No / Eicosanoids play an integral part in homeostatic mechanisms related to skin health and structural integrity. They also mediate inflammatory events developed in response to environmental factors, such as exposure to ultraviolet radiation, and inflammatory and allergic disorders, including psoriasis and atopic dermatitis. This review article discusses biochemical aspects related to cutaneous eicosanoid metabolism, the contribution of these potent autacoids to skin inflammation and related conditions, and considers the importance of nutritional supplementation with bioactives such as omega-3 and omega-6 polyunsaturated fatty acids and plant-derived antioxidants as means of addressing skin health issues. / The Wellcome Trust and BBSRC-DRINC

No / The skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation, and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides, and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, and 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids was reflected in the lipid mediators produced, whereas similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin and hint at their contribution to signaling, cross-support, and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl– system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.

Eicosanoids are potent mediators of inflammatory response whose role has been well established in inflammatory disorders. Release of arachidonic acid by group IVA cytosolic phospholipase A2 α (cPLA2α) is the initial rate limiting step for the production of eicosonoids in response to inflammatory mediators. Previous findings from our laboratory have demonstrated that cPLA2α is directly activated by the emerging bioactive sphingolipid, ceramide-1-phosphate (C1P). In this study, we have developed a modified Triton X-100/phosphatidylcholine (PC) mixed micelle assay which was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the activity of the enzyme increased in a dose dependent manner with increasing amount of C1P in the mixed micelle and the stoichiometry of this interaction was found to be 2 molecules of C1P to achieve full activation. This activation was found to be lipid specific as other phospholipids such as PE, PS, PA, DAG, and S1P had insignificant effect on cPLA2α activity. Furthermore, based on previous studies we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (R57, K58, R59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated ((R57A/K58A/R59A), (R57A/R59A), (K58A/R59A), (R57A/K58A), (R57A), (K58A), and (R59A)) and examined for C1P affinity by surface plasmon resonance (SPR). The triple, the double mutants, and the single mutant (R59A) demonstrated significantly reduced affinity for C1P containing vesicles compared to wild-type cPLA2α. Examining these five mutants for enzymatic activity demonstrated significant reduction in the ability of C1P to increase the Vmax of the reaction and significantly decreased the dissociation constant (KSA) of the reaction as compared to the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for PC and PtdIns(4,5)P2 compared to wild-type cPLA2α. Finally, we demonstrated these amino acids were critical for translocation of cPLA2α in A549 lung adenocarcinoma cells in response to inflammatory agonists like A23187 and IL-1β. Lastly, we also demonstrated the mechanistic difference between activation of cPLA2α by the two anionic lipids, C1P and PI(4,5)P2.

Orientadores: Admar Costa de Oliveira, Roberto Carlos Burini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-11T21:09:22Z (GMT). No. of bitstreams: 1 Haidamus_LeandroLopes_D.pdf: 785768 bytes, checksum: a63544a849a7db50b64812194be804ca (MD5) Previous issue date: 2007 / Resumo: O uso de drogas antiinflamatórias é comum entre atletas nos quais a prevalência de lesões musculares é elevada tanto nos períodos de treinamento e competição. Entretanto, algumas dessas drogas possuem ação imunossupressiva, que quando utilizadas em grande quantidade ou por tempo prolongado pode provocar queda nas respostas imunológicas. O objetivo da presente Tese foi verificar se a suplementação com ácidos graxos poliinsaturados ômega-3 ocasionaria uma redução, tanto nas concentrações plasmáticas dos indicadores de lesão muscular (enzima lactato desidrogenase e creatina quinase) como dos mediadores químicos da inflamação da série ¿2¿ (prostaglandina E2, tromboxano B2 e leucotrieno B4) derivados do ácido araquidônico (C20:4, ?-6) e das alterações morfológicas do músculo esquelético. O experimento foi desenvolvido com 36 ratos Wistar distribuídos ao acaso em quatro grupos experimentais com nove ratos. Os grupos formados eram os seguintes: Grupo 1 - ratos sedentários sem suplementação; Grupo 2 - ratos sedentários com suplementação; Grupo 3 - ratos submetidos às sessões de natação (60 minutos diários), com carga de peso extra crescente, sem suplementação e Grupo 4 ¿ ratos submetidos às sessões de natação, com carga de peso extra crescente, com suplementação. A suplementação era feita por gavagem utilizando-se 3,0 g diárias de óleo de peixe contendo AGPI ?-3 (EPA: 187,0 mg/g e DHA: 140,0 mg/g) durante 28 dias. Os grupos não suplementados receberam 3,0 g de azeite de oliva diariamente (grupo controle). Os ratos receberam dieta não purificada de fórmula fechada (MP-77- Primor) e água à vontade, sendo os pesos avaliados semanalmente. Os indicadores de lesão muscular (LDH e CK), análise histológica e morfométrica das fibras musculares esqueléticas bem como os mediadores do processo inflamatório (PGE2, TXB2 e LTB4) foram determinados no final do experimento. Os resultados obtidos indicam que a suplementação com AGPI ?-3 reduziu a concentração plasmática da enzima LDH, tanto nos animais sedentários como nos submetidos ao treinamento físico (p<0,05). Com relação aos mediadores químicos da inflamação (PGE2, TXB2 e LTB4) as concentrações plasmáticas reduziram significativamente (p<0,05) nos animais sedentários, mas não reduziram nos animais submetidos ao exercício físico. No entanto, ao se observar esse resultado, verificou-se que em todos os mediadores químicos houve uma redução nos valores absolutos nos animais suplementados com AGPI ?-3. Pela análise histológica das fibras musculares mostrou que nos grupos treinados, os ratos não suplementados apresentaram as seguintes alterações morfológicas: presença de processo de fagocitose de fibra muscular, fibra atrófica e fibras polimórficas, enquanto que nos ratos suplementados, a análise histológica mostrou uma redução acentuada dessas alterações o que resultou na quase inexistência de lesões musculares. Tendo em vista os resultados obtidos, pode ser sugerido de que ação antiinflamatória dos AGPI ?-3 nos ratos e por extensão em atletas, seria maior se a suplementação fosse realizada diariamente independentemente do período de treinamento / Abstract: The use of antiinflammatory drugs is common among athletes in which the prevalence of muscular lesions is elevated in training and competition periods. However, some of those drugs have an immunosuppression action, and when used in high dosages or for long time can provoke a decrease in the immunological responses. The objective of the present Thesis was to verify if the supplementation with polyunsaturated fatty acids omega-3 would cause a reduction, in the plasmatic concentrations of the indicators of muscular lesion (lactate dehidrogenase enzyme and creatine kinase) as well as in the chemical mediators of the inflammation of the series ¿2¿ (prostaglandin E2, thromboxane B2 and leukotriene B4) derived of arachidonic acid (C20:4, ?-6) and the morphologic alterations of skeletal muscle. The experiment was developed with 36 Wistar rats distributed at random in four experimental groups with nine rats. The formed groups were the following: Group 1 - sedentary rats without supplementation; Group 2 - sedentary rats with supplementation; Group 3 - rats submitted to swimming sessions (60 minutes daily), with load of gaining extra weight, without supplementation and Group 4 - rats submitted to swimming sessions, with load of gaining extra weight, with supplementation. The supplementation was made daily by ¿gavage¿ using 3,0g of fish oil containing AGPI ?-3 (EPA: 187,0 mg/g and DHA: 140,0 mg/g) for 28 days. The groups not supplemented received 3,0g of olive oil daily (control group). The rats received a non-purified diet of closed formula (MP-77-Primor) and water was ad libitum, with their weight being checked weekly. The indicators of muscular lesion (LDH and CK), histological and morphometric analyses of the skeletal muscular fibers as well as the mediators of the inflammatory process (PGE2, TXB2 and LTB4) were determinated at the end of the experiment. The obtained results indicate that the supplementation with AGPI ?-3 did reduce the plasmatic concentration of the LDH enzyme in the sedentary animals as well as in the animals submitted to the physical training (p<0,05). With respect to the chemical mediators of the inflammation (PGE2, TXB2 and LTB4) the plasmatic concentrations were reduced significantly (p < 0,05) in the sedentary animals, but were not reduced in the animals submitted to the physical exercise. However, by observing this results, it was verified that in all of the chemical mediators there was a reduction in the absolute values in the supplemented animals with AGPI ?-3. By the histological analysis of the muscular fibers showed that in the trained groups, the no supplemented rats had the following morphologic alterations: presence of the process of phagocytosis of muscular fiber, atrophic fiber, polymorphic fibers, while that in the supplemented rats, the histological analysis showed an accentuated reduction of those alterations that resulted in an almost inexistence of muscular lesions. Having in mind the obtained results, it can be suggested that the antiinflammatory action of AGPI ?-3 in the rats and for extension in athletes, would be improved if the supplementation was administered daily independently of the training period / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutor em Alimentos e Nutrição

A galectina-1 (Gal-1) pertence a uma família de lectinas endógenas que reconhecem ß-galactosídeos e atuam em vários processos biológicos. A Gal-1 pode modular a resposta imunológica por vários mecanismos incluindo o controle da liberação de citocinas pró e anti-inflamatórias e o direcionamento dessa resposta para um padrão do tipo TH2. Apesar da Gal-1 participar de vários processos fisiopatológicos, na literatura não existe relatos sobre o papel dessa lectina em infecções fúngicas. O objetivo deste trabalho foi investigar o impacto biológico da Gal-1 no modelo experimental de histoplasmose murina. Os camundongos (Gal-1-/- e Gal-1+/+) foram inoculados, por via intratraqueal, com uma carga fúngica sub-letal (5x105 leveduras) e a sobrevida desses animais foi avaliada até o 30o dia de infecção. Considerando que o início da mortalidade dos animais ocorreu após duas semanas de infecção, as demais análises foram realizadas em amostras obtidas no 15o dia. O grau de disseminação do H. capsulatum foi analisado pela contagem do número de unidades formadoras de colônia, em partes de pulmões ou baços dos animais infectados. Os cortes de pulmões foram corados por hematoxilina eosina ou por prata (GMS), para investigação histopatológica e quantificação de neutrófilos ou fungos, respectivamente. Nos fluídos bronco-alveolares (BAL) foram realizadas contagens globais e diferenciais de leucócitos. As dosagens de citocinas e de prostagladina E2 foram feitas por ELISA, em homogeneizados de pulmões. A coloração por tetróxido de ósmio foi usada na avaliação da capacidade da Gal-1 de induzir ou modular a formação de corpúsculos lipídicos, in vitro, por componentes do fungo. Nos soros dos animais de experimentação foi determinada a concentração total de nitrito, como indicador da produção de óxido nítrico. A análise dos resultados de sobrevivência indicou que 100% dos animais Gal-1+/+ resistiram à infecção por H. capsulatum; ao contrário, apenas 33% dos animais Gal-1-/- sobreviveram. Os números médios de unidades formadoras de colônias recuperadas no pulmão e no baço de camundongos Gal-1-/- foram de 2,7 e 3,8 vezes maiores do que os obtidos de animais Gal-1+/+, respectivamente. De modo semelhante, os números médios de neutrófilos e fungos no pulmão de animais Gal-1-/-, foram superiores aos valores encontrados nos pulmões de animais grupo Gal-1+/+. Curiosamente, nos homogeneizados pulmonares dos e o dobro da concentração de nitrito total sérico. Além disso, nos homogeneizados de pulmão dos animais Gal-1desafiados com o fungo, detectou-se elevadas concentrações de citocinas do tipo T1 e inflamatórias (IFN-, IL-1 e IL-12) em comparação com amostras de animais selvagens. Em ensaios in vitro, esta lectina não foi capaz de induzir corpúsculos em células do lavado peritoneal de camundongos Gal-1e Gal-1, entretanto inibiu parcialmente a formação induzida por F1 e -glucana. Finalmente, sugerimos que a Gal-1 pode participar da montagem de uma resposta imunológica protetora contra o Histoplasma capsulatum, por modular a liberação de citocinas inflamatórias, síntese de eicosanóides, geração de óxido nitrito; e por controlar a migração e/ou as funções de leucócitos. Além disso, os dados obtidos desse trabalho poderão auxiliar no melhor entendimento da fisiopatologia da histoplasmose e no desenvolvimento de novas estratégias terapêuticas envolvendo o reconhecimento de carboidratos na resposta imunológica. / Galectin-1 (Gal-1) belongs an endogenous lectins family that recognizes -galactoside and participates of various biological activities. This lectin can modulate the innate and adaptative immune responses. Although, Gal-1 participates of various pathophysiological processes, in literature we did not find reports related to the participation of Gal-1 in fungal infections. The aim of this work was to investigate the biological impact of Gal-1 in the experimental histoplasmosis. The mice (GAL-1-/- and GAL-1+/+) were injected (i.t.) with 5 x 105 yeast cell and at 15 days post-infection, BALF cells and lungs cytokine and PGE2 were measured by ELISA. The Recovery of H. capsulatum was made in lung and spleen and the fungal burden was assessed as the CFU per organ. The lung slices were stained by hematoxiline eosin or with Gomoris methanemine silver (GMS) and submitted to histopathological investigation and quantification of neutrophil or fungus, respectively. Total and differential cell counts of the bronchoalveolar lavage (BAL).were performed using diluting solution in Neubauer chamber and Rosenfeld-stained smear. The capacity of Gal-1 to induce or modulate the lipids bodies formation by fungus components was analyzed by staining treated cells with osmium tetroxide. The total nitrite (NO2) concentration in the animals serum was measured by Griess reaction. All H. capsulatum-infected wild type mice survived until 30 days post-infection, whereas only 33% of the Gal-1-/- infected mice died during of this period of observation. At 15 days post-infection, CFU were found to be higher in the spleens or lung from infected-Gal1-/- mice. The number of neutrophils in the lung of the infected-Gal-1-/- mice higher than infected Gal-1+/+ animals. Curiously, H. capsulatum infected Gal-1-/- mice, presented higher levels of PGE2 and TH1 inflammatory cytokines (IFN-, IL-1 e IL-12) in comparison with wild type infected-mice. Adherent peritoneal cells peritoneal derived from Gal-1+/+ and Gal-1-/- mice and treated with Gal-1 did not induce lipid bodies. However, the capacity of F1 e -glucan to induce lipid bodies on the peritoneal cells was inhibited by gal-1 treatment. We suggest that the Gal-1 could participate of the development of a protective immune response to H. capsulatum.

Orientador: Marcos Renato de Assis / Resumo: A expectativa de vida diminui com artrite reumatoide (AR) devido ao aumento da mortalidade por doenças cardiovasculares. Isto se deve, possivelmente, à disfunção endotelial resultante da intensa atividade inflamatória relacionada à AR. Evidências tem apontado para um possível envolvimento do sistema renina-angiotensina (SRA) nos danos cardiovasculares inerentes à AR. Acredita-se que a participação do SRA agrave o comprometimento endotelial decorrente de inflamações sistêmicas através da ativação do receptor de angiotensina II tipo 1 (AT1) pela angiotensina II (Ang II). Por outro lado, a literatura também reporta a influência dos hormônios andrógenos, principalmente da testosterona, nas ações da Ang II sobre os tecidos vasculares. Assim, o objetivo do presente estudo é investigar os efeitos da artrite induzida por adjuvante (AIA) sobre o equilíbrio redox, a função endotelial e as respostas da aorta de ratos à Ang II, bem como, verificar se esses efeitos podem ser influenciados pela redução dos níveis circulantes de testosterona. Para isto, ratos Wistar machos adultos foram submetidos à falsa-castração e falsa-imunização (Controles), castração seguida de falsa-imunização (ORX), falsa-castração seguida de imunização (ORX) e castração seguida de imunização (ORX+AIA). Ao final do experimento, segmentos de aorta torácica foram desafiadas em cubas de órgãos isolados com acetilcolina (ACh), Ang II, KCl e nitroprussiato de sódio e, das curvas concentração resposta obtidas, calculou-se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Life expectancy of patients with rheumatoid arthritis (RA) is lower due to increased mortality in consequence of cardiovascular diseases. Presumably, this occurs due to endothelial dysfunction resulting from severe inflammatory activity related to RA. Evidence has demonstrated a possible involvement of the renin-angiotensin system (RAS) in the cardiovascular injury inherent to RA. The RAS involvement is considered to worsen the endothelial impairment due to systemic inflammation through angiotensin II type 1 receptor (AT1) activation of Ang II. (Ang II). Additionally, previous studies also observed that androgen hormones, mainly the testosterone, modulate the Ang II actions in cardiovascular tissues. Thus, the aim of the present study is to investigate the effects of adjuvant-induced arthritis (AIA) on systemic redox balance, endothelial function and rat aorta responses to Ang II, as well as, whether these effects may be influenced by the reduction of circulating levels of testosterone. For this, adult male Wistar rats were submitted to false-castration and false-immunization (Controls), castration followed by false-immunization (ORX), false-castration followed by immunization (ORX) and castration followed by immunization (ORX + AIA) . At the end of the experiment, thoracic aorta segments were challenged in isolated organ bath with acetylcholine (ACh), Ang II, KCl and sodium nitroprusside and, from the concentrantion-response curves obtained were calculated pEC50 and maximal ... (Complete abstract click electronic access below) / Doutor

Le syndrome d’apnées obstructives du sommeil (SAOS) affecte 5 à 20% de la population générale et est associée à des complications cardiovasculaires, ce qui en fait un véritable problème de santé publique. Les épisodes itératifs d’obstruction pharyngée nocturne qui le caractérisent aboutissent à une hypoxie intermittente, elle-même impliquée dans ces complications cardiovasculaires. A ce jour, les mécanismes reliant SAOS et athérosclérose restent méconnus. De plus, le traitement de référence du SAOS par pression positive continue présente dans certaines populations une efficacité limitée sur les conséquences cardiovasculaires du SAOS, d’où la nécessité de développer de nouvelles thérapeutiques ciblant spécifiquement le processus athéromateux. Des perturbations du métabolisme de l’acide arachidonique à l’origine de la synthèse d’eicosanoïdes pro-inflammatoires ont déjà été décrites au cours du SAOS et ont été associées au processus athéromateux. Le but de ce travail a donc été de préciser par une approche translationnelle le rôle de certains de ces eicosanoïdes, à savoir le thromboxane A2 et les cystéinyl-leucotriènes dans l’athérogénèse associée au SAOS et de les évaluer en tant que potentielles cibles thérapeutiques. Ces travaux ont montré non seulement une activation des voies métaboliques du thromboxane A2 et des cystéinyl-leucotriènes au cours du SAOS, mais également leur association avec le processus athéromateux. Enfin, des traitements pharmacologiques ciblant spécifiquement ces médiateurs étaient capables de ralentir la progression de l’athérosclérose en lien avec le SAOS dans un modèle murin de SAOS. Ces travaux démontrent donc le rôle du thromboxane A2 et des cystéinyl-leucotriènes dans l’athérosclérose associée au SAOS, et positionnent ces deux voies métaboliques comme des cibles thérapeutiques potentielles pour traiter les conséquences cardiovasculaires du SAOS. / Obstructive sleep apnea (OSA) is a really public health problem since it affects 5 to 20% of general population and is associated with increased cardiovascular morbidity and mortality. Repetitive nocturnal pharyngeal obstruction leads to intermittent hypoxia, which is responsible of premature atherosclerosis and also cardiovascular complications.Nevertheless, mechanims linking OSA and atherosclerosis remains poorly understood. In addition, continuous positive airway pressure (CPAP) application, which is the gold standard treatment of OSA, has poor effect on OSA cardiovascular consequences in some populations, highlighting the need of alternative therapeutic strategies. Alterations of several eicosanoids resulting to arachidonate metabolism had already been described during OSA, these latter being associated with vascular remodeling. The aim of this work was to precise trough a translational approach the contribution of some eicosanoids, especially thromboxane A2 and cysteinyl-leukotriene on OSA related-atherogenesis and also to evaluate these latter as therapeutic target.We had shown an activation of both thromboxane A2 and cysteinyl-leukotriene pathways in OSA, this latter being associated with vascular remodeling. In addition, pharmacological treatment by cyclooxygenase type 1 inhibitor or CysLT1 receptor antagonist reduced OSA-related atherosclerosis progression in an OSA mouse model.Finally, these works had demonstrated the implication of both thromboxane A2 and cysteinyl-leukotrienes activation in OSA-related atherosclerosis, but also the potential therapeutic of these targets to treat cardiovascular consequences of OSA.

A galectina-1 (Gal-1) pertence a uma família de lectinas endógenas que reconhecem ß-galactosídeos e atuam em vários processos biológicos. A Gal-1 pode modular a resposta imunológica por vários mecanismos incluindo o controle da liberação de citocinas pró e anti-inflamatórias e o direcionamento dessa resposta para um padrão do tipo TH2. Apesar da Gal-1 participar de vários processos fisiopatológicos, na literatura não existe relatos sobre o papel dessa lectina em infecções fúngicas. O objetivo deste trabalho foi investigar o impacto biológico da Gal-1 no modelo experimental de histoplasmose murina. Os camundongos (Gal-1-/- e Gal-1+/+) foram inoculados, por via intratraqueal, com uma carga fúngica sub-letal (5x105 leveduras) e a sobrevida desses animais foi avaliada até o 30o dia de infecção. Considerando que o início da mortalidade dos animais ocorreu após duas semanas de infecção, as demais análises foram realizadas em amostras obtidas no 15o dia. O grau de disseminação do H. capsulatum foi analisado pela contagem do número de unidades formadoras de colônia, em partes de pulmões ou baços dos animais infectados. Os cortes de pulmões foram corados por hematoxilina eosina ou por prata (GMS), para investigação histopatológica e quantificação de neutrófilos ou fungos, respectivamente. Nos fluídos bronco-alveolares (BAL) foram realizadas contagens globais e diferenciais de leucócitos. As dosagens de citocinas e de prostagladina E2 foram feitas por ELISA, em homogeneizados de pulmões. A coloração por tetróxido de ósmio foi usada na avaliação da capacidade da Gal-1 de induzir ou modular a formação de corpúsculos lipídicos, in vitro, por componentes do fungo. Nos soros dos animais de experimentação foi determinada a concentração total de nitrito, como indicador da produção de óxido nítrico. A análise dos resultados de sobrevivência indicou que 100% dos animais Gal-1+/+ resistiram à infecção por H. capsulatum; ao contrário, apenas 33% dos animais Gal-1-/- sobreviveram. Os números médios de unidades formadoras de colônias recuperadas no pulmão e no baço de camundongos Gal-1-/- foram de 2,7 e 3,8 vezes maiores do que os obtidos de animais Gal-1+/+, respectivamente. De modo semelhante, os números médios de neutrófilos e fungos no pulmão de animais Gal-1-/-, foram superiores aos valores encontrados nos pulmões de animais grupo Gal-1+/+. Curiosamente, nos homogeneizados pulmonares dos e o dobro da concentração de nitrito total sérico. Além disso, nos homogeneizados de pulmão dos animais Gal-1desafiados com o fungo, detectou-se elevadas concentrações de citocinas do tipo T1 e inflamatórias (IFN-, IL-1 e IL-12) em comparação com amostras de animais selvagens. Em ensaios in vitro, esta lectina não foi capaz de induzir corpúsculos em células do lavado peritoneal de camundongos Gal-1e Gal-1, entretanto inibiu parcialmente a formação induzida por F1 e -glucana. Finalmente, sugerimos que a Gal-1 pode participar da montagem de uma resposta imunológica protetora contra o Histoplasma capsulatum, por modular a liberação de citocinas inflamatórias, síntese de eicosanóides, geração de óxido nitrito; e por controlar a migração e/ou as funções de leucócitos. Além disso, os dados obtidos desse trabalho poderão auxiliar no melhor entendimento da fisiopatologia da histoplasmose e no desenvolvimento de novas estratégias terapêuticas envolvendo o reconhecimento de carboidratos na resposta imunológica. / Galectin-1 (Gal-1) belongs an endogenous lectins family that recognizes -galactoside and participates of various biological activities. This lectin can modulate the innate and adaptative immune responses. Although, Gal-1 participates of various pathophysiological processes, in literature we did not find reports related to the participation of Gal-1 in fungal infections. The aim of this work was to investigate the biological impact of Gal-1 in the experimental histoplasmosis. The mice (GAL-1-/- and GAL-1+/+) were injected (i.t.) with 5 x 105 yeast cell and at 15 days post-infection, BALF cells and lungs cytokine and PGE2 were measured by ELISA. The Recovery of H. capsulatum was made in lung and spleen and the fungal burden was assessed as the CFU per organ. The lung slices were stained by hematoxiline eosin or with Gomoris methanemine silver (GMS) and submitted to histopathological investigation and quantification of neutrophil or fungus, respectively. Total and differential cell counts of the bronchoalveolar lavage (BAL).were performed using diluting solution in Neubauer chamber and Rosenfeld-stained smear. The capacity of Gal-1 to induce or modulate the lipids bodies formation by fungus components was analyzed by staining treated cells with osmium tetroxide. The total nitrite (NO2) concentration in the animals serum was measured by Griess reaction. All H. capsulatum-infected wild type mice survived until 30 days post-infection, whereas only 33% of the Gal-1-/- infected mice died during of this period of observation. At 15 days post-infection, CFU were found to be higher in the spleens or lung from infected-Gal1-/- mice. The number of neutrophils in the lung of the infected-Gal-1-/- mice higher than infected Gal-1+/+ animals. Curiously, H. capsulatum infected Gal-1-/- mice, presented higher levels of PGE2 and TH1 inflammatory cytokines (IFN-, IL-1 e IL-12) in comparison with wild type infected-mice. Adherent peritoneal cells peritoneal derived from Gal-1+/+ and Gal-1-/- mice and treated with Gal-1 did not induce lipid bodies. However, the capacity of F1 e -glucan to induce lipid bodies on the peritoneal cells was inhibited by gal-1 treatment. We suggest that the Gal-1 could participate of the development of a protective immune response to H. capsulatum.

L’époxyde hydrolase soluble (sEH) est une enzyme ubiquitaire, bifonctionnelle, codée par le gène EPHX2. La partie hydrolase (sEH-H) est responsable de la dégradation de facteurs endothéliaux vasodilatateurs, les acides époxyeicosatriénoïques (EETs), alors que la partie phosphatase (sEH-P) est impliquée dans le métabolisme des acides lysophosphatidiques (LPAs).L’objectif de ce travail a été de développer des outils méthodologiques permettant d'évaluer le rôle de la sEH dans la physiopathologie des maladies cardiovasculaires.Nous avons développé une méthode de quantification par CLHP-MS² des EETs et de leurs métabolites, les acides dihydroxyeicosatrienoic acids (DHETs). L'application de cette méthode montre que la dysfonction endothéliale des patients atteints d’hypertension artérielle et de diabète de type 2 est associée à une diminution de la libération locale des EETs lors de l'augmentation du débit sanguin, notamment liée à une augmentation d’activité de la sEH-H. L’inhibition pharmacologique de la sEH-H a permis de diminuer l’inflammation et l’atteinte glomérulaire dans un modèle murin d’insulino-résistance. De plus, l’étude des polymorphismes génétiques du gène EPHX2, codant la sEH, a permis de démontrer que la fonction sEH-H joue probablement un rôle important dans le contrôle de la fonction rénale et vasculaire des patients transplantés rénaux. Enfin, les résultats expérimentaux obtenus dans un modèle d’inactivation génétique de la sEH-P et l'étude des polymorphismes génétiques d'EPHX2 chez les patients insuffisants cardiaques suggèrent un rôle important de cette partie dans la régulation du métabolisme des lipides ainsi que dans le contrôle de l’homéostasie cardiovasculaire.Ainsi, les résultats obtenus au cours de ce travail soutiennent l’intérêt de développer des inhibiteurs pharmacologiques de la sEH-H pour traiter les maladies cardiovasculaires, rénales et métaboliques chez l’homme et suggèrent que la modulation de la sEH-P pourrait également constituer une nouvelle cible d'intérêt dans la prise en charge de ces pathologies. / Soluble epoxide hydrolase (sEH) is an ubiquitous bifunctional enzyme that is encoded by the EPHX2 gene. The hydrolase activity (sEH-H) is responsible for the conversion of the endothelial vasodilator epoxyeicosatrienoic acids whereas the phosphatase activity (sEH-P) is involved in the metabolism of lysophosphatidic acids (LPAs).The aim of this work was to develop chromatographic methods and molecular biology techniques to evaluate sEH activities in cardiovascular diseases.We developed a LC-MS/MS method to quantify EETs and their metabolites, the dihydroxyeicosatrienoic acids (DHETs). Using this method, we showed that the endothelial dysfunction of hypertensive and type 2 diabetic patients is associated with a decrease in the local production of EETs during flow increase notably due to increased sEH-H activity. In a murine model of insulin resistance, pharmacological inhibition of sEH-H improved renal function by decreasing inflammation, oxidative stress and glomerular lesions. Moreover, genetic investigations of EPHX2 revealed that sEH-H may play a substantial role in the control of renal and vascular function in kidney recipients. Finally, experimental results obtained in knock-in sEH-P deficient rats and genetics findings in patients with heart failure strongly suggest that sEH-P is involved in lipid metabolism and cardiovascular homeostasis.Taken together, these results strengthen the interest of developing pharmacological inhibitors of sEH-H to be tested in patients with cardiovascular, renal or metabolic diseases and suggest that the modulation of sEH-P represents a new therapeutic target to treat these pathologies.