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Elevated levels of the inflammatory cytokine interleukin-6, IL-6, have been linked with poor prognosis in ovarian cancer patients by influencing tumour growth, invasion, angiogenesis and chemo-resistance. A clinical trial conducted in parallel with pre-clinical studies showed an anti-IL-6 antibody to have some activity in ovarian cancer patients and in xenograft models, via reduction in pro-inflammatory and angiogenic factors such as TNF-α, IL-8 and VEGF. Anti-IL-6 treatment also showed significant reductions in vascular area with decreased expression of an angiogenic factor Jagged1. The aim of my study was to investigate the effects of IL-6 on normal and tumour angiogenesis. I found that recombinant IL-6 stimulates angiogenesis in mouse and rat aortic ring assays and that it can also stimulate growth and migration of endothelial cells in vitro. IL-6 has similar potency as VEGF in inducing vessel sprouting. IL-6 itself does not induce VEGF in the endothelial cells I tested. Investigation of the effects of IL-6 on vessel maturation revealed a significant reduction in pericyte coverage of vessels treated with IL-6 compared with VEGF. Collectively, these data led to my hypothesis that ‘IL-6 drives aberrant angiogenesis, independent of VEGF signalling’. Investigating the mechanism by which IL-6 drives angiogenesis and leads to defective pericyte formation, I showed a link between IL-6 and the Notch ligands, Jagged1 and DLL4. My data suggested that IL-6 could stimulate Jagged1 in endothelial cells, whereas VEGF induces DLL4, the Notch ligand known to be involved in inducing stalk phenotype. Exploring previous findings to get a better understanding of the interaction of Notch ligands and pericyte recruitment also suggested a role of Angiopoeitin-2 in relation to IL-6 signalling. These observations were extended in IGROV-1 ovarian cancer xenografts treated with an anti-IL-6 antibody and by analysis of gene expression datasets from ovarian cancer biopsies. My results suggest therapeutic potential of combining inhibitors of IL-6 and VEGF in ovarian cancer.

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3-/-) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3-/- and WT mice were subjected to myocardial ischemia (45min) followed by reperfusion for up to 3days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3-/- mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3-/- mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.

Dysregulation between pro- and anti-inflammatory cytokines activity in rheumatoid arthritis (RA) contributes to immune dysregulation, chronic inflammation and subsequent joint destruction. Interleukin-37 (IL-37) has been described as an anti-inflammatory cytokine in several autoimmune diseases. The main aim of this work was to determine the levels of IL-37 in serum and synovial fluid (SF) of RA patients and to compare them with the levels in patients with osteoarthritis (OA) and further explore the association of IL-37 with disease activity and other clinical parameters. Subsequent goal was to study its anti-inflammatory function on RA synovial fibroblasts and describe other cells types of synovial tissue contributing to its production. IL-37 levels were detected using enzyme-linked immunosorbent assay (ELISA). Synovial fibroblasts were stimulated by lipopolysaccharide (LPS) and recombinant IL-37 (rIL-37). The levels of studied genes were detected by PCR. Synovial tissues and immune cells were visualized by immunohistochemical and by immunofluorescence staining. We found increased levels of IL-37 in SF of patients with RA in comparison to OA patients. There was a significant correlation between serum and SF levels of IL-37. RA as well as OA patients showed increased levels of IL-37 in serum than in...

<p>  </p> <p>Osteoarthritis (OA) is prevalent in equine and can be career-ending for performance horses due to lameness limitations and decreased quality of life. OA is a progressive, multifactorial disease that compromises the synovial joints' normal function, resulting in subchondral bone and articular cartilage deterioration over time. OA is a complex disease that impacts the entire joint, wherein activation of the innate immune system has an essential role in the disease progression and the development of pain. The synovial membrane, or the synovium, is a crucial contributor to the inflammation of diseased joints, regardless of the intra-articular tissue type initially affected. Synoviocytes are a predominant cell type of the synovium and contribute to inflammation by releasing key mediators and degradative enzymes, such as interleukin (IL)-6, IL-1β, a disintegrin, and metalloproteinase (ADAM) domains, and matrix metalloproteinases (MMPs). The production of pro-inflammatory molecules sequentially influences the expression of degradative enzymes and cartilage destruction. Therefore, the pathophysiological processes within synovial joints afflicted by OA can be further understood by studying the characteristics of synoviocytes.</p> <p>We aimed to investigate the inflammatory component of OA in an <em>in vitro</em> model using a primary cell line of equine fibroblast-like synoviocytes (eqFLS) stimulated with tumor necrosis factor-alpha (TNF-α) to represent an initial inflammatory stimulus. Our studies have shown that stimulating eqFLS with TNF-α for 24 hours significantly increased the gene expression of pro-inflammatory biomarkers. Among several pro-inflammatory candidate genes assayed, only pro-inflammatory cytokine IL-6 gene expression could be detected reproducibly following stimulation with the TNF-α gene in eqFLS. We characterized the pro-inflammatory response of eqFLS and utilized this system to examine the impact of novel therapeutic compounds designed <em>in-silico</em> with the goal of reducing the inflammatory response of eqFLS. A piperazine-based compound (C3) and its derivative (02-09) were primarily designed to mimic the interactions of the growth factor pigment epithelium-derived factor (PEDF) with its receptor, the non-integrin laminin receptor 1 (LAMR1). Based on previous <em>in vitro</em> studies in the laboratory, C3 and 02-09 had been proposed to have a strong potential for inhibiting inflammation while reducing angiogenesis and chondrocyte hypertrophy. The efficacy of these two novel compounds on eqFLS was examined in the present work by assessing the gene expression levels of inflammatory biomarkers, including IL-6, IL-1β, IL-8, ADAMs, and MMPs relative to a control housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in various study designs. An <em>in-vitro</em> screen with the IL-1β promoter driving a reporter green fluorescent protein (GFP) was also designed to detect and track the inflammatory response of eqFLS by imaging following stimulation with or without (+/-) TNF-α relative to controls. This screen will be utilized in future studies to potentially identify more effective compounds in the LAMR1-interacting series. The current findings suggest that the novel compounds, especially 02-09, might exhibit an anti-inflammatory effect on eqFLS; therefore, it is a potential therapeutic agent in modulating inflammation during OA development. </p> <p><br></p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24880号 / 医博第5014号 / 新制||医||1068(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 長船 健二, 教授 江木 盛時, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Skeletal muscle is a primary contributor to body mass and whole-body energy metabolism. It is an adaptive tissue with the ability to fluctuate in size and mechanical properties in response to stimulus. Health conditions involving chronic elevated inflammation levels often feature metabolic inflexibility and losses in skeletal muscle mass. Mononuclear stem cells, termed satellite cells, are mitotic and serve to donate nuclei to muscle fibers to enable skeletal muscle adaptation. Despite the well-characterized nature of satellite cell activation, proliferation, and differentiation, the underlying mechanisms regulating this process is not fully understood. Recent characterization of cytokines secreted by skeletal muscle in an endocrine type fashion has led to discoveries of inflammatory cytokines influencing satellite cell function. However, how the autocrine production and secretion of these cytokines during proliferation and differentiation in humans and their correlation with myogenic transcription factors is not well understood. Our study used satellite cells cultured from the vastus lateralis of 12 male human research subjects, and ELISA analysis to measure levels of TNF-α and IL-6 across proliferation, early differentiation, and late differentiation. Additionally, mRNA levels of Pax7, MyoD, myogenin, IL-6, TNF-α, and TGF-β were assessed in satellite cells cultured from a subset of two endurance trained and two sedentary individuals from the larger group of 12 human subjects. The novelty of our study is the large number of human research subjects and simultaneous analysis of inflammatory cytokine secretion, mRNA inflammatory cytokine expression, and myogenic transcription factor mRNA expression. Results showed an 83% decrease in IL-6 protein secretion 24 hours after exposure to differentiation media (p-value <0.05) before increasing 50-fold after 7 day of exposure to differentiation media (p-value < 0.05). Myogenin and TGF-β mRNA expression levels were positively correlated (R2 = 0.5814, p-value < 0.0001). A negative correlation was found between IL-6 and MyoD (R2 = 0.2473, p – value = 0.0257). After 1 day of exposure to differentiation media, satellite cells from endurance trained subjects exhibited higher levels of TGF-β mRNA expression compared to sedentary satellite cells of sedentary subjects of the same age and levels of adiposity (p-value < 0.05). Results support a potential relationship in humans satellite cells between myogenic transcription factors and inflammatory cytokines, however, further study is necessary in order to investigate the underlying mechanisms behind the correlations. / Master of Science / Skeletal muscle is responsible for conscious, voluntary movement. In addition, the tissue is responsible for the majority of energy expenditure in the human body. Skeletal muscle is able to adapt to exercise programs through the fusion of undifferentiated stem cells – called satellite cells – in the skeletal muscle fiber. In long-term diseased conditions, the immune response involves chronic rises in inflammation and results in the loss of skeletal muscle and corresponding loss of ability to move. A shorter rise in inflammation is also linked with the positive exercise response. Our study features satellite cells harvested from muscle samples of 12 male human research participants. We were interested in evaluating the relationships between the expression and secretion of two proteins associated with inflammation and regulation of the satellite cell cycle. The two proteins of interest in our study are tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We also measure the gene expression of another inflammatory protein, transforming growth factor beta (TGF-β). In order to know where the cells were in their life cycle, we measured expression of genes associated with the division (Pax7), early fusion (MyoD), and late fusion of satellite cells (myogenin). Our study found a decrease in IL-6 secretion and expression as the process of satellite cells turning into muscle fibers was initiated. Additionally, a 50-fold increase in IL-6 expression was found at day 7 compared to day 0 of the satellite cell cycle. Additionally, we found a positive correlation between TGF-β and myogenin and a negative correlation between IL-6 and MyoD. Although we found correlations between satellite cell cycle genes and inflammation genes, more research is necessary to see if there is a pathway causing this relationship.

Made available in DSpace on 2016-08-10T10:38:42Z (GMT). No. of bitstreams: 1 CELINA KASSUMI KUNIEDA SUZUKI.pdf: 2013145 bytes, checksum: c807ebe49c872194bb7af7cd6bb5d17f (MD5) Previous issue date: 2012-06-29 / The increased prevalence of childhood obesity in recent years suggests the existence of a genetic predisposition or susceptibility to conditioning factors for obesity which act on the environmental factors related to lifestyle involving diet and physical activity. Food education programs and physical activities have been developed in order to reverse this situation. The chewing is considered one of the early stages of the digestive process and therefore primordial important because performing correctly promotes natural hunger satiety as well as a healthy digestion. The aim of this study is to investigate the polymorphism of the TNF-&#945; and DRD2 genes and the influence of masticatory profile in childhood obesity to enable the preparation of proposals for more effective interventions to control weight gain. For this purpose the study has been divided into two articles. ARTICLE 1: This study evaluated the profile of the obese child chewing through survey and evaluation of clinical history miofunctional from the protocols of MgBr and AMIOFE-A (attached) in 11 normal children representing the control population (Group I) and 16 obese children (Group B) participating in the CAIS Group of Obesity in Goiânia Municipal and School of Clinical Speech Therapy PUC Goiás aged between 6 and 13 years for both sexes. According to the findings obtained in the research, it is concluded that obese group (OB) presents an incision chewing food made with the central and lateral incisors with the majority labial sealing with or without excessive movement, unilateral pattern, with higher presence of altered behaviors in chewing and shorter chewing time. ARTICLE 2: This study evaluated the genetic polymorphism of TNF-&#945; gene (G308A) and DRD2 (Taq1&#945; - C32806T) in 27 patients with childhood obesity (16) and controls (11) using the method ARMS-PCR and PCR-RFLP, respectively, in peripheral blood samples. The results suggest that the genetic polymorphism of Taq1&#945; (C32806T) in the DRD2 gene was associated with the increase of obesity in the childhood. Still genetic variants of the SNP G308A TNF-&#945; gene is not possible to confirm it s influence on children s weight gain. Understanding how genetic variations affect the tendency to become or remain obese is an important step to comprehend the mechanisms that lead to obesity. / O aumento da prevalência da obesidade infantil nestes últimos anos sugere a existência de predisposição ou suscetibilidade genética como fatores condicionantes para a obesidade, sobre a qual atuam fatores ambientais relacionados ao estilo de vida, que envolvem hábitos alimentares e atividade física. Programas de educação alimentar e de atividades físicas foram desenvolvidos visando reverter este quadro. A mastigação é considerada uma das fases iniciais do processo digestório e de primordial importância, pois quando realizada de forma correta, promove a saciedade natural da fome, bem como uma digestão saudável. O objetivo deste estudo foi investigar o polimorfismo dos genes TNF-&#945; e DRD2 e a influência da mastigação na obesidade infantil para possibilitar a elaboração de propostas de intervenções mais eficazes para o controle do ganho de peso Para esta finalidade, o estudo foi dividido em dois artigos. ARTIGO 1: Este estudo avaliou o perfil mastigatório da criança obesa, por meio de levantamento de histórico clínico e avaliação miofuncional orofacial a partir dos protocolos do MGBR e AMIOFE-A (anexos), em 11 crianças eutróficas representando a população controle (Grupo EU) e 16 crianças obesas (Grupo OB) participantes de Grupo de Obesidade dos CAIS Municipais da cidade de Goiânia e da Clínica- Escola de Fonoaudiologia da PUC Goiás, com idade entre 6 e 13 anos de ambos os sexos. Conforme os achados obtidos na pesquisa, conclui-se que o grupo obeso (OB) apresenta uma mastigação com incisão do alimento feito com os incisivos centrais e laterais, sendo a maioria com vedamento labial sem ou com movimentação excessiva, padrão unilateral, com maior presença de comportamentos alterados na mastigação e tempo de mastigação reduzido. ARTIGO 2: Este estudo avaliou o polimorfismo genético dos genes TNF-&#945; (G308A) e DRD2 (Taq1&#945; - C32806T) em 27 pacientes com obesidade infantil (16) e de controle (11), utilizando o método de ARMS-PCR e PCR-RFLP, respectivamente, em amostras do sangue periférico. Os resultados sugerem que o polimorfismo genético em Taq1&#945; (C32806T) no gene DRD2 apresentou associação com o incremento na obesidade infantil. Por outro lado, as variantes genéticas do SNP G308A do gene TNF-&#945; não permitiram confirmar sua participação no ganho de peso em crianças. Entender como variações genéticas afetam a obesidade consiste em um passo importante na compreensão dos mecanismos desencadeadores da obesidade.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-17T11:14:30Z No. of bitstreams: 2 Dissertação - Barbara Hess Rodrigues Gonçalves - 2016.pdf: 1742093 bytes, checksum: 80bd9955d0b421c59b1e6faf6d41f773 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-17T11:14:56Z (GMT) No. of bitstreams: 2 Dissertação - Barbara Hess Rodrigues Gonçalves - 2016.pdf: 1742093 bytes, checksum: 80bd9955d0b421c59b1e6faf6d41f773 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-17T11:14:56Z (GMT). No. of bitstreams: 2 Dissertação - Barbara Hess Rodrigues Gonçalves - 2016.pdf: 1742093 bytes, checksum: 80bd9955d0b421c59b1e6faf6d41f773 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Canine atopic dermatitis (CAD) is the second most frequent disease in dermatological clinical routine of dogs. It is defined as a pruritic allergic skin disease, with genetic predisposition and clinical features, being related to the response to environmental allergens. Interleukin 31 (IL) 31 is a cytokine that participates in inflammatory processes and it is associated with pruritic diseases, especially those involving chronic inflammation such as allergic dermatitis. Produced by mononuclear cells, IL-31 is described to play an important role in the pathogenesis of atopic dermatitis. Two studies were performed in order to correlate clinical features, histopathological changes and the presence of IL-31 in the skin of dogs with atopic dermatitis. 34 dogs were selected from clinical routine, which 31 animals were diagnosed with CAD and three were healthy. The animals were evaluated for pruritus level by the owners' report and by clinical examination according to the CAD extent and severity index (CADESI-4). The dogs were grouped in discreetly, moderately and markedly compromised by CAD following the sum of scores’ values assigned in the clinical examination. Cutaneous samples from the axillary and interdigital regions of each dog were collected and submitted to histopathological (HE and toluidine blue) and immunohistochemical analyzes (IL-31). There was a correlation between the clinical score and the microscopic changes. As well, there was correlation among all the microscopic changes, but not between the degree of pruritus and the clinical score of CAD. Also, the inflammatory infiltrate was more intense in the axillary region in relation to interdigital skin. An increased numbers of cells immunostained for IL-31 was observed in dogs severely compromised by CAD. There was a correlation between the clinical score and the amount of interdigital mast cells, with an amount of cells immunostained for IL-31 in the axilla. It was also verified correlation between the amount of mast cells and cells immunostained for IL-31 in the axilla, as well as between acanthosis and all other histopathological alterations of the skin in the axillary region. We concluded that there is a correlation between severity of dermathological lesions, IL-31 immunostaining, mast cell count and histopathological changes in dogs with atopic dermatitis, but there is no correlation between the degree of pruritus reported by owners and severity of cutaneous lesions in animals with CAD. Moreover, the intensity of the inflammatory process may vary depending on the anatomical site of the lesion. / A dermatite atópica canina (DAC) é a segunda doença mais incidente na rotina de atendimento clínico dermatológico de cães. É definida como doença cutânea alérgica pruriginosa, de predisposição genética e características clínicas definidas, estando relacionada a resposta a alérgenos ambientais. A interleucina 31 (IL) 31 é uma citocina que participa de processos inflamatórios e está associada a doenças pruriginosas, principalmente as que envolvem inflamação crônica como as dermatites alérgicas. Produzida por células mononucleares, a IL-31 é descrita por desempenhar papel importante na patogênese da dermatite atópica.Com o objetivo de correlacionar as alterações clínicas, histopatológicas e a presença da IL-31 na pele de cães com dermatite atópica foram realizados dois estudos. Para isso, foram selecionados 34 cães da rotina de atendimento clínico, sendo 31 com diagnóstico de DAC e três hígidos. Os animais foram avaliados quanto ao nível de prurido a partir do relato de seus proprietários e quanto ao índice de extensão e severidade da DAC (CADESI-4) a partir do exame clínico. Os cães foram agrupados em discretamente, moderadamente e acentuadamente comprometidos pela DAC após somatório dos valores de escores atribuídos no exame clínico. Amostras cutâneas da região axilar e interdigital de cada cão foram colhidas e submetidas às análises histopatológica (HE e azul de toluidina) e imunoistoquímica (IL-31). Houve correlação entre o escore clínico e as alterações microscópicas, assim como entre as alterações microscópicas, mas não entre o grau de prurido e o escore clínico da DAC. Também o infiltrado inflamatório foi mais intenso na região axilar em relação a interdigital. Maior número de células imunomarcadas para IL-31 foi observado nos cães acentuadamente comprometidos pela DAC. Houve correlação entre o escore clínico e a quantidade de mastócitos no interdígito, do mesmo modo com a quantidade de células imunomarcadas para IL31 na axila. Também foi verificada correlação entre a quantidade de mastócitos e células imunomarcadas para IL-31 na axila, assim como entre acantose e todas as demais alterações histopatológicas da pele na região axilar. Conclui-se que há correlação entre a gravidade das lesões, a imunomarcação de IL-31, a contagem de mastócitos e as alterações histopatológicas em cães com dermatite atópica, mas não há correlação entre o grau de prurido relatado pelos proprietários e a gravidade das lesões cutâneas em animais com DAC. Ainda, a intensidade do processo inflamatório pode variar em função do sítio anatômico da lesão.