A asma é uma doença inflamatória crônica das vias aéreas responsável por considerável morbimortalidade em todo o mundo. O tratamento atualmente disponível que inclui broncodilatadores e corticosteróides, apresenta respostas variáveis e alguns pacientes não atingem o controle preconizado. Além disso, esses medicamentos apresentam efeitos colaterais indesejáveis. Assim, é necessário o desenvolvimento de novas drogas para o tratamento da asma. A Pyrostegi venusta (KerGahl.) Miers (\"cipó-de-são-joão\", Bignoniaceae), uma trepadeira amplamente distribuída no mundo inteiro e no Brasil principalmente no cerrado brasileiro, apresenta atividade anti-inflamatória e antioxidativa. Até o momento, não há descrição de estudos de P. venusta no tratamento da asma. O objetivo do estudo foi avaliar os efeitos da administração de dois extratos de P. venusta nas doses de 100 e 300mg/kg no tratamento da asma em um modelo animal e os mecanismos envolvidos. Foram realizados dois protocolos com períodos de tratamento e doses diferentes. No protocolo 1, camundongos Balb/c foram sensibilizados duas vezes com ovalbumina (OVA) intraperitoneal (ip), com uma semana cada de intervalo; após uma semana, foram desafiados com OVA intranasal (in) por três dias consecutivos, os camundongos receberam tratamento com extrato aquoso ou extrato hidroetanólico de P. venusta (100 mg/kg) por via ip, também por três dias consecutivos, durante os desafios com OVA. No protocolo 2, camundongos Balb/c foram sensibilizados duas vezes com OVA ip, com uma semana de intervalo e, após uma semana, desafiados com OVA in por quatro dias alternados. Os animais foram tratados com os extratos aquoso ou extrato hidroetanólico de P. venusta (300 mg/kg), ip, por sete dias consecutivos. Camundongos controles receberam salina nos mesmos dias. Após a sensibilização e desafios, os animais foram ventilados e foram realizadas medidas in vivo da hiper-responsividade brônquica com concentrações crescentes de metacolina. Após, foi coletado lavado broncoalveolar para contagem total e diferencial de células. O sangue foi coletado para dosagem de IgE específica para OVA e os pulmões foram retirados para dosagem de citocinas e capacidade antioxidante no homogenato pulmonar e análise histológica. No protocolo 1, houve redução de células totais, eosinófilos e da hiper- responsividade brônquica no grupo tratado com extrato aquoso quando comparado ao grupo controle. O extrato hidroetanólico não reduziu a inflamação das vias aéreas. Em relação ao protocolo 2, o grupo asmático que recebeu tratamento com extrato aquoso apresentou diminuição de células inflamatórias totais, eosinofílicas e no tecido pulmonar, além de diminuição da hiper-responsividade brônquica e aumento da capacidade antioxidante. O grupo tratado com extrato hidroetanólico apresentou redução apenas de células totais e de eosinófilos. Os extratos aquoso e hidroetanólico da P. venusta não reduziram os níveis de citocinas inflamatórias. Conclui-se que a administração do extrato aquoso de P. venusta na dose de 300mg/kg atenuou as principais características da asma em um modelo animal, provavelmente por um mecanismo antioxidante. / Asthma is a chronic inflammatory disease of the airways responsible for considerable morbidity and mortality worldwide. With the currently available treatment, including bronchodilators and corticosteroids, some patients do not reach the recommended control. Furthermore, these drugs have undesirable side effects. Thus, the development of new drugs for the treatment of asthma is needed. P. venusta (KerGahl.) Miers (\"liana-of St. John,\" Bignoniaceae), a widely distributed climbing worldwide and in Brazil mainly in the Brazilian cerrado, has anti-inflammatory and antioxidative activity. To date, there is no study of P. venusta in the treatment of asthma. The aim of the study was to evaluate the effects of administration of two extracts of P. venusta with different doses (100 and 300 mg/kg) in the treatment of asthma in an animal model and the mechanisms involved. Two protocols were conducted with different period of treatment. In protocol 1, Balb/c mice were sensitized twice with ovalbumin (OVA) intraperitoneally (ip) one week apart. After one week, mice were challenged with intranasal OVA for three consecutive days and treated with aqueous extract or hydroethanolic extract of P. venusta (100mg/kg) ip for three consecutive days, during OVA challenges. In protocol 2, Balb/c mice were sensitized ip with OVA twice with an interval of one week and after one week challenged four times with OVA intranasally in alternate days. The animals were treated with the aqueous extract or hydroethanolic extract of P. venusta (300 mg/kg) ip for seven consecutive days. Control mice received saline on the same days. After sensitization and challenge, the animals were ventilated and in vivo measurement of bronchial hyperresponsiveness was performed wiht increasing concentrations of methacholine. After, bronchoalveolar lavage was collected for total and differential cell count. The blood was collected to measure OVA specific IgE and lungs were removed for cytokines quantification in the pulmonary homogenates and histological analysis. In protocol 1, aqueous extract administration significantly reduced total and differential cells number, and bronchial hyperresponsiveness compared to the group that received no treatment. Hydroethanolic extract did not significant reduce airway inflammation. In relation to Protocol 2, the asthmatic group treated with aqueous extract had a significant decrease in total and differential inflammatory cells, lung inflammation, and bronchial hyperresponsiveness. Moreover, aqueous extract administration increased significantly antioxidant capacity. The hydroethanol extract decreased significantly only total cells and eosinophils. The aqueous and hydroethanolic extracts of P. venusta did not reduce the levels of inflammatory cytokines. We conclude that the administration P. venusta aqueous extract at a dose of 300mg/kg attenuated the main features of asthma in an animal model, probably via an antioxidant mechanism.

A piometra canina é uma infecção bacteriana que acomete o útero de cadelas sexualmente maduras e gera a síndrome clínica conhecida como sepse e síndrome da resposta inflamatória sistêmica. Quando não tratada adequadamente ocorre a progressão da doença para estágios mais graves como a sepse grave e o choque séptico. Embora as alterações ocasionadas pela infecção serem conhecidas, o correto estadiamento da gravidade do quadro ainda é um obstáculo. O processo de inflamação sistêmica é complexo, o fator de necrose tumoral (TNF-α e a interleucina 1β (IL-1&#946) aparecem como mediadores inflamatórios centrais nos casos de sepse. Estudos recentes do DNA de pacientes em choque séptico têm demonstrado a presença de mutações ou polimorfismos que exercem grande influência na produção do TNF-α e da IL-1&#946. Com exceção da avaliação sérica de lactato, a proteína C reativa (CRP) é o biomarcador mais utilizado em testes clínicos para o diagnóstico e tratamento da sepse. Os objetivos principais do estudo foram avaliar a gravidade do caso; as alterações sistêmicas e metabólicas do paciente no período pré-operatório; as concentrações plasmáticas do TNF-α, IL-1β, IL-6 e CRP no momento do diagnóstico; a presença de polimorfismos na região decodificadora do TNF-α e da IL-1&#946. Foram incluídas 9 cadelas sem piometra e 85 com piometra, afecção diagnosticada a partir do histórico e dos sinais clínicos identificados por meio do exame físico e ultrassonografia abdominal; e foram estratificadas para a gravidade do caso em sepse, sepse grave ou choque séptico. As variáveis clinicas e laboratoriais foram coletadas no momento do diagnóstico e confrontadas estatisticamente pelo método de comparações múltiplas. Os resultados mostraram 23 (24,4%) animais em sepse, 60 (63,82%) em sepse grave e dois (2,12%) em choque séptico. As alterações mais significativas foram observadas nos animais com sepse grave e em choque séptico e pode-se destacar a variação no tempo de preenchimento capilar, na pressão arterial, na contagem leucocitária, na albuminemia, na fosfatase alcalina, na creatinina, no pH, na CRP e na IL-6. O exame do gene da IL-1β demonstrou que os animais em sepse com o alelo GG apresentam níveis séricos de IL-1β mais elevados. Foi possível concluir que a estratificação da gravidade do caso para os animais em sepse grave é muito branda e não distingue os animais em estado mais grave dos menos grave dentro do mesmo grupo; que o estadiamento da gravidade poder ser realizado com avaliações corriqueiras e empregadas de forma rotineira no atendimento dos pacientes; que a proteína C reativa e a IL-6 apresentam correlação direta com a gravidade do caso; e que o polimorfismo do gene da IL-1β e o tempo decorrido entre o inicio dos sintomas até que os proprietários procurassem ajuda médica foram os de maior influência sobre a gravidade do caso / Canine pyometra is a bacterial infection that affects the uterus of sexually mature bitches and generates the clinical syndrome known as sepsis and systemic inflammatory response syndrome. When not treated properly the disease progression to more severe stages as severe sepsis and septic shock occurs. Although the changes caused by the infection are known, the correct staging of the severity of the condition is still an obstacle. The process of systemic inflammation is complex, tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) appear as central inflammatory markers in sepsis. Recent studies of DNA from patients in septic shock have shown the presence of mutations or polymorphisms that exert great influence on the production of TNF-α and IL-1β. Except the assessment of serum lactate, C-reactive protein (CRP) is the most widely used biomarker in clinical trials for the diagnosis and treatment of sepsis. The main objectives of the study were to evaluate the severity of the case, systemics and metabolics changes in the patient preoperatively, plasma concentrations of TNF-α, IL-1β, IL-6 and CRP at the time of diagnosis; the presence of polymorphisms in the decoding region of the TNF-α and IL-1β. In the study was included 9 bitches without pyometra and 85 bitches with pyometra, disease was diagnosed from the history and clinical signs identified through physical examination and abdominal ultrasonography; and were stratified according to the severity of the case in sepsis, severe sepsis or septic shock. The clinical and laboratory variables were collected at diagnosis and compared statistically by the method of multiple comparisons. The results showed 23 (24.4%) animals with sepsis, 60 (63.82%) in two and severe sepsis (2.12%) in septic shock. The most significant changes were observed in animals with severe sepsis and septic shock and can highlight the variation in capillary refill time, blood pressure, leukocyte count, the albumin, alkaline phosphatase, creatinine, pH, CRP and IL-6. Examination of the IL-1β gene showed that animals with sepsis with GG allele have higher levels of seric IL-1β. It was concluded that the stratification of the severity of the case for animals in severe sepsis is very bland and does not distinguish the animals in a more severe state of less severe within the same group; the staging of gravity can be done with ordinary ratings and employed routinely in patient care; the C reactive protein and IL-6 show a direct correlation with the severity of the case; and the polymorphism of the IL-1β and the time elapsed between the onset of symptoms until the owners sought medical help were the most influence on the severity of the case

A asma é uma doença inflamatória crônica com alta prevalência e caracterizada por hiperresponsividade da via aérea, inflamação, e remodelamento brônquico, e é responsável por considerável morbimortalidade em todo mundo. Os tratamentos disponíveis para a asma podem apresentar diferentes respostas e vários efeitos colaterais. Por isso, o desenvolvimento de novas drogas para o tratamento da asma é muito importante. A Uncaria guianensis (Aubl.) J. F. Gmel. (\"unha de gato\", Rubiaceae) (UG), é uma planta encontrada principalmente na Amazônia e na América Central, e apresenta atividade anti-inflamatória e antioxidante. Até o momento, não há relato de estudos da U. guianensis no tratamento da asma. O objetivo deste estudo foi avaliar os efeitos da administração de dois extratos das folhas da U. guianensis no tratamento da asma em um modelo animal, e os mecanismos envolvidos. Camundongons Balb/c foram sensibilizados duas vezes com ovalbumina (OVA) por via interperitoneal (ip) com uma semana de intervalo entre as sensibilizações, após uma semana, os camundongos foram desafiados com OVA via intranasal por três dias consecutivos e tratados com os extratos aquoso ou hidroetanólico da U. guianensis (100 mg/kg) via intraperitoneal por três dias consecutivos, durante os desafios com OVA. Os camundongos controles receberam solução salina nos mesmos dias. Após a sensibilização e desafios, os animais foram ventilados e medidas in vivo da hiper-responsividade brônquica foram realizadas com a administração de aerossois com concentrações crescentes de metacolina. Após, o lavado broncoalveolar (LBA) foi coletado para contagem de células totais e diferenciais. Sangue foi colhido para dosagem de IgE específica para OVA e os pulmões foram retirados para quantificação de citocinas inflamatórias no homogenato, além de determinação da capacidade antioxidante total e análise histológica. A administração do extrato hidroetanólico diminuiu significativamente o número de células totais e diferenciais no LBA e a hiper-responsividade brônquica, quando comparados ao grupo que não recebeu o tratamento. O extrato aquoso não foi capaz de diminuir a contagem de células totais e diferenciais e mostrou uma pequena diminuição na hiper-reatividade brônquica. Tanto o extrato aquoso quanto o hidroetanólico diminuíram as concentrações de interleucina (IL)-13 no homogenato pulmonar, mas não foram capazes de diminuir os níveis de IgE no soro, o número de células inflamatórias no tecido pulmonar, nem foram capazes de aumentar a capacidade antioxidante total. Com isso, concluímos que a administração dos dois extratos (aquoso e hidroetanólico) das folhas da espécie U. guianensis foi efetiva no tratamento da asma em um modelo animal, tanto em termos de mecânica pulmonar quanto em marcadores inflamatórios, com superioridade terapêutica do extrato hidroetanólico. / Asthma is a highly prevalent chronic inflammatory disease characterized by airway hyperresponsiveness, inflammation, and remodeling, with considerable morbidity and mortality worldwide. Available asthma treatments can elicit different responses and numerous side effects. Therefore, the development of new drugs for asthma treatment is highly desirable. Uncaria guianensis (Aubl.) J. F. Gmel. (\"cat\'s claw\", Rubiaceae) (UG), a plant found mainly in the Amazon and the Central America, has anti-inflammatory and antioxidant activities. To date, there is no study of U. guianensis in the treatment of asthma The aim of the study was to evaluate the effects of the administration of two extracts from leaves of U. guianensis in the treatment of asthma in an animal model and the mechanisms involved. Balb/c mice were sensitized twice with ovalbumin (OVA) intraperitoneally (ip) one week apart. After one week, mice were challenged with intranasal OVA for three consecutive days and treated with either aqueous or hydroethanolic extract of U. guianensis (100 mg/kg) ip for three consecutive days, during OVA challenges. Control mice received saline solution on the same days. After sensitization and challenge, the animals were ventilated and in vivo measurement of bronchial hyperresponsiveness was performed with increasing concentrations of aerosolized methacholine. After, bronchoalveolar lavage (BAL) was collected for total and differential cell count. The blood was collected to measure OVA specific IgE, and the lungs were removed for measurement of cytokine levels and total antioxidant capacity in the pulmonary homogenate, and for histological analysis. The hidroethanolic extract administration significantly reduced total and differential cells number and bronchial hyperresponsiveness, compared to the group that received no treatment. The aqueous extract did not decrease the total and differential cell count and showed a small decrease in bronchial hyperresponsiveness. Both extracts decreased interleukin (IL)-13 levels in the pulmonary homogenate, but did not decrease serum IgE levels nor the number of inflammatory cells in lung tissue, nor were they able to increase total antioxidant capacity. We concluded that the administration of two extracts (aqueous and hydroethanolic) from leaves of U. guianensis was effective in the treatment of asthma in animal models, both in pulmonary mechanics and in inflammatory markers, with therapeutic superiority of the hydroethanolic extract.

abstract: The prevalence of obesity and obesity-related disorders have increased world-wide. In the last decade, the intestinal microbiome has become a major indicator of metabolic and gastrointestinal health. Previous research has shown that high-fat diet (HFD) consumption can alter the microbial composition of the gut by increasing the abundance of gram-positive bacteria associated with the onset of obesity and type 2 diabetes. Although, the most common form of obesity and metabolic syndrome intervention is exercise and diet, these recommendations may not improve severe cases of obesity. Thus, an important relevance of my project was to investigate whether the intake of an organometallic complex (OMC) would prevent the onset of metabolic and gastrointestinal complications associated with high-fat diet intake. I hypothesized that the consumption of a HFD for 6 weeks would promote the development of metabolic and gastrointestinal disease risk factors. Next, it was hypothesized that OMC treatment would decrease metabolic risk factors by improving insulin sensitivity and decreasing weight gain. Finally, I hypothesized that HFD-intake would increase the abundance of gram-positive bacteria associated with gastrointestinal disease. My preliminary data investigated the effects of a 6-week HFD on the development of hepatic steatosis, intestinal permeability and inflammation in male Sprague Dawley rats. I found that a 6-week HFD increases hepatic triglyceride concentrations, plasma endotoxins and promotes the production of pro-inflammatory cytokines in the cecum wall. I then investigated whether OMC treatment could prevent metabolic risk factors in male Sprague-Dawley rats fed a HFD for 10 weeks and found that OMC can mitigate risk factors such hyperglycemia, liver disease, impaired endothelial function, and inflammation. Lastly, I investigated the effects of a 10-week HFD on the gastrointestinal system and found an increase in liver triglycerides and free glycerol and alterations of the distal gut microbiome. My results support the hypothesis that a HFD can promote metabolic risk factors, alter the gut microbiome and increase systemic inflammation and that OMC treatment may help mitigate some of these effects. Together, these studies are among the first to demonstrate the effects of a soil-derived compound on metabolic complications. Additionally, these conclusions also provide an essential basis for future gastrointestinal and microbiome studies of OMC treatment. / Dissertation/Thesis / Doctoral Dissertation Biology 2019

Le réseau lymphatique permet le drainage des liquides interstitiels, le transport des cellules immunitaires et intervient dans le métabolisme lipidique. La dérégulation de ce système est impliquée dans de nombreuses pathologies comme les lymphœdèmes, le rejet de greffe ou encore l'échappement tumoral. Le cœur est pourvu d'un réseau lymphatique abondant dont l'importance n'est apparue que très récemment dans les pathologies ischémiques. En effet, suite à un infarctus du myocarde, la dysfonction lymphatique observée favorise l'œdème et l'inflammation tissulaire, révélant le réseau lymphatique comme un acteur majeur du développement de cette pathologie. Les processus régulant la formation de vaisseaux lymphatiques chez l'adulte sont largement dépendants de facteurs de croissance aux effets pro- ou anti- lymphangiogéniques. Les cellules immunes et plus particulièrement les macrophages sécrètent un grand nombre de ces facteurs et leur importance dans la réponse lymphangiogénique a été montrée dans différentes conditions physio-pathologiques, telles que la lymphangiogenèse tumorale et inflammatoire. Il a longtemps été admis que les macrophages tissulaires provenaient exclusivement de la différenciation de monocytes sanguins. Cependant, l'existence de macrophages provenant de progéniteurs embryonnaires a été récemment mise en évidence. Dorénavant, les macrophages tissulaires résidents sont perçus comme des populations hétérogènes pouvant prétendre à des différences fonctionnelles. A ce jour, les modifications du réseau lymphatique et les mécanismes permettant sa régulation, au cours de pathologies non ischémiques, n'ont pas été abordées. Ainsi, nous avons entrepris d'identifier et de caractériser les différentes populations de macrophages cardiaques participant à la régulation du réseau lymphatique suite à une surcharge barométrique. Nos résultats ont montré une diminution précoce du réseau lymphatique cardiaque dans le cœur murin hypertrophié au cours d'une surcharge de pression induite par constriction de l'aorte transverse. Cette réduction du réseau est associée à la perte d'une population majoritaire de macrophages cardiaques portant le récepteur 1 à l'acide hyaluronique (Lyve-1). Cette population résidente diminue dans le cœur insuffisant, au profit de macrophages infiltrants dérivés de monocytes sanguins. Par ailleurs, en plus d'être corrélée à la baisse du réseau lymphatique cardiaque, la diminution du nombre de macrophages Lyve-1 est proportionnelle à la détérioration de la fonction cardiaque. La prévention de l'infiltration monocytaire a permis de maintenir le pool de macrophages exprimant Lyve-1, le réseau lymphatique et la fonction cardiaques lors d'une surcharge de pression. La caractérisation par RT-PCR des différentes populations de macrophages récupérés par tri cellulaire nous a permis de montrer que les macrophages Lyve-1 présentent des caractéristiques particulières, avec une forte expression de VEGFR3 et NRP2, molécules de signalisation lymphangiogène. De plus, ces macrophages Lyve-1 à la polarisation mixte et à l'activité phagocytaire importante, expriment de nombreux facteurs pro-lymphangiogéniques (VEGFc, VEGFd, IGF1). Ils ont montré une activité pro-lymphangiogène in vitro sur des explants de canal thoracique et sur des cellules endothéliales lymphatiques et in vivo avec la formation de vaisseaux lymphatiques naissants lors de l'injection de ces macrophages Lyve-1 dans le cœur. Leur localisation à proximité des vaisseaux lymphatiques et leur capacité pro-lymphangiogène leurs confèrent un rôle évident dans le maintien du réseau lymphatique lors d'un remodelage cardiaque de surcharge de pression. Ces travaux ont permis l'identification d'une population originale de macrophages cardiaques pouvant entrer dans la régulation du système lymphatique au cours d'une surcharge de pression. / The lymphatic system has recently emerged as an important regulator of the cardiac interstitial fluid compartment and function. Experimental obstruction of lymphatic vessel leads to cardiac œdema, myocardial stiffness, fibrosis and ventricle dysfunction. Following myocardial infarction, stimulation of lymphangiogenesis was found to reduce fibrosis and inflammation and to improve cardiac function. Macrophages have been largely described as important contributors of lymphangiogenesis in inflammatory situations such as cancer. Recently, genetic fate mapping demonstrated that distinct populations of macrophages coexist in the adult heart. In addition to monocyte derived-macrophages that massively colonize injured heart, a subpopulation of tissue-resident macrophages that originates from embryonic precursors persists into adulthood by means of local self- renewal. To date the distinct involvement of cardiac macrophage subpopulations in cardiac lymphatic remodeling and heart failure progression induced by pressure overload is largely unknown. In our study, we observed that expression of Lyve-1 identifies a resident macrophage subset abundant in mouse heart. This Lyve-1 positive macrophage subset decreased rapidly in cardiac remodeling induced by chronic pressure overload. In addition, the number of cells found in heart was positively correlated with the preservation of cardiac lymphatic network and function after transverse aortic constriction (TAC). Blocking recruitment of monocyte derived macrophages expanded Lyve-1 positive macrophages, attenuated cardiac lymphatic remodeling and contractile dysfunction of pressure overloaded heart. Lyve-1 positive macrophages express pro-lymphangiogenic factors and sustain lymphangiogenesis in vitro and in vivo. In conclusion, resident macrophage subset expressing the Lyve-1 receptor participates to maintain cardiac function after chronic pressure overload by mechanisms that may involve the preservation of cardiac lymphatic system. These results provide insight into the regulation of lymphatic homeostasis by tissue resident macrophage during heart failure induced by pressure overload.

Interleukin-1-beta (IL-1β) is a potent proinflammatory cytokine. A primary outcome of IL-1β signaling is the activation of NFκB, a transcription factor that induces a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors. Recent work has demonstrated that the activation of NFκB involves a multistep redox-signaling cascade that requires endocytosis of the interleukin receptor (IL-1R1)/ligand pair and superoxide production by NADPH oxidase 2 (Nox2) within the resulting newly formed early endosome. Hydrogen peroxide produced by the rapid dismutation of superoxide is necessary for the subsequent downstream recruitment of IL-1R1 effectors (TRAF6, IKK kinases) and ultimately the activation of NFκB. In this thesis, I have further dissected the spatial and temporal events that coordinate signaling processes of the IL-1β pathway. Using a combination of biophotonic imaging, immunofluorescence imaging, and lipid raft density gradient isolation, I demonstrate that both Nox2 and IL-1R1 are constitutively present in lipid raft microdomains on the plasma membrane. Stimulation by IL-1β induces endocytosis of Nox2 and IL-1R1 from the plasma membrane into caveolin-1, lipid raft positive early endosomes. Further, inhibition of lipid raft mediated endocytosis or deletion of caveolin-1 inhibits activation of NFκB, by IL-1β. We have also identified Vav1 as the Rac1 guanine exchange factor that is recruited to caveolin-1 positive lipid rafts following IL-1β stimulation, and demonstrated that dominant negative Vav1 inhibits NFκB activation by IL-1β. Following this work, I utilized assays for redox sensitivity and mass spectrometry to demonstrate that C70, C73, and C105 are hydrogen peroxide sensitive cysteines within the RING domain of TRAF6. I further demonstrate that hydrogen peroxide does not alter the E3 ubiquitin ligase activity associated with the TRAF6 RING domain. My findings suggest that the redox sensitivity of the RING domain mediates TRAF6 recruitment to the receptor complex. This is supported by the observation that hydrogen peroxide treatment of TRAF6, but not early signaling effectors (IL-1R1, IRAK1, IRAK4, MyD88) mediates TRAF6 recruitment to the IL-1 receptor complex. Further, mutation of the identified redox sensitive cysteines inhibits IL-1β signaling and NFκB activation. This research has helped to refine the understanding of the IL-1β signaling pathway, and may ultimately lead to new therapeutic targets for controlling inflammation.

Orientador: Flávio Ruas de Moraes / Coorientador: Marco Antonio de Andrade Belo / Banca: Rogério Salvador / Banca: Maurício Laterça Martins / Resumo: Este ensaio teve por objetivos avaliar o efeito da administração parenteral de dexametasona e fosfato de levamisol sobre a cinética de acúmulo de macrófagos e formação de gigantócitos em lamínulas de vidro, implantadas no tecido subcutâneo de pacus (Piaractus mesopotamicus), na tentativa de simular um estímulo estressante com imunossupressão e a possibilidade de compensa-la usando um imuoestimulante. Foram utilizados 147 peixes juvenis, distribuídos aleatoriamente em vinte e uma caixas de água plásticas de 250 L, contendo sete animais cada, constituindo os tratamentos: NV = não tratado; CL = controle; L 25 = levamisol (25 mg/kg), L 50 = levamisol (50 mg/kg) , D2 = dexametasona (2 mg/kg), D2+L25 = dexametasona (2 mg/kg + levamisol 25 mg/kg), D2+L50 = dexametasona (2 mg/kg + levamisol 50 mg/kg). Os pacus foram anestesiados em solução alcoólica de benzocaína (1:10000 v/v) e submetidos ao implante de lamínulas de vidro esterilizadas no tecido subcutâneo, quando administrou-se, por via intra-muscular, os fármacos de acordo com cada tratamento. Decorridos dois, sete e 15 dias pós-implante (DPI) os peixes foram novamente anestesiados para coleta de sangue e eutanasiados (solução alcoólica de benzocaína, 1:500 v/v) para a retirada das lamínulas. O sangue destinou-se aos exames hematológicos e as lamínulas à avaliação da cinética do acúmulo de macrófagos e formação de gigantócitos. Os resultados foram analisados pelo teste de Tukey, a 5% de probabilidade e demonstraram que ao longo do período experimental observou-se redução do número de eritrócitos e taxa de hemoglobina associadas ao aumento do volume corpuscular médio, quadro que sugere ligeira anemia macrocítica. Na avaliação da cinética de acúmulo celular sobre a lamínula, as maiores contagens de macrófagos isolados ocorreram sete dias após... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to investigate the effect of parenteral administration of dexamethasone and levamisole phosphate on the kinetics of accumulation of macrophages and formation of giant cells in glass cover slips implanted in subcutaneous tissue of pacu (Piaractus mesopotamicus) to simulate a stressful stimulus with immunosuppression and the possibility to compensate for it using an immunostimulant. We used 147 juvenile fish, randomly divided in twenty-one plastic water tanks of 250 L, containing seven animals each: NV = not treated, and CL = control, L25 = levamisole (25 mg / kg), L 50 = levamisole (50 mg / kg), D2 = dexamethasone (2 mg / kg), D2 + L25 = dexamethasone (2 mg / kg + levamisole 25 mg / kg), D2 + L50 = dexamethasone (2 mg / kg + levamisole 50 mg / kg). Fish were anesthetized with an alcoholic solution of benzocaine (1:10,000 v / v), implanted with sterile glass cover slips at subcutaneous tissue and were administered intra-muscular drugs, according of each treatment. After two, seven and 15 days post-implantation (DPI) fish were anesthetized for blood collection and euthanized (alcoholic solution of benzocaine, 1:500 v / v) to remove the glass cover slips. The blood was used for hematological tests and the glass cover slips to assess the kinetics of accumulation of macrophages and formation of giant cells. Statistical analyses were performed using 5% Tukey test and was observed reduction in the number of erythrocytes and hemoglobin associated with increase of mean corpuscular volume, suggesting slight macrocytic anemia. Kinetic evaluation of cell accumulation on glass cover slip, the highest number of isolated macrophages occurred at seven days after implantation in all groups, associated with increased formation of policariontes cells regardless of the number of cores. The dexamethasone... (Complete abstract click electronic access below) / Mestre

Curcumin is the yellow pigment found in the Indian spice curry. It has anti-inflammatory, ant-oxidant, anti-cancer, anti-viral, anti-bacterial and wound healing properties. It is widely used in industry for its flavor as a spice and as a coloring agent because of its brilliant yellow color. It is also used as a dye for textiles and as an additive to cosmetics. Dendritic cells (DCs) are the sentinels of the immune system and functions as the bridge between the innate and adaptive immune response. The effect of curcumin on DCs is poorly understood. A study shows curcumin prevents the immuno-stimulatory function of bone marrow-derived murine DCs, but no study examines the effects on human DCs. This study investigates the effects of curcumin on immature human DC maturation and function in response to immune stimulants lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C). Human CD14+ monocytes isolated from the peripheral blood of donors are cultured with GM-CSF and IL-4 supplemented media to generate immature DCs. The cultures are treated with curcumin, stimulated with the above mentioned stimulants then functional assays performed. These assays include homotypic cluster formation, surface marker expression, cytokine production, chemotaxis, endocytosis, DC-induced allogeneic CD4+ T cell proliferation after mixed lymphocyte reaction, gene expression analysis and immuno-fluorescence labeling and imaging. Curcumin-induced changes in gene expression indicate the actin cytoskeleton signaling pathway is a target. Immuno-fluorescence labeling and imaging of f-actin was carried out. Curcumin reduces DC maturation in response to the stimulants used in the study. Expression of surface markers, cytokines and chemokines is reduced as well as DC-induced stimulation of allogeneic CD4+ cells after MLR. Curcumin prevents chemotaxis without affecting chemokine receptor expression and significantly reduces endocytosis in non-stimulated cells. Curcumin-treated DCs do not induce a Th1 or Th2 population in allogeneic MLR but induces a CD25+Foxp3+ regulatory cell population. Immuno-fluorescence imaging shows curcumin causes the cell to become more rounded. These data imply that curcumin inhibits f-actin polymerization and thereby prevents DC maturation and function in response to stimulation. This outlines a novel role for curcumin as an immune suppressant and shows its therapeutic potential as an anti-inflammatory agent.

Many college students exercise individually or participate in collegiate and intramural sports in addition to fulfilling their stressful academic requirements. The combination of accumulated stress and vigorous exercise could result in an impaired immune system, prompting the onset of disease and absences in class and sports practice. Twenty-six male and female participants aged 18 to 23 were recruited for this study. Over the course of an academic semester, participants completed weekly electronic surveys documenting stress levels, aerobic exercise, and symptoms related to upper respiratory tract infections. Participants were evaluated at four different time points (Baseline, Post-Midterm Exam, Baseline Reassessment, and Post-Final Exam) for body fat percentage, cardiovascular fitness, heart rate, blood pressure, and a 10mL blood draw. Blood samples were used to measure blood glucose, cortisol, IL-6, and CD11b levels. Analysis of cortisol and IL-6 concentrations required ELISA kits for protein quantification in plasma samples. CD11b levels in peripheral blood mononuclear cell samples were measured by Western Blot analysis. There was a significant increase in blood pressure during the final exam compared to rest for systolic (p=0.005) and diastolic (p=0.004) blood pressures. There was a significant decrease in anxiety during the final exam compared to anxiety during the mid-term exam (p=0.022). The acute stress of an exam was strong enough to illicit physiologic blood pressure change, but the chronic stress throughout the semester was not intense enough did not illicit physiologic or immune responses. The volume of aerobic exercise in the vigorous workout group was not great enough to influence immune responses nor disease incidence.

Studies on repeated exposure to opioids have been carried out for decades yet the mechanisms for certain phenomena such as tolerance are still not fully understood. Furthermore, different medications, such as frequently prescribed benzodiazepines, or different disease states, such as HIV, have their own effects and interactions with chronic opioid exposure that are not fully understood. The overall objective of this dissertation was to investigate the complexities of chronic opioid exposure and how different disease states and medications may modulate the effects of chronic opioids. Our findings demonstrate that the administration of diazepam, at doses that are not antinociceptive or have any motor effects, reverse both antinociceptive and locomotor tolerance to orally active opioids. These doses of diazepam did not potentiate the acute effects of these prescription opioids. We also found that HIV-1 Tat expression significantly attenuated the antinociceptive potency of acute morphine in non-tolerant mice while not significantly altering the antinociceptive tolerance to morphine. Consistent with this, Tat attenuated withdrawal symptoms among morphine-tolerant mice. Pretreatment with maraviroc, a CCR5 antagonist blocked the effects of Tat, reinstating morphine potency in non-tolerant mice and restoring withdrawal symptomology in morphine-tolerant mice. Protein array analyses revealed only minor changes to cytokine profiles whether morphine was administered acutely or repeatedly; however, 24 h post repeated morphine administration, the expression of several cytokines was greatly increased. Tat further elevated levels of several cytokines and maraviroc pretreatment attenuated these effects. With the understanding that gap junctions may be involved in both HIV-Tat effects on opioid antinociception as well as tolerance, we investigated the role of gap junctions in opioid antinociceptive tolerance. We observed that carbenoxolone, a gap junction inhibitor, administered systemically attenuated the development of opioid antinociceptive tolerance. Furthermore, we observed a small percentage of carbenoxolone in brain tissue compared to the amount found in blood, suggesting a peripheral site of action. Finally, we show preliminary evidence that in vivo administration of carbenoxolone is able to attenuate tolerance to morphine in DRG neurons.