understanding of these results. And finally, although these are still preliminary data, we observed a possible association between high concentrations of organochlorines in subcutaneous adipose tissue of a portion of P. blainvillei studied in this research and the occurrence of splenic lymphoid depletion in these animals. These data confirm the importance of developing more detailed analysis on these topics in future scientific investigations.

No description available.

A hanseníase causada pelo Mycobacterium leprae é a infecção que mais gera danos ao sistema nervoso periférico. Este microrganismo tem como alvo a célula de Schwann, o qual se liga à laminina, um dos constituintes da lâmina basal. Através desta ligação o Mycobacterium leprae penetra nas células de Schwann onde se multiplica, infectando o sistema nervoso periférico e desse modo comprometendo sua estrutura e funções. Provavelmente tal como ocorre em outras neuropatias degenerativas, inflamatórias ou neoplásicas, no processo de agressão nervosa na hanseníase, participam outras moléculas como a integrina beta1 e a proteína S-100. O presente trabalho estudou 44 doentes de hanseníase, classificados de acordo com Ridley e Jopling, distribuídos em: 12 doentes indeterminados, 7 doentes tuberculóides, 17 doentes dimorfo-tuberculóides, 2 doentes dimorfo-dimorfos, 2 doentes dimorfo virchowianos e 4 doentes virchowianos. Os propósitos foram estudar o dano nervoso nas terminações nervosas da pele por meio da expressão da integrina beta1 e da proteína S100; e ainda a análise da relação entre as manifestações dermatológica, neurológica, reação de Mitsuda, bacterioscopia e os achados imunohistoquímicos. A alteração da expressão da integrina beta1 nas terminações nervosas da pele foi variável, precoce e constante em 100% dos doentes. A alteração da proteína S-100 nas terminações nervosas da pele nos doentes foi de 88,6%. Apesar da significativa correlação entre elas, a sensibilidade da integrina beta1 foi maior. Encontrou-se correlação entre a clínica dermatológica e neurológica, bem como com a bacteriscopia e a reação intradérmica de Mitsuda. Não houve correlação das reações imunohistoquímicas com os dados clínicos, provavelmente em decorrência das alterações moleculares ocorrerem antes das manifestações clínicas / Leprosy caused by Mycobacterium Leprae is the infection that most causes damage to the peripheral nervous system. This microorganism has its principal target in the Schwann cells, which bind themselves to laminin, one of the constituints of the basic lamina. The Mycobacterium Leprae, by way of this link, penetrates the Schwann cells, where they multiply, infecting the peripheral nervous system and thus compromising its structure and functions. Probably, as happens in other degenerative neuropathies, inflammatory or neoplastic, other molecules participate in the process of nervous aggression of leprosy, such as beta1 integrin and S-100 protein. This paper studied 44 patients with leprosy, classified according to Ridley and Jopling, distributed as: 12 indetermined patients, 7 tuberculoid patients, 17 borderline-tuberculoid patients, 2 mid-borderline patients, 2 borderline-lepromatous patients and 4 lepromatous patients. The aims were to study the damage to the skin nerve endings by way of the levels of beta1-integrin and S-100 protein; and also the analysis of relation between dermatological, neurological clinical manifestations, the Mitsuda reactions, bacterioscopical and the immunohistochemical findings. The alterations in the amounts of beta1 integrin in the skin nerve endings were variable, premature and constant in 100 % of the patients. The alteration in the S-100 level in the skin nerve endings in the patients was 88,6 %. In spite of the correlation between them, the sensibility of the beta1 integrin was greater. There was found to be correlation between dermatological and neurological clinical manifestations as well as with the bacterioscopy and the Mitsuda intradermal reactions. There was no correlation between immunohistochemical reactions with the clinical data, probably because of the molecular alterations that occur before the clinical manifestations