O objetivo deste estudo foi avaliar o efeito da competição de triathlon na função e morte de neutrófilos de atletas de elite e investigar o possível envolvimento dos ácidos graxos livres (AGs) neste processo. Os neutrófilos foram obtidos do sangue coletado de 11 sedentários e de 12 triatletas em repouso e após competição de triathlon (Half Ironman, 2 Km de natação, 80 Km de ciclismo e 20 Km de corrida). A competição de triathlon aumentou a capacidade dos neutrófilos de migrar e de realizar burst oxidativo, porém inibiu a fagociose realizada por estas células. Além disso, induziu aumento da fragmentação de DNA e externalização de fosfatidilserina. A competição de triathlon aumentou a concentração de AGs no plasma dos triatletas e esta foi correlacionada positivamente com a proporção de neutrófilos com DNA fragmentado e fosfatidilserina externalizada. A elevação da concentração plasmática dos ácidos oléico, linoléico e esteárico induzida pela competição parece estar envolvida nas alterações funcionais e na apoptose verificadas após a competição de triathlon. / The objective of this study was to evaluate the effect of triathlon competition on function and death of neutrophils from elite athletes and to investigate the involvement of fatty acids in this process. Neutrophils were obtained from blood collected from eleven sedentary volunteers and twelve triathletes under rest and after a Half Ironman triathlon competition (2 Km swimming, 80 Km cycling and 20 Km running). The triathlon competition increased the migration and reactive oxygen species production in neutrophils, however it reduced the phagocytosis activity. Moreover, it induced neutrophils death possibly by apoptosis as indicated by DNA fragmentation and phosphatidylserine externalization. The increase in plasma levels of oleic, linoleic and stearic acids induced by the competition may be involved in modulation on neutrophil function and death.

Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T11:46:04Z (GMT). No. of bitstreams: 1 DiasJunior_PedroPaulo_M.pdf: 932681 bytes, checksum: a8ceae0940e26759a4abe344de43b8f4 (MD5) Previous issue date: 2012 / Resumo: A resposta vascular inflamatória envolve a interação complexa entre células. A adesão dos neutrófilos aos sítios inflamatórios é constituído de várias etapas que envolvem a interação das moléculas de adesão com os neutrófilos, intermediado pela L-selectina (CD62L) e pelas integrinas ?2, LFA-1 e Mac-1 (CD11a/CD18 e CD11b/CD18) com ligantes sobre o endotélio. Os eritrócitos podem aderir ao endotélio vascular usando as moléculas de adesão CD 36 e integrina VLA-4 entre outras moléculas de adesão. O mecanismo de adesão das plaquetas envolve o sequestro celular no local da lesão tecidual através da interação de quatro receptores sinérgicos: a glicoproteína GPIb/IX (CD42b/CD42a), a integrina ?2?1 (GPIa/IIa; CD49b/CD41a), a integrina ?IIb?3 (GPIIb/IIIa; CD41a/CD61), e a integrina ?5?1 (GPIc/IIIa; CD51/CD61). Em doenças associadas com a inflamação vascular, tais como a doença falciforme e aterosclerose, alterações na adesão de leucócitos à parede vascular desempenham um papel central na fisiopatologia da doença. O CD40L e o LIGHT são proteínas citocinas pertencente ao fator de necrose tumoral (TNF), tendo como receptores o CD40L, LTBR e o HVEM, respectivamente. Diante disso, este estudo objetivou avaliar os efeitos das proteínas inflamatórias CD40L e LIGHT nas propriedades adesivas dos neutrófilos, hemácias e plaquetas. Os neutrófilos, hemácias e plaquetas foram separados do sangue periférico e foram realizados ensaios de adesão estática e citometria de fluxo após estimulo destas células com as proteínas recombinantes, CD40L e LIGHT. Após incubação com CD40L ou LIGHT, os neutrófilos e hemácias da circulação periférica apresentaram alterações quanto às propriedades adesivas em relação aos neutrófilos e hemácias não estimulados. As plaquetas estimuladas com as citocinas não demonstraram alteração nas propriedades adesivas. Neutrófilos incubados com anticorpos bloqueadores das integrinas Mac-1 e LFA-1 apresentaram uma reversão no aumento das propriedades adesivas após estimulo com CD40L ou LIGHT. Os neutrófilos estimulados com as citocinas apresentaram uma diminuição na expressão proteíca de CD62L, característica de ativação celular. Nao foi observado nenhuma diferença quanto a expressão das integrinas Mac-1 e LFA-1 nos neutrófilos após estímulo com as citocinas. Esses resultados sugerem que essas proteínas inflamatórias podem aumentar as propriedades adesivas de neutrofilos (intermediado por um aumento na afinidade das integrinas) e hemácias. A presença de altas concentrações destas citocinas na circulação, como encontrados em algumas patologias caracterizadas por inflamação vascular, pode resultar em conseqüências importantes, como a indução da adesão dos leucócitos e hemácias à parede vascular / Abstract: The vascular inflammatory response involves a complex interaction between cells. The adhesion of neutrophils to inflammatory sites is composed of several stages involving the interaction of adhesion molecules on neutrophils and on the endothelium. These interactions are mediated by Lselectin (CD62L) and the ?2 integrins, LFA-1 and Mac-1 (CD11a/CD18 and CD11b / CD18, respectively) with ligands on the endothelium. The red cells may adhere to vascular endothelial adhesion molecules using CD 36 and the VLA-4 integrin and other adhesion molecules. The mechanism of adhesion of platelets involves the adhesion of these cells at the site of tissue injury through the interaction of four synergistic receptors: glycoprotein GPIb/IX (CD42b/CD42a), integrin ?2?1 (GPIa/IIa; CD49b/CD41a), integrin ?IIb?3 (GPIIb / IIIa, CD41a/CD61), and integrin ?5?1 (GPIC/ IIIa; CD51/CD61). In diseases associated with vascular inflammation, such as sickle cell disease and atherosclerosis, changes in leukocyte adhesion to the vascular wall plays a central role in the pathophysiology of the disease. The CD40L and LIGHT cytokines are proteins belonging to the tumor necrosis factor (TNF) family, and interact with the receptors, CD40L, LTBR and HVEM, respectively. Thus, this study aimed to evaluate the effects of the inflammatory proteins, CD40L and LIGHT, on the adhesive properties of neutrophils, erythrocytes and platelets. Neutrophils, erythrocytes and platelets were separated from peripheral blood and static adhesion assays and flow cytometry were performed after stimulation of these cells with the recombinant proteins, CD40L and LIGHT. After incubation with CD40L or LIGHT, neutrophils and red blood cells from the peripheral circulation showed alterations in adhesive properties compared to unstimulated neutrophils and erythrocytes. Platelets stimulated with cytokines showed no changes in adhesive properties. Neutrophils incubated with antibodies that block the functions of integrins Mac-1 and LFA-1 reversed the increase in adhesive properties after stimulation with CD40L or LIGHT. Neutrophils stimulated with cytokines showed a decrease in the protein expression of CD62L, a characteristic of cellular activation. No difference was observed in the expression of the integrins Mac-1 and LFA-1 on neutrophils after stimulation with cytokines. These results suggest that these inflammatory proteins can increase the adhesive properties of neutrophils (mediated by an increase in the affinity of the integrin) and erythrocytes. The presence of high concentrations of these cytokines in the circulation, as found in certain vascular diseases characterized by inflammation, can result in important consequences, such as the induction of the adhesion of leukocytes and red blood cells to the vascular wall / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Sepsis is an exaggerated inflammatory syndrome involving cell, organ and system dysregulation. This dysregulation is detrimental to the patient, mainly as it causes imbalances in the immune system. Monocytes and neutrophils are key cell players in the pathogenesis of sepsis; Two populations that follow divergent apoptotic fates; most importantly delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. These two subpopulations have recently been referred to as specific targets of leukocyte filtration, a noveltherapeutic approach in septic patients. As the molecular mechanisms driving the survival/death switches of these cells in sepsis is still controversial, we decided to investigate the role of a new family of proteins predicted to be involved in inflammation and cell death mechanisms; the ApoLs. We studied ApoL expression in whole blood leukocytes and purified populations of neutrophils and monocytes from healthy volunteers and patients with or without sepsis. We then correlated ApoL expression to the degree of inflammation (CRP levels) and cellular apoptosis. We showed a down regulation in ApoL expression in septic and non-septic patients as compared to healthy volunteers, whereas purified monocytes showed an up regulation of ApoL1 and ApoL2 expression in septic patients as compared to non-septic patients. Given that these cells behave differently in the septic frame, we aim to investigate and invalidate the role of ApoLs in conditions related to cell activation and death. Given the high degree of variability in human samples, we resorted to an in-vitro cell line, the PLB 985 myelomonoblastic cell line with granulocytic differentiation potential. Culturing these cells in RPMI 1640 in the presence of specific differentiation agents resulted in mature terminally differentiated cells with phenotypic and functional similarities to human neutrophils. This was determined by monitoring specific leukemic and granulocytic markers. This cell model will help us to better understand the role ofApoLs in regulating neutrophil half-life particularly in inflammatory conditions. It can also be employed to characterize “The cytokine(s)” that is inducing a change in ApoL expression and ultimately uncover the signaling pathway dictating both ApoL expression and apoptosis/survival. / Le sepsis est defini comme une reponse inflammatoire exageree, associee a une dysfonction d'organes faisant suite a un desequilibre du systeme immunitaire. En effet, les monocytes et les neutrophiles representent les acteurs cle dans la physiopathogenese du sepsis. Etant donne que les mecanismes moleculaires regulant la balance de survie / mort cellulaire de ces deux populations de cellules restent jusqu'a present controverses, nous nous sommes interesses a etudier l'implication d'une nouvelle famille de proteines: les apols, susceptibles d'intervenir dans le processus inflammatoire et de la mort cellulaire. Nous avons explore l'expression des apols dans les leucocytes totaux et dans les populations de neutrophiles et de monocytes purifiees a partir de donneurs sains et de patients septiques et non septiques. Par la suite, nous avons etablit le lien de correlation entre l'expression des apols, le degre d'inflammation systemique (dosage de la crp) et l'apoptose. Nos resultats ont demontre une baisse de l'expression des apols dans les leucocytes totaux el les neutrophiles purifies, chez les patients septiques et non septiques, en comparaison avec les donneurs sains. En revanche, nous remarquons une surexpression de l'apol1 et l'apol2 chez les patients septiques par rapport aux patients non septiques. Etant donne la grande variabilite dans les echantillons preleves, nous avons procede a la differenciation d'une lignee myelomonoblastique ayant un pouvoir de differenciation granulocytaire. La mise en culture de cette lignee dans les conditions de differenciation adequates, nous a permis d'obtenir des cellules ayant des similarites fonctionnelles et phenotypiques aux neutrophiles humains. Ce modele cellulaire va nous aider d'une part, a mieux comprendre l'implication des apols dans le controle de la viabilite cellulaire dans les conditions inflammatoires; et d'autre part, a caracteriser la / (les) cytokine(s) susceptibles de moduler l'expression des apols; et finalement a decortiquer la voie de signalisation impliquant les apols dans le controle du processus de survie ou de mort cellulaire. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The objective was to establish the safety and therapeutic efficacy of oral potassium humate in reducing the signs and symptoms of hayfever. Potassium humate was randomly assigned to 40 atopic patients with acute symptoms of hayfever. Blood and nasal samples were used to determine the safety and the effects of potassium humate on basophil activation, cytokine levels and eosinophil migration. A skin prick test was used to determine its anti-allergic effects. An in vitro neutrophil adhesion was also used. A significant decrease in the skin prick test results and eosinophil counts was observed. No significant differences were observed with regard to neutrophil adhesion nor were any differences observed with regard to the stimulation of basophils. Decreases were observed in the expression of IL-4, IL-5, IL-8 and IL-1â after treatment, although not reaching significance. This study confirmed that this product possesses anti-inflammatory and anti-allergic properties possibly due to a decreased recruitment of eosinopils to the inflammatory site the recruitment and activation of eosinophils by decreasing the expression of IL-4, IL-5, IL-8 and IL-1â, although not reaching statistical significance. / Dissertation (MSc (Pharmacology))--University of Pretoria, 2008. / Pharmacology / unrestricted

Moxifloxacin is considered to be a broad-spectrum fluoroquinolone due to its activity against both gram positive and gram negative bacteria. Importantly this agent is currently being evaluated in ongoing clinical trials in South Africa and South America as a treatment for Moxifloxacin is considered to be a broad-spectrum fluoroquinolone due to its activity against both gram positive and gram negative bacteria. Importantly this agent is currently being evaluated in ongoing clinical trials in South Africa and South America as a treatment for pulmonary tuberculosis, with the specific objective of decreasing the duration of chemotherapy. However, relatively little is known about the effects of moxifloxacin on host defenses, particularly innate protective mechanisms, involving neutrophils. The primary theme of the laboratory research presented in this dissertation was to investigate the role of moxifloxacin in modulating the host immune system, specifically neutrophil protective functions, as well as lymphocyte proliferation and cytokine production (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL13, IL-17, IFN-γ, GM-CSF, G-CSF, TNF-α, and MCP-1). The generation of reactive oxidants and elastase release by neutrophils activated with the chemoattractant, fMLP, or the phorbol ester, PMA, were assayed using luminol- and lucigenin-enhanced chemiluminescence (LECL) and colorimetric procedures, while alterations in cytosolic Ca2+ concentrations were monitored by radiometric (45Ca2+) procedures. Moxifloxacin (1-20 ㎍/ml) was found to have no significant priming or inhibitory effects on oxidant generation by human neutrophils activated with fMLP or PMA, while elastase release was increased at the highest concentrations of the antibiotic. The magnitude of efflux or store-operated Ca2+ influx was unaffected following activation of neutrophils with fMLP. Moxifloxacin at all concentrations tested, did not affect either lymphocyte proliferation or CD25 expression by PHA-activated mononuclear leukocytes (MNLs). Similarly, none of the cytokines measured were significantly affected by moxifloxacin, either in the absence or presence of PHA, compatible with a lack of effect of this agent on Th1 and Th2 lymphocytes. In conclusion, this study suggests that moxifloxacin, at therapeutic doses, does not affect the protective functions of human neutrophils and lymphocytes. / Dissertation (MSc (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted

Dans le cancer, l'activation de la cascade de coagulation et des plaquettes participent à la formation de thromboses, à la croissance tumorale, et les métastases. Ces thromboses représentent une complication clinique chez les patients atteints du cancer du pancréas. Cet état serait dû à expression par la tumeur et leurs microparticules (MPs) de facteur tissulaire (FT). Dans une première partie, nous avons identifié le FT et le FT pathway inhibitor exprimés par les MPs cancéreuses et la P-sélectine plaquettaire impliqués dans la progression tumorale, les métastases et la thrombose associée au cancer du pancréas .Nous avons montré le rôle des intégrines αvβ3 et αvβ et des "neutrophils extracellular traps" dans l’interaction des MPs cancéreuses avec les plaquettes dans des modèles murins de « deep vein thrombosis » et de blessure au laser. Nous avons alors évalué l’efficacité du clopidogrel qui présente une action anti tumorale et thrombotique. Cette étude a permis d’initier une étude clinique de phase III pour d’évaluer le potentiel thérapeutique du clopidogrel chez des patients atteints de cancer du pancréas. Dans une seconde partie, nous avons montré que des neutrophiles exprimant le FT agissent comme « starter » de la formation de thrombi. A l’inverse, dans un modèle d’inflammation stérile, nos travaux montrent le rôle de la P-sélectine plaquettaire dans le slow rolling, l’adhésion et la transmigration des neutrophiles a des temps précoses. L’ensemble de nos résultats suggère que les coopérations cellulaires entre l’endothélium, les plaquettes, les MPs et les neutrophiles constituent des mécanismes essentiels à la thrombose et l'inflammation. / In cancers, the blood coagulation cascade and platelets can be activated to form thrombosis. This state will mainly due by the tumor and their microparticles (MPs) expression of tissue factor (TF), key protein of the coagulation cascade. In the first part of this study, we demonstrated that TF and the TF pathway inhibitor expressed by cancer MPs and the platelet P-selectin are involved in tumor progression, metastasis and the associated thrombosis in pancreatic cancer in mice. We showed the key role-play by αvβ3 and αvβ1 integrins and neutrophils extracellular traps in the interaction between cancer cells-derived MPs and platelets. We also evaluated the effect of clopidogrel, but not aspirin, treatment exhibits an anti-tumor action and limits thrombosis formation in preclinical models of pancreatic cancer. This study initiates a national investigation of a multicenter clinical phase III study to evaluate the therapeutic potential of clopidogrel in pancreatic cancer patients. In the second part of this study, we identified a “population of neutrophils expressing TF” that acts like a starter of the thrombus formation. At the reverse, in a sterile inflammatory model, our work showed the primordial role of platelet P-selectin in the slow rolling, the adhesion and the transmigration of neutrophils. All together our results suggest that the cooperation between the endothelium, platelets, MPs and neutrophils constitute essential mechanisms acting in the thrombosis and the inflammation.

The Streptococcus pneumoniae-derived toxin, pneumolysin, has been reported to augment neutrophil-mediated inflammatory responses in murine models of experimental infection of the airways, and to favour invasive pneumococcal disease. The laboratory research presented in this thesis has been designed to investigate the possible proinflammatory interactions of pneumolysin with human neutrophils in vitro, as well as the underlying mechanisms of these. Addition of pneumolysin (0.0167 - 41.75 ng/ml) to neutrophils caused dose-related enhancement of the following proinflammatory activities of these cells: superoxide generation, elastase release, expression of the β2-integrin CR3, phospholipase A2 activity and production of leukotriene B4 and prostaglandin E2, oxidative inactivation of α-1-proteinase inhibitor, and synthesis and release of interleukin-8. Pneumolysin-mediated enhancement of these neutrophil activities was observed in the absence of detectable cytotoxicity and was most striking when the toxin was added together with the bacterial chemoattractant N-formyI-L-methionyl-L-leucyl-L-pnenylalanine (FMLP, 1 µM). Treatment of neutrophils with pneumolysin also resulted in uncontrolled influx of Ca2+ into the cells in the setting of membrane depolarisation and efflux of K+, which appeared to be a consequence of the pore forming actions of the toxin. Importantly, the proinflammatory interactions of pneumolysin with neutrophils were completely attenuated by exclusion of Ca2+ from the cell-suspending medium. These observations identify novel proinflammatory properties of pneumolysin which result from pore formation in the plasma membrane, influx of Ca2+ and augmentation of Ca2+ -activitable neutrophil functions. / Thesis (DPhil)--University of Pretoria, 2005. / Immunology / unrestricted

Background: COPD is characterised by increased neutrophilic inflammation which further increases during exacerbations. Corticosteroids are currently one of the mainstays of treatment but they have limited effectiveness; there is a great need to develop new anti-inflammatory pharmacotherapies for use in COPD. Inhaled LPS has been used as a model of increased neutrophilic inflammation in healthy patients, smokers and asthmatics. Its use in patients with COPD as a model of exacerbations has not yet been evaluated. PI3 kinase is a vital intracellular enzyme, which upon activation leads to a number of cellular processes; the γ and δ isoforms of the enzyme are of particular importance in leucocyte migration, development and activation. There is increasing evidence for upregulation of this pathway in COPD.Aims: (1) To test the safety of the use of inhaled LPS in patients with COPD for use as a model of exacerbation and to investigate the systemic and airway inflammatory response in vivo. (2) To investigate the action of PI3 kinase enzyme inhibitors and dexamethasone in vitro on neutrophilic inflammation in COPD patients during the stable state and exacerbations. Methods: (1) 12 patients with mild to moderate COPD inhaled 5µg LPS; safety measurements and airway and systemic biomarkers were collected up to 24 hours post inhalation. (2) The effect of PI3 kinase enzyme inhibitors and dexamethasone on MMP-9 and ROS release from peripheral and airway neutrophils from stable COPD and exacerbations was examined in vitro. The effect of PI3 kinase enzyme inhibitors and dexamethasone on cytokine release from peripheral neutrophils from stable COPD patients was also investigated. Results: (1) Inhaled LPS (5µg) caused a significant fall in FEV1 and increase in sputum neutrophil numbers. There was an associated increase in systemic IL-6, CRP and CC-16, all with differing temporal patterns. No patients reported any significant symptoms. (2) PI3 kinase enzyme inhibitors significantly reduced MMP-9 and ROS release from airway and peripheral neutrophils from COPD patients in the stable state and during exacerbations; dexamethasone had minimal effect. Cytokine release from peripheral neutrophils from COPD patients in the stable state was also significantly inhibited by PI3 kinase enzyme inhibitors and dexamethasone. Conclusions: (1) Inhaled LPS in patients with COPD is a safe model to induce acute on chronic neutrophilic inflammation and therefore could be used as a model to study COPD exacerbations. (2) PI3 kinase enzyme inhibitors reduce COPD neutrophil MMP-9, ROS and cytokine release in vitro and are therefore are a promising new anti-inflammatory pharmacotherapy.

Orientador: Carlos Amilcar Parada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T14:12:30Z (GMT). No. of bitstreams: 1 Rocha_LilianWunsch_D.pdf: 2264409 bytes, checksum: 95c935aebbbef2ad5593f1d56eb53bed (MD5) Previous issue date: 2015 / Resumo: Vimang® é um medicamento fitoterápico constituído pelo extrato aquoso da planta Mangifera indica L. Este medicamento é utilizado em Cuba para o tratamento de estresse e de doenças imunopatológicas, pois possui propriedade antioxidante reconhecida. O componente majoritário deste extrato é a mangiferina (cerca de 7%) e dados da literatura apontam que ambos, extrato e mangiferina, possuem atividade antinociceptiva. O objetivo deste trabalho foi verificar o potencial anti-hiperalgésico da mangiferina no tecido periférico e investigar seu possível mecanismo de ação em ratos. Para isso foram utilizados modelos de hiperalgesia inflamatória, tais como: hiperalgesia induzida pela carragenina (100 ?g/pata), epinefrina (1 ?g/pata) e pelos mediadores inflamatórios PGE2 (100 ng/pata), IL-1? (0,5 pg/pata) e CINC-1 (1 pg/pata). A hiperalgesia também foi induzida por ?,?-meATP (agonista de receptor P2X3)(50 ?g/pata) e 8-Br-cAMP (ativador de PKA) (0,01 ?g/pata). A avaliação da hiperalgesia mecânica foi realizada através de Von Frey eletrônico. O modelo de nocicepção aguda induzido pela capsaicina (0,5 ?g/pata) também foi realizado. Para complementar os experimentos comportamentais, foi realizada a dosagem de citocinas pró-inflamatórias IL-1?, TNF-? e CINC-1, a avaliação da migração de neutrófilos através da dosagem de mieloperoxidase, Western Blotting para COX-1 e COX-2 e ainda o teste de calcium imaging para verificar a possível interferência da mangiferina em receptores purinérgicos. Em todos os testes moleculares, a inflamação foi induzida pela carragenina e o tecido plantar da pata traseira dos ratos foi coletado 3 horas após a injeção intraplantar. Em todos os experimentos em que houve pré-tratamento com mangiferina, o mesmo foi realizado através de administração local, intraplantar. Para verificar a participação dos receptores purinérgicos no mecanismo de ação da mangiferina, no teste de calcium imaging, o influxo de cálcio in vitro foi induzido por ?,?-meATP. Os resultados demonstram que a mangiferina nas doses de 300, 600 e 1200 ?g/pata reduziu a hiperalgesia induzida pela carragenina de forma dose-dependente e sem apresentar efeito sistêmico, o qual foi avaliado através do limiar na pata contralateral. Para os demais experimentos foi utilizada a dose submáxima de 600 ?g/pata. A hiperalgesia mecânica induzida por epinefrina, IL-1? e CINC-1, mas não por PGE2, foi prevenida pela administração local de mangiferina. Também foi observado efeito da mangiferina reduzindo a nocicepção induzida pela capsaicina. No teste de ELISA, o pré-tratamento com mangiferina foi capaz de reduzir a concentração local da citocina TNF-? e da quimiocina CINC-1 induzida pela carragenina; porém, os níveis de IL-1? mantiveram-se inalterados. A migração de neutrófilos também foi avaliada e o tratamento local com mangiferina reduziu a migração de neutrófilos induzida pela carragenina para o tecido plantar. Os resultados obtidos através da dosagem das proteínas COX-1 e COX-2 por Western Blotting demonstraram que o tratamento local com mangiferina não altera a expressão destas proteínas. Na avaliação in vitro da atividade da mangiferina em inibir o influxo de cálcio induzido pelo ?,?-meATP não houveram resultados positivos, ou seja, a mangiferina não causou dessensibilização nos neurônios. Tomados em conjunto, os resultados demonstram que a mangiferina possui atividade anti-hiperalgésica e possivelmente esta ação esteja relacionada com a sua habilidade em reduzir citocinas que são essenciais para o desenvolvimento da hiperalgesia inflamatória / Abstract: Vimang® is an herbal medicine constituted by the aqueous extract of Mangifera indica L plant. It is used in Cuba for the treatment of stress and immunopathological diseases because it has recognized antioxidant properties. The major component of this extract is mangiferin (about 7%) and literature data indicates that both, extract and mangiferin, have antinociceptive activity. The aim of this study was to evaluate the antihyperalgesic potential of mangiferin in peripheral tissue and investigate its possible mechanism of action in rats. Were used inflammatory hyperalgesia models such as carrageenan-induced hyperalgesia (100 ?g/paw), epinephrine (1 ?g/paw) and the inflammatory mediators PGE2 (100 ng/paw), IL-1? (0.5 pg/ paw) and CINC-1 (1 pg/paw). Hyperalgesia was also induced by ?,?-meATP (P2X3 receptor agonist) (50 ?g/paw) and 8-Br-cAMP (PKA activator) (0.01 ?g/paw). The evaluation of mechanical hyperalgesia was performed using Electronic Von Frey. The acute model of nociception induced by capsaicin (0.5 ?g/paw) was also performed. In addition to the behavioral experiments, the dosage of pro-inflammatory cytokines IL-1?, TNF-? and CINC-1 was performed, evaluation of the migration of neutrophils through the myeloperoxidase dosage, Western Blotting for COX-1 and COX-2, and the calcium imaging test to check possible interference of mangiferin in purinergic receptors. In all molecular tests, inflammation was induced by carrageenan and the plantar tissue of the hind paw of the rats was collected 3 hours after intraplantar injection. In all experiments in which there was pretreatment with mangiferin, it was conducted by local administration, intraplantar. To verify the involvement of the purinergic receptors in the mangiferin mechanism of action, using calcium imaging test, in vitro calcium influx was induced by ?, ?-meATP. The results demonstrate that mangiferin at doses of 300, 600 and 1200 ?g/paw reduced carrageenan-induced hyperalgesia in a dose-dependent manner and without providing systemic effect, which was evaluated through the threshold of the contralateral paw. For other experiments the submaximal dose of 600 ?g/paw was used. Mechanical hyperalgesia induced by epinephrine, IL-1? and CINC-1, but not by PGE2, was prevented by local administration of mangiferin. It was also observed mangiferin effect of reducing the capsaicin-induced nociception. In the ELISA test, the mangiferin pretreatment reduced the local concentration of the cytokine TNF-? and chemokine CINC-1, however, IL-1? levels were unchanged. The neutrophil migration was also assessed and local treatment with mangiferin reduced neutrophil migration induced by carrageenan into the plantar tissue. The results obtained with the COX-1 and COX-2 proteins dosage by Western Blotting demonstrated that local treatment with mangiferin did not alter the expression of these proteins. In vitro evaluation of the activity of mangiferin in inhibiting calcium influx induced by ?, ?-meATP there were no positive results, mangiferin did not cause desensitization in neurons. Taken together, the results show that mangiferin has anti-hyperalgesic activity and possibly this action is related to its ability to reduce cytokines that are essential to the development of inflammatory hyperalgesia / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22120号 / 医博第4533号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM