IL-15 is a pleiotropic cytokine which influences a variety of immune and inflammatory responses. Our lab has previously demonstrated that IL-15 and IL-15Rα promote atherosclerosis. However, the mechanisms by which IL-15 affect atherosclerosis development were not fully defined. In this study, we reported that overexpression of the IL-15 gene resulted in an increase of granzyme B level in the atherosclerotic plaque of ApoE deficient mice. Furthermore, we observed that leukocytes-specific genetic deletion of IL-15Rα reduced the granzyme B level within atherosclerotic lesions from LDLr deficient mice. Collectively, our data shows one of the mechanistic pathways by which IL-15 promotes atherosclerosis development. Moreover, we tested the role of IL-15 in carotid artery disease. It has been reported previously that immunization of low density lipoprotein receptor deficient mice against IL-15, by inoculating them with bacteria harboring an IL-15 expression plasmid, led to reduced development of diet induced atherosclerosis in carotid arteries whose diameter was restricted to induce a hemodynamic stress. Others, however, reported that injection of wild type mice with an antibody against IL-15 triggered increased neointima formation in carotid arteries that were partially ligated. In our study, we found no differences in the amount of collar/diet induced atherosclerosis in control apoE KO mice and in IL-15/apoE dKO mice.Therefore,inactivation of IL-15 gene expression does not appear to affect the rapid onset of atherosclerosis in carotid arteries of ApoE KO mice induced by a combination of high fat diet and hemodynamic stress. / Thesis / Master of Science (MSc)

Orientador: Maria Terezinha Serrão Peraçoli / Banca: Ângela Maria Victoriano de Campos Soares / Banca: Gil Benard / Resumo: A interleucina-15 (IL-15) é uma citocina pró-inflamatória produzida principalmente por monócitos e macrófagos em resposta a agentes infecciosos, desempenhando importante papel modulador na imunidade inata e adaptativa. O objetivo do presente trabalho foi avaliar o efeito da IL-15 sobre a atividade fungicida, metabolismo oxidativo e a produção de citocinas por monócitos humanos, infectados in vitro com cepa virulenta de Paracoccidioides brasiliensis (Pb18). Monócitos de sangue periférico, obtidos de indivíduos saudáveis, foram préincubados na ausência ou presença de IL-15 (12,5, 25 e 50 ng/mL) por 24 h a 37oC e infectados com Pb18 na proporção de 50 monócitos para uma célula fúngica durante 4 h e 18 h. A atividade fungicida de monócitos foi determinada após 4 h pela recuperação de fungos viáveis por plaqueamento das co-culturas em meio BHI-ágar. O metabolismo oxidativo foi avaliado pela liberação de peróxido de hidrogênio (H2O2) e de ânion superóxido (O2 -) nas culturas desafiadas com Pb18 e estimuladas com phorbol myristate acetate (PMA) durante 60 mim. A produção de fator de necrose tumoral-alfa (TNF-a), IL-6, IL-10 e IL-15 foi determinada por ensaio imunoenzimático (ELISA) nos sobrenadantes das coculturas obtidos após 4 e 18 h de incubação. Os resultados mostraram que a préincubação de monócitos com IL-15 induziu aumento significativo na atividade fungicida contra Pb18 de maneira dose-dependente, sendo esse efeito neutralizado pela adição de anticorpo monoclonal anti-IL-15. O tratamento com IL- 15 não interferiu na capacidade de liberação de H2O2 e O2 - por monócitos desafiados com Pb18 sugerindo que a atividade fungicida estimulada por IL-15 ocorre por mecanismos independentes do metabolismo oxidativo. Monócitos infectados com o fungo, na ausência de IL-15, produziram níveis de TNF-a, IL-6 e IL-10... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Interleukin-15 (IL-15) is a pro-inflammatory cytokine especially produced by monocytes and macrophages against infectious agents and that play a pivotal role in teh innatte and adaptive immune response. The aim of this study was to analyze the effects of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with a virulente strain of Paracoccidioides brasiliensis (Pb18). Peripheral blood monocytes obtained from healthy individuals were pre-incubated for 24h with or without human recombinant IL-15 (12.5,25 and 50 ng/mL), and then challenged with Pb18 in a ratio of 50:1 monocytes:fungi. Fungicidal activity of monocytes against Pb18 was assessed by viable fungi recovery from 4 h co-cultures after plating in BHI-agar. Oxidative metabolism was evaluated by hydrogen peroxide (H2O2) and superoxide anion (O2 -) release in the monocyte cultures challenged by Pb18 and stimulated with phorbol myristate acetate (PMA) for 60 min. Tumor necrosis factor-alpha (TNF-a), IL-6, IL-10 and IL-15 production by monocytes were determined in culture supernatants by enzyme immunoassay (ELISA). The results showed that IL-15 ehanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by additon of anti-IL-15 monoclonal antibody to the co-cultures. No significant effect of IL-15 on H2O2 and O2 - release by monocytes was observed suggesting that the fungicidal activity was independent of oxidative metabolism activation. Monocytes infected with P. brasiliensis in the absence of IL-15, produced significantly higher levels of TNF-a, IL-6 and IL-10 after 18 h of coculture in comparison to experiments of 4h-incubation with the fungus. The pretreatment of monocytes with IL-15 induced significant higher levels of TNF-a, IL-10 and IL-15 production by these cells challenged with the fungus... (Complete abstract click eletronic address below) / Mestre

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-15Bitstream added on 2014-06-13T18:50:11Z : No. of bitstreams: 1 bannwart_cf_me_botfm.pdf: 462065 bytes, checksum: 2e3988993f9db6f1ef7a8c8058e75e6a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A interleucina-15 (IL-15) é uma citocina pró-inflamatória produzida principalmente por monócitos e macrófagos em resposta a agentes infecciosos, desempenhando importante papel modulador na imunidade inata e adaptativa. O objetivo do presente trabalho foi avaliar o efeito da IL-15 sobre a atividade fungicida, metabolismo oxidativo e a produção de citocinas por monócitos humanos, infectados in vitro com cepa virulenta de Paracoccidioides brasiliensis (Pb18). Monócitos de sangue periférico, obtidos de indivíduos saudáveis, foram préincubados na ausência ou presença de IL-15 (12,5, 25 e 50 ng/mL) por 24 h a 37oC e infectados com Pb18 na proporção de 50 monócitos para uma célula fúngica durante 4 h e 18 h. A atividade fungicida de monócitos foi determinada após 4 h pela recuperação de fungos viáveis por plaqueamento das co-culturas em meio BHI-ágar. O metabolismo oxidativo foi avaliado pela liberação de peróxido de hidrogênio (H2O2) e de ânion superóxido (O2 -) nas culturas desafiadas com Pb18 e estimuladas com phorbol myristate acetate (PMA) durante 60 mim. A produção de fator de necrose tumoral-alfa (TNF-a), IL-6, IL-10 e IL-15 foi determinada por ensaio imunoenzimático (ELISA) nos sobrenadantes das coculturas obtidos após 4 e 18 h de incubação. Os resultados mostraram que a préincubação de monócitos com IL-15 induziu aumento significativo na atividade fungicida contra Pb18 de maneira dose-dependente, sendo esse efeito neutralizado pela adição de anticorpo monoclonal anti-IL-15. O tratamento com IL- 15 não interferiu na capacidade de liberação de H2O2 e O2 - por monócitos desafiados com Pb18 sugerindo que a atividade fungicida estimulada por IL-15 ocorre por mecanismos independentes do metabolismo oxidativo. Monócitos infectados com o fungo, na ausência de IL-15, produziram níveis de TNF-a, IL-6 e IL-10... / Interleukin-15 (IL-15) is a pro-inflammatory cytokine especially produced by monocytes and macrophages against infectious agents and that play a pivotal role in teh innatte and adaptive immune response. The aim of this study was to analyze the effects of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with a virulente strain of Paracoccidioides brasiliensis (Pb18). Peripheral blood monocytes obtained from healthy individuals were pre-incubated for 24h with or without human recombinant IL-15 (12.5,25 and 50 ng/mL), and then challenged with Pb18 in a ratio of 50:1 monocytes:fungi. Fungicidal activity of monocytes against Pb18 was assessed by viable fungi recovery from 4 h co-cultures after plating in BHI-agar. Oxidative metabolism was evaluated by hydrogen peroxide (H2O2) and superoxide anion (O2 -) release in the monocyte cultures challenged by Pb18 and stimulated with phorbol myristate acetate (PMA) for 60 min. Tumor necrosis factor-alpha (TNF-a), IL-6, IL-10 and IL-15 production by monocytes were determined in culture supernatants by enzyme immunoassay (ELISA). The results showed that IL-15 ehanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by additon of anti-IL-15 monoclonal antibody to the co-cultures. No significant effect of IL-15 on H2O2 and O2 - release by monocytes was observed suggesting that the fungicidal activity was independent of oxidative metabolism activation. Monocytes infected with P. brasiliensis in the absence of IL-15, produced significantly higher levels of TNF-a, IL-6 and IL-10 after 18 h of coculture in comparison to experiments of 4h-incubation with the fungus. The pretreatment of monocytes with IL-15 induced significant higher levels of TNF-a, IL-10 and IL-15 production by these cells challenged with the fungus... (Complete abstract click eletronic address below)

Interleukin 15 has great application potential such as in the biological treatment of cancer. It is involved in a variety of immunological processes, the most important of these involve influencing and induction of NK cells and T-lymphocytes proliferation. However, its therapeutic usages are limited by a low stability and short half-life. For this reason, there are various approaches of stabilization and expansion of its biological activity being explored. In this work, we analysed and developed a new approach, which uses viral nanostructures derived from major capsid VP1 protein of mouse polyomavirus as a carrier of IL-15. Moreover, VP1 proteins can be relatively easily modified and they are also capable to penetrate into the tumour cells. There were prepared two variants of IL-15 together with control nanostructures in the baculovirus expression system, one was composed of IL-15 and the other of the IL-15 fusion protein and truncated variant of VP1. Protein constructs were characterized by electron microscopy and biochemical methods. The total protein yield of VP1ΔC-IL15-HIS fusion variant was higher (up to 53 mg/L of complete medium) than IL-15 alone (8,5 mg/L). However, testing of the biological activity of the prepared proteins in vitro did not show any induction of proliferation on Jurkat...

No description available.

Skeletal muscle can perceive cellular energy status and substrate availability and demonstrates remarkable plasticity in response to environmental changes. Nonetheless, how skeletal muscle and its resident stem cells (satellite cells; SCs) sense and respond to nutrient flux remains largely undefined. The dynamic post-translational modification O-GlcNAcylation has been shown to serve as a cellular nutrient sensor in a wide range of cells and tissues, yet its role in skeletal muscle and SCs remains unexplored. Here, we ablated skeletal muscle O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, and found the knockout mice exhibited enhanced glucose uptake, insulin sensitivity, and resistance to high-fat diet induced obesity. Additionally, mKO mice had a 3-fold increase in circulating levels of interleukin-15 (IL-15), a potent anti-obesity cytokine, potentially through epigenetic regulation of Il15 by OGT. To further investigate if there was a causal relationship between OGT ablation and the lean phenotype, we generated muscle specific OGT and interleukin-15 receptor alpha (IL-15ra) double knockout mice (mDKO). As a result, mDKO mice had blunted IL-15 secretion and minimal protection against HFD-induced obesity. Together, these data indicate the skeletal muscle OGT-IL15 axis plays an essential role in the maintenance of skeletal muscle and whole-body metabolic homeostasis. As satellite cells (SCs) play an indispensable role in postnatal muscle growth and adult regenerative myogenesis, we investigated the role of O-GlcNAcylation in SC function. To this end, we conditionally ablated OGT in SCs (cKO) and found cKO mice had impaired SC proliferation, in vivo cycling properties, population stability, metabolic regulation, and adult regenerative myogenesis. Together these findings show that SCs require O-GlcNAcylation, presumably to gauge nutritional signals, for proper function and metabolic homeostasis. Another critical yet often neglected player in myogenesis are mitochondria. Traditionally depicted as a power plant in cells, mitochondria are critical for numerous nonconventional, energy-independent cellular process. To investigate the role of both mitochondrial energy production and alternative mitochondrial functions in myogenic regulation, we ablated ATP synthase subunit beta (ATP5b) and ubiquinol-cytochrome c reductase (UQCRFS1) in C2C12 myoblasts to disrupt mitochondrial ATP production and mitochondrial membrane potential, respectively. Ablation of UQCRFS1, but not ATP5b, impaired myoblast proliferation, although lack of either gene compromised myoblast fusion. Interestingly, addition of the potent myogenic stimulator IGF-1 rescued ATP5b fusion but could not override UQCRFS1 knockout effects on proliferation or differentiation. These data demonstrate mitochondrial ATP production is not the "metabolic switch" that governs myogenic progression but rather an alternative mitochondrial function. In summary, skeletal muscle and their resident stem cell population (SCs) both use O-GlcNAcylation, feasibly to sense and respond to nutritional cues, for the maintenance of metabolic homeostasis and normal physiology. A deeper understand of both muscle and SC metabolic regulation may provide therapeutic targets to improve global metabolism and muscle growth. / Doctor of Philosophy / Skeletal muscle is responsible for approximately 20% of basal energy expenditure and 70-90% of insulin-mediated glucose disposal, and as such changes in skeletal muscle metabolism and insulin sensitivity have profound impacts on whole body metabolism. Skeletal muscle is a plastic tissue that can perceive nutrient availability, which permits metabolic adaptations to environmental changes. Deletion of the nutrient sensing pathway O-GlcNAcylation in skeletal muscle (mKO) protected mice from high-fat diet induced obesity and ameliorates whole-body insulin sensitivity. Skeletal muscle can secrete myokines to elicit endocrine effects on other tissues in the body, and as such, we proposed perturbation of this nutrient sensing pathway in skeletal muscle alters myokine secretion to elicit responses in other metabolically active tissues to support its energy requirements. Indeed, circulating levels of interleukin-15, a potent anti-obesity myokine, increased 3-fold in mKO mice. To determine the contribution of IL-15 to the mKO phenotype, we used a genetic approach to blunt IL-15 secretion from skeletal muscle (mDKO), which partially negated the lean mKO phenotype. Our findings show the ability of skeletal muscle to "sense" changes in nutrients through O-GlcNAcylation is necessary for proper muscle and whole-body metabolism. Moreover, this nutrient sensing mechanism is also important for proper muscle stem cell function, also known as satellite cells (SCs). Loss of O-GlcNAcylation in SCs impairs their ability to regenerate muscle after injury, which can be attributed to a reduced capacity to proliferate and an inability to maintain a healthy SC population. Interestingly, SCs lacking O-GlcNAcylation have a greater mitochondrial content. Using a myoblast cell line, we investigated the contribution of mitochondria to myogenesis, the formation of muscle, and found mitochondrial energy production is dispensable in the myogenic process. Our studies show skeletal muscle and SCs rely on highly integrated signaling cascades that sense and respond to intrinsic metabolic changes and extrinsic nutritional cues to function properly.

Orientador: Valéria Marçal Felix de Lima / Resumo: A Leishmaniose Visceral (LV) é causada nas Américas, pelo protozoário intracelular obrigatório Leishmania infantum e os cães domésticos são os principais reservatórios urbanos do parasita e em áreas endêmicas, o aumento da LV em humanos tem sido associado ao aumento da infecção canina. Os atuais medicamentos disponíveis para a Leishmaniose Canina (CanL) não são completamente eficientes e meses após o tratamento a maioria dos cães apresentam recidiva, indicando a necessidade de buscar formas alternativas de tratamento. Na CanL, cães desenvolvem uma resposta imune celular (Th1) ineficiente para combater o parasita e a estimulação das vias de citocinas em células de defesa com proteínas recombinantes, tem o potencial de se tornar parte de métodos imunoterapêuticos eficazes. Neste estudo, as citocinas recombinantes caninas (IL-12, IL-2, IL-15 e IL-7) e o receptor solúvel de IL-10R1 (sIL-10R1), com atividade antagonista, foram avaliadas pela primeira vez em combinações (IL-12/IL- 2, IL-12/IL-15, IL-12/sIL-10R1, IL-15/IL-7) ou isoladamente (sIL-10R1) quanto à capacidade imunomodulatória em células mononucleares do sangue periférico (sigla em inglês PBMC) de cães com leishmaniose. Todas as combinações de proteínas recombinantes testadas mostraram melhorar a resposta linfoproliferativa. Além disso, as combinações de IL-12/IL-2 e IL-12/IL-15 promoveram a diminuição na expressão da proteína “Programed Cell Death 1” (PD-1) nos linfócitos. Estas mesmas combinações de citocinas e IL-12/sI... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral Leishmaniasis (LV) is caused in the Americas by the obligate intracellular protozoan Leishmania infantum and domestic dogs are the major urban reservoirs of the parasite and in endemic areas, and increase LV in humans has been associated with increased canine infection. The current medications available for Canine Leishmaniasis (CanL) are not completely effective and months after treatment most dogs present with relapse, indicating the necessity to looking for alternative forms of treatment. In CanL, dogs develop an ineffective cellular immune response (Th1) to combat the parasite. Then, the stimulation of cytokine pathways in defense cells with recombinant proteins, has the potential to become part of effective immunotherapeutic methods. In this study, the canine recombinant cytokines (IL-12, IL-2, IL-15 and IL-7) and the soluble receptor of IL-10R1 (sIL-10R1) with antagonistic activity, were evaluated for the first time in combinations IL-12/IL-2, IL-12/IL-15, IL12/sIL-10R1, IL-15/IL-7) or alone (sIL-10R1) for their immunomodulatory capacity in peripheral blood mononuclear cells (PBMC) from dogs with leishmaniasis. All combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, combinations of IL-12/IL-2 and IL-12/IL-15 promoted decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and IL-12/casIL-10R1 induced IFN-y production in PBMC. Furthermore, the combinat... (Complete abstract click electronic access below) / Mestre

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 189 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-15T16:35:40Z No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) / Made available in DSpace on 2013-10-15T16:35:40Z (GMT). No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A maioria dos cães com leishmaniose visceral submetida à quimioterapia convencional apresenta recaída após a interrupção do tratamento, talvez, pelo fato desses animais desenvolverem resposta imune celular específica apenas transitoriamente. Uma vez que as citocinas IL-7 e IL-15 são descritas na literatura como sendo capazes de promover resposta imune celular de longa duração (memória), o presente trabalho teve como objetivo a clonagem, a expressão e a avaliação da atividade biológica de IL7 e IL-15 recombinantes caninas (rca-IL-7 e rcaIL-15). Diversos estudos têm demonstrado o papel essencial da IL-7 e IL-15 na homeostase de células-T. A Interleucina-7 é um fator de crescimento e anti-apoptótico de linfócitos T, sendo essencial para a sobrevida de células T maduras, naïve e células de memória, especialmente CD4+. IL-15 apresenta um amplo espectro de atividades biológicas. É crucial para o desenvolvimento, proliferação, sobrevivência e diferenciação de múltiplas células tanto da imunidade inata, quanto da imunidade adaptativa, e apresenta papel essencial na manutenção de células T CD8+ de memória. Nesse trabalho descreveu-se a clonagem do DNA complementar (cDNA) e a expressão de IL-7 e IL-15 recombinantes caninas biologicamente ativas em Escherichia coli. Para expressão em E. coli foram realizadas as clonagens do cDNA de IL-7 e de IL-15 no vetor pRSET, gerando as construções pRSET-caIL-7 e pRSET-caIL-15. O sucesso da clonagem em ambos os vetores de expressão foi confirmado a partir do sequenciamento. As proteínas foram expressas como proteína de fusão com seis moléculas de histidina na extremidade amina da cadeia polipeptídica e após serem solubilizadas com uréia, as proteínas foram purificadas por cromatografia de afinidade e renaturadas por diálise. A avaliação da atividade biológica de IL-15 canina purificada foi realizada em células CTLL-2 e foi demonstrada uma relação dose dependente na faixa de concentração proteica de 0,9 ng/mL até 1 µg/mL. O mesmo efeito foi demonstrado quando essas células foram cultivadas na presença de diferentes diluições de T-STIM (controle positivo do ensaio). Visando determinar as condições em que rca-IL-7 e rca-IL-15 são capazes de induzir efeitos biológicos in vitro, foram realizados experimentos com diferentes doses de ambas as citocinas e demonstrado pela primeira vez que tanto IL-7 quanto IL-15 são capazes de induzir a proliferação de CMNSP de cães pré-ativadas com PHA. Adicionalmente, as citocinas foram avaliadas quanto à capacidade de estimulação da proliferação de CMNSP de cães sem estímulo prévio ou concomitante e foi possível observar que rca-IL-7 foi capaz de manter a proliferação dessas células por até 12 dias de cultivo, enquanto rca-IL-15 foi capaz de fazê-lo por até 14 dias. Diante dos resultados obtidos a rca-IL-7 e rca-IL-15 poderão ser utilizadas no futuro em estudos que visam o estabelecimento de uma resposta de longa duração em cães com LVC submetidos à quimioterapia convencional. / Most dogs with LV subjected to conventional chemotherapy has relapsed after discontinuation of treatment, possibly due these animals develop specific cellular immune response transiently. Since IL-7 and IL-15 are described in the literature asbeing capable of promoting cellular immune response of long-term (memory), this study aimed at cloning, expression and biological activity evaluation of rca-IL7 and rca IL-15. Many studies have demonstrated the essential role of IL-7 and IL- 15 in the homeostasis of T-cells. The Interleukin-7 is a growth and anti-apoptotic factor of T lymphocytes, essential for the occurrence of mature, naive, and memory T cells, especially CD4+. IL-15 presents a wide spectrum of biological activities. It is crucial for the development, proliferation, survival, and differentiation of multiple cells of innate immunity as well as of adaptive immunity, and presents an essential role in the maintenance of CD8+ memory T cells. This work describes the cloning of complementary DNA (cDNA) and the expression of IL-7 and IL-15 canine recombinants biologically active in Escherichia coli. For expression in E. coli, we cloned cDNA of IL-7 and IL- 15 in the vector pRSET, generating the constructions pRSET-caIL7 and pRSETcaIL- 15. The success of the cloning in both the expression vectors was confirmed from the sequencing. The proteins were expressed as fusion protein with six molecules of histidine in the amine extremity of the polypeptide chain and afterwards were solubilized with urea, the proteins were purified by affinity chromatography and renatured by dialysis. The evaluation of the biological activity of purified canine IL-15 was achieved in CTLL-2 cells and a dose-dependent relation in the range of protein concentration of 0.9 ng/mL to 1 ƒÊg/mL was demonstrated. The same effect was demonstrated when these cells were cultivated in the presence of different dilutions of T-STIM (positive control of the test). With the goal of determining the conditions in which rca-IL-7 and rca-IL-15 are capable of inducing biological effects in vitro, we carried out experiments with different doses with both the cytokines and demonstrated for the first time that IL-7 as well as IL- 15 are capable of inducing proliferation of PBMC of dogs pre-activated with PHA. Also, the cytokines were evaluated as to the capacity of stimulation of the proliferation of PBMC of dogs without previous or concomitant stimulation and it was possible to observe that rca-IL-7 was capable of maintaining the proliferation of these cells for up to 12 days of cultivation, while rca-IL-15 was capable of doing it for 14 days. Considering the results obtained rca-rca-7 and IL-IL-15 may be used in future studies that aim to establish a long-termresponse in dogs with CVL undergoing conventional chemotherapy

Orientador: Maria Heloisa Souza Lima Blotta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T00:09:24Z (GMT). No. of bitstreams: 1 Soares_LannyCristinaBurlandy_D.pdf: 1993850 bytes, checksum: c3f425ae548a166847fe87f0b24ad2b0 (MD5) Previous issue date: 2008 / Resumo: Em doenças causadas por microorganismos intracelulares como a tuberculose, criptococose e listeriose foi demonstrado que as células T CD8+ contribuem de forma relevante para o controle da infecção. Em trabalho anterior verificamos um aumento do número de linfócitos T CD8+ no lavado broncoalveolar de pacientes com paracoccidioidomicose (PCM) pulmonar, sugerindo um papel para estas células, cuja ação efetora se dá por meio da produção de citocinas como o IFN-? e atividade citotóxica. O presente estudo teve por objetivo verificar o estado de ativação e a produção de moléculas citotóxicas por linfócitos do sangue periférico (CD4+ e CD8+) de pacientes com PCM, indivíduos com PCM-Infecção e controles, tanto ex-vivo como in vitro, após estimulação com leveduras de P. brasiliensis. A expressão dos marcadores de ativação e moléculas citotóxicas foi avaliada por citometria de fluxo. A análise ex-vivo mostrou que, de maneira geral, os pacientes apresentam menor freqüência de células positivas para moléculas citotóxicas (granzima A, B e perforina), em relação aos indivíduos com PCM-infecção. A estimulação com leveduras de P. brasiliensis levou a um aumento discreto de células ativadas (CD69+) e uma redução na expressão de grânulos citotóxicos. A adição de IL-15 às culturas mostrou elevação da freqüência de células CD69+ apenas no grupo com PCM-infecção e controles. Já as células T CD4+ e CD8+ dos pacientes foram ativadas apenas na ausência do fungo. O efeito da adição de IL-15 na expressão dos grânulos foi pouco expressivo em relação à granzima A e B, mas maior freqüência de células CD8+perforina+ foi observada em indivíduos com PCM-infecção, em relação aos pacientes. Menor expressão do receptor para IL-15 (IL-15Ra) foi detectada em células T CD4+ de pacientes com PCM comparada ao grupo PCM-infecção e aos controles. A dosagem da granulisina sérica pela metodologia de ELISA mostrou níveis inferiores nos pacientes com PCM, comparado aos outros grupos. Além disso, os resultados mostraram uma tendência a um aumento de granulisina nos pacientes após terapia antifúngica. Em conjunto os resultados mostraram que os linfócitos de pacientes com PCM encontram-se em um estado de menor ativação, expressam menores quantidades do receptor para IL-15 e produzem níveis basais de grânulos citotóxicos (granzima A, B, perforina e granulisina). Estes fatores, ao lado de outros mecanismos que comprometem a imunidade celular, poderiam resultar em atividade citotóxica deficitária e, portanto, menor capacidade de lisar o fungo / Abstract: CD8+ T cells play a pivotal role in host defense against diseases caused by intracellular pathogens such as tuberculosis, cryptococcosis and listeriosis. In a previous study we verified an increased number of T CD8+ cells in bronchoalveolar lavage of patients with pulmonary paracoccidioidomycosis (PCM), suggesting a role for them in the local immune response. CD8+ T cells effector functions include cytokines production, mainly IFN-? and cytotoxic activity. The aims of this study were to verify the activation state as well as the production of cytotoxic molecules by peripheral blood lymphocytes (CD8+ and CD4+) from patients with PCM, individuals with PCM-infection and controls, both ex-vivo and in vitro after stimulation with P. brasiliensis yeast cells. The expression of activation and cytotoxic molecules was evaluated by flow cytometry. The ex-vivo analysis showed that, in general, the patients presented a lower frequency of granzime A, B and perforin-positive cells as compared to PCM-infection individuals. P. brasiliensis yeast cells stimulation led to a discrete increase in CD69+ cells and a reduction in cytotoxic granules expression in all groups. The addition of IL-15 to the cultures induced an increase in the frequency of CD69+ cells only in individuals with PCM-infection and controls. Differently, CD8+ and CD4+ T cells from PCM patients were activated only in the absence of fungal cells. The effect of IL-15 in granzyme A and B expression was low but a higher frequency of CD8+perforin+ was detected in individuals with PCM-infection than in patients with PCM. IL-15Ra expression was lower in CD4+ T cells from patients in relation to individuals with PCM-infection and controls. The detection of granulysin levels by ELISA showed lower levels in PCM patients than in individuals with PCM-infection and controls. Moreover, a tendency to a rise in granulysin levels was observed after antifungal therapy. Altogether the results showed that lymphocytes from PCM patients are poorly activated, express low levels of IL-15Ra and produce basal levels of cytotoxic granules (granzyme A, B, perforin and granulysin). These findings, in addition to other mechanisms that impair cellular immunity, may account to defective cytotoxic activity and consequently low capacity to kill the fungus / Doutorado / Ciencias Medicas / Doutor em Ciências Médicas