Oral and oropharyngeal squamous cell carcinoma (OOSCC) is a malignant neoplasm of epithelial origin that accounts for 90 to 95% of tumors in oral cavity. Alcohol and tobacco consumption are main risk factors, but the formation of reactive oxygen species (ROS) and presence of chronic inflammatory processes have been shown to favor the carcinogenesis process. Human serum paraoxonase 1 (PON1) is a protein with an important antioxidant action and prevents oxidative stress induced by ROS, in turn, high levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) are commonly found in inflammatory processes, and changes in these proteins have been related to development of different neoplasms. Present study aims to investigate the prognostic value of functional polymorphisms in PON1, TNF-α and TGF-β genes in OOSCC. This is a prospective cohort study with patients attended at the Advanced Oncology Center of the North-Riograndense League against Cancer. Data collection of clinical variables was done through the form: gender, age, tumor site, TNM (primary tumor, regional nodule and distant metastasis) classification, clinical stage; and through interview: smoking habits and alcohol intake by the patient. A total of 163 samples from patients with OOSCC and 146 control samples were genotyped by real-time PCR. It was observed that 76.1% of the population was males, 84% older than 52 years, with a more frequent intra-oral clinical presentation (53.4%), in the tongue region (21.5%), tumors greater than 4cm (56.45%), presence of nodal involvement (58.9%) and stages III and IV (79.15%). There was a positive association between drinking and smoking habits in patients with OOSCC and between clinical stage and tumor site (p <0.05). The polymorphisms were in Hardy-Weinberg equilibrium, with exception of rs662 of PON1. TNF-α wild-type GG homozygous genotype (rs1800629) was associated with intra-oral lesions, clinical stage of the most advanced disease (III and IV), and decreased overall disease survival, whereas the polymorphic AA genotype was associated with lip lesion, clinical stage I and II and a longer overall disease survival (p <0.05). It was observed association of TGF-β polymorphic AG and AA genotypes (rs1800469) with larger diameter tumors (T3 and T4) (p <0.05). Finally, the polymorphic TT genotype of PON1 (rs662) in recessive model was associated with a shorter disease survival time within threshold of significance (p = 0.05). In view of the findings, it is suggested that wild-type GG homozygous genotype of TNF-α rs1800629 and TGF-β rs1800469 polymorphic AG and AA genotypes may exert an influence on more aggressive biological behavior of OOSCC and that AG genotype of TNF-α rs1800629 and TT genotype of PON1 rs662 could be prognostic markers in OOSCC. In the clinical practice of oncology, these genotypes can be used to perform early diagnosis, knowledge of the biological behavior of the tumor and choice of appropriate individualized therapy / O carcinoma de células escamosas oral e orofaríngeo (CCEO) é uma neoplasia maligna de origem epitelial que representa 90 a 95% dos tumores da cavidade oral. O consumo de álcool e de tabaco são os principais fatores de risco, mas a formação de espécies reativas de oxigênio (EROs) e a presença de processos inflamatórios crônicos têm-se mostrado favorável ao processo de carcinogênese. A paraoxonase de soro humano 1 (PON1) é uma proteína com importante ação antioxidante e previne o estresse oxidativo induzido pelas EROs, por sua vez, elevados níveis do fator de necrose tumoral-alfa (TNF-α) e do fator de crescimento transformante-beta (TGF-β) são comumente encontrados em processos inflamatórios, e alterações nessas proteínas têm sido relacionadas com o desenvolvimento de diferentes neoplasias. O presente estudo tem por objetivo investigar o valor prognóstico dos polimorfismos funcionais nos genes da PON1, TNF-α e TGF-β no CCEO. Trata-se de um estudo coorte prospectivo com pacientes atendidos no Centro Avançado de Oncologia da Liga Norte-Riograndense contra o Câncer. Realizou-se a coleta de dados das variáveis clínicas por meio de formulário: gênero, idade, localização do tumor, classificação TNM (Tumor primário, nódulo regional e metástase à distância), estadiamento clínico (EC); e por meio de entrevista: hábitos de fumo e ingestão alcoólica pelo paciente. Foram genotipadas 163 amostras de pacientes com CCEO e 146 amostras controle por meio de PCR em tempo real. Observou-se que 76,1% da população era do gênero masculino, sendo 84% com mais de 52 anos, com apresentação clínica mais frequente intra-oral (53,4%), na região da língua (21,5%), tumores maiores que 4cm (56,45%), presença de envolvimento nodal (58,9%) e em estágios III e IV (79,15%). Evidenciou-se associação positiva entre os hábitos de beber e fumar com pacientes portadores de CCEO e entre o EC e a localização do tumor (p<0,05). Os polimorfismos encontravam-se em equilíbrio de Hardy-Weinberg, com exceção do rs662 da PON1. O genótipo homozigoto selvagem GG do TNF-α (rs1800629) associou-se com lesões intra-orais, EC da doença mais avançado (III e IV) e menor sobrevida global da doença, enquanto o genótipo AA polimórfico associou-se a lesão de lábio, EC I e II e maior sobrevida global da doença (p<0,05). Observou-se associação dos genótipos AG e AA polimórfico do TGF-β (rs1800469) com tumores de maior diâmetro (T3 e T4) (p < 0,05). Por fim, o genótipo TT polimórfico da PON1 (rs662) no modelo recessivo apresentou associação com menor tempo de sobrevida da doença dentro do limiar de significância (p=0,05). Diante dos achados, sugere-se que o genótipo GG selvagem do rs1800629 do TNF-α e os genótipos AG e AA polimórfico do rs1800469 do TGF-β podem exercer influência no comportamento biológico mais agressivo do CCEO e que o genótipo AG do rs1800629 do TNF-α e o genótipo TT polimórfico do rs662 da PON1 poderiam ser marcadores com valor prognóstico no CCEO. Na prática clínica da oncologia, esses genótipos podem ser utilizados para realização de diagnóstico precoce, conhecimento do comportamento biológico do tumor e escolha da terapêutica individualizada adequada. / Lagarto, SE

Orientador: Ibsen Bellini Coimbra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T21:15:58Z (GMT). No. of bitstreams: 1 Sartori_AngelicaRossi_D.pdf: 3838547 bytes, checksum: 0be815456ed0b3b8faf496ae6738da31 (MD5) Previous issue date: 2009 / Resumo: O Fator Induzido por Hipóxia-1 (Hypoxia Inducible Factor-1 - HIF-1) é um fator de transcrição responsável por transcrever genes relacionados às alterações nas concentrações de oxigênio e sobrevivência celular. A cartilagem articular é um tecido avascular e o ambiente dos condrócitos é caracterizado por condições de hipóxia dentro da matriz. Nestas condições a proteína HIF-1alfa do Fator de transcrição Induzido por Hipóxia-1alfa (HIF-1alfa) é necessário para controlar o metabolismo e a integridade funcional da cartilagem. Além da hipóxia algumas citocinas como IL-1? e TNF? são também capazes de estabilizar HIF-1?, além de serem consideradas as principais mediadoras da osteoartrite (OA). Objetivo: Verificar a participação da IL-1? na regulação do HIF-1? em condições normais de oxigênio; Verificar a utilização da via da Fosfatidil-Inositol-3-Kinase (PI-3K) pelo HIF-1? e analisar a participação HIF-1? na expressão de colágeno tipo II e agrecano e a sua regulação pelas citocinas TNF-? e da IL-1?. Material e Métodos: Condrócitos humanos provenientes de pacientes em OA, submetidos à artroplastia de joelho, foram cutivados em suspensão e em monocamada, submetidos ou não ao silenciamento do gene do HIF-1? pela técnica de Interferência por RNA e estimulados com IL-1?, TNF? e LY294002, o inibidor da via da PI-3K em condições normais de oxigênio e em hipóxia e foram submetidos à extração de proteína nuclear, extração de RNA e precipitação das proteínas do meio de cultura das células. A análise das proteínas foi feita por meio da técnica de Western Blotting para a detecção da proteína nuclear HIF-1? e do colágeno tipo II no meio de cultura das células. O RNA foi analisado pela técnica de PCR em Tempo Real para a quantificaçãos dos genes do HIF-1?, Colágeno tipo II e Agrecano. Resultados: IL-1? aumentou a expressão da proteína nuclear HIF-1?, mas não alterou a expressão do RNAm. Essa modulação utilizou a via da PI-3K. A hipóxia aumentou as concentrações do RNAm do HIF-1? em comparação com as condições normais de oxigênio, mas IL-1? e TNF? não alteraram o RNAm do HIF- 1? em condições normais de oxigênio e em associação com a hipóxia, inibiram o efeito regulatório positivo desta sobre o HIF-1?. A hipóxia isoladamente também aumentou RNAm de colágeno tipo II, o que foi anulado pelo estímulo associado com a IL-1?. Nos grupos de condrócitos com deficiência do HIF-1? (silenciados), em quaisquer condições de oxigênio, IL-1? e TNF? não alteraram significativamente a expressão de HIF-1?, mas em hipóxia, a expressão de colágeno tipo II foi regulada negativamente nesses grupos. A associação entre a falta do HIF-1? e as citocinas diminuiu ainda mais a expressão do colágeno tipo II em condições de hipóxia. Em todas as análises não foram observadas diferenças significativas nas expressões do RNAm do agrecano e na análise do colágeno tipo II das proteínas precipitadas dos meios de cultura das células. Conclusão: IL-1? aumentou a expressão da proteína nuclear HIF-1? pós-transcrição. A regulação do HIF-1? pela IL-1? em situação normal de oxigênio ocorreu, ao menos em parte, pela PI-3K. O HIF-1? se relacionou positivamente com a expressão do gene do colágeno tipo II, principalmente em hipóxia, mas não com os níveis da proteína. Não houve associação com a expressão do gene do agrecano / Abstract: Introduction: Hypoxia Inducible Factor -1 (HIF-1) is a transcription factor that regulates the expression of genes related with oxygen concentration e cellular survive. Articular cartilage is a non-vascular tissue and the condrocytes microenviroment are caracterized by hypoxic conditions inside the extracelluar matrix. In this condition, the protein HIF-1?, from HIF-1 is necessary to the metabolism control and cartilage functional intengrity. Some citokines like IL-1? and TNF?, as well as hypoxia, are capable to stabilize HIF-1? and are essential in oateoarthritis (OA) progression disease. Objective: To verify the participation of IL-1? in the HIF-1? regulation under normal conditions of oxygen; To verify if the pathway of phosphatidilynositol-3-Kinase (PI-3K) is used in this modulation; To analyse HIF-1? participation in the collagen type II and aggrecan gene expression and your regulation by TNF-? and IL-1?. Material and Methods: Human OA chondrocytes were obtained from patients with OA that underwent total knee joint replacement surgery, were cultured either in suspension or monolayer. They were submitted to HIF-1? gene silencing by RNA Interference and lately stimulated with IL-1?, TNF? and LY294002, a specific inhibitor to Phosphatidilinosiltol - 3- Kinase (PI-3K) pathway in normal conditions of oxygen and in hypoxia. The condrocytes were submitted to protein nuclear extraction, RNA extraction and protein precipitation of culture media from the experiments. The proteins were analyzed by Western Blotting to HIF-1? detection in nuclear extraction and collagen type II detection in precipitated media. The RNA were analized by Real Time PCR to HIF- 1?, collagen type II and aggrecan gene quantification. Results: HIF-1? expression is up-regulated by IL-1? at the protein level but not in the mRNA expression. This modulation used, at least in part, the PI-3K pathway. Hypoxia enhanced the mRNA concentrations of HIF-1? when compared with normal conditions of oxygen, but in this cases IL-1? and TNF? did not change mRNA expression of HIF-1?. In association with hypoxia, these citokines inhibited the positive regulatory effect under HIF-1? mRNA expression. Hypoxia up-regulated collagen type II gene expression and that was inhibited by the association with IL-1?. In the groups with lack of HIF-1? (silenced), in both oxygen conditions, IL-1? and TNF? did not cause any significant change of the HIF-1? mRNA expression, but in hypoxia, collagen type II was up-regulated in those groups. The association between the lack of HIF- 1? and citokines down-regulated more strongly collagen type II in hypoxia. In all analyzes it was not observed significant differences in the aggrecan mRNA expression and collagen tipe II protein from cultured media of the cells Conclusion: IL-1? post-trancriptionaly up-regulated the HIF-1? protein level. HIF- 1? regulation by IL-1? in normal conditions of oxygen used, at least in part, the PI- 3K pathway. HIF-1? positively related with gene expression, but not with the protein levels, of collagen type II, mainly in hypoxia. There was no relation in the HIF-1? and aggrecan gene expression / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A mucosite intestinal (MI) à um efeito colateral comum e limitante da quimioterapia com irinotecano. Estudos sugerem a participaÃÃo de citocinas, como TNF-&#945;, IL-1&#946; e IL-18 na fisiopatologia da MI, alÃm de demonstrar que inibidores nÃo seletivos de citocinas, como pentoxifilina, atenuam a MI por irinotecano. O papel das citocinas està bem estabelecido no Ãmbito das doenÃas inflamatÃrias intestinais, inclusive com utilizaÃÃo de tratamentos visando inibir seletivamente componentes da casacata inflamatÃria, como, por exemplo, o anticorpo monoclonal anti-TNF&#945;, infliximabe, que tem um efeito benÃfico comprovado no tratamento da doenÃa de Crohn, artrite reumatÃide e espondilite anquilosante. Hà escassos estudos na literatura de terapia alvo anti-citocinas inflamatÃrias no contexto da mucosite intestinal induzida por quimioterÃpico. Objetivo: Avaliar o papel do Infliximabe sobre as alteraÃÃes inflamatÃrias e disfunÃÃo intestinal associados à MI induzida por Irinotecano. MÃtodos: Camundongos C57BL&#61487;6 foram divididos em grupos (n=5-9) e tratados por 4 dias com salina (5 ml&#61487;kg, i.p) ou irinotecano (75 mg&#61487;kg, i.p) ou prÃ-tratados com infliximabe (5 mg&#61487;kg, e.v, dose Ãnica) 1 hora antes da primeira injeÃÃo de irinotecano. O peso e mortalidade foram avaliados diariamente. No 5o dia avaliou-se a diarrÃia por escores, o leucograma e, apÃs sacrifÃcio, coletou-se o duodeno para dosagem da atividade de mieloperoxidase (MPO) e de IL-1&#946;, expressÃo de Ãxido nÃtrico sintase induzida (iNOS), anÃlise de escores histopatolÃgicos, morfometria e contratilidade in vitro ao carbacol. Resultados: Os animais tratados com irinotecano apresentaram mucosite intestinal, evidenciada por aumento significativo (p<0,05) nos escores de diarrÃia, diminuiÃÃo do comprimento e Ãrea do vilo, aumento da atividade de MPO, aumento no nÃvel tecidual de IL-1&#946; e de expressÃo de iNOS e hipercontratilidade da musculatura lisa duodenal, alÃm de terem apresentado leucopenia, quando comparado com o grupo tratado com salina. O grupo de animais que recebeu infliximabe previamente ao tratamento com irinotecano apresentou melhora significativa (p<0,05) nos parÃmetros inflamatÃrios avaliados (atividade de MPO, nÃvel tecidual de IL-1&#946;, expressÃo de iNOS), comparado com o grupo tratado apenas com irinotecano. Contudo, a perda ponderal, a mortalidade e a disfunÃÃo intestinal (diarrÃia e contratilidade intestinal in vitro) nÃo foram afetadas pelo tratamento com infliximabe (p>0,05). ConclusÃes: Os resultados mostram o proeminente efeito antiinflamatÃrio da terapia anti TNF-&#945; com infliximabe na MI induzida por irinotecano. Tal droga, no entanto, nÃo foi capaz de alterar a sobrevida e de proteger contra a disfunÃÃo na MI experimental por irinotecano. Adicionalmente, acentuou a leucopenia induzida pelo irinotecano, o que pode aumentar o risco de complicaÃÃes infecciosas. / IntroduÃÃo: A mucosite intestinal (MI) à um efeito colateral comum e limitante da quimioterapia com irinotecano. Estudos sugerem a participaÃÃo de citocinas, como TNF-&#945;, IL-1&#946; e IL-18 na fisiopatologia da MI, alÃm de demonstrar que inibidores nÃo seletivos de citocinas, como pentoxifilina, atenuam a MI por irinotecano. O papel das citocinas està bem estabelecido no Ãmbito das doenÃas inflamatÃrias intestinais, inclusive com utilizaÃÃo de tratamentos visando inibir seletivamente componentes da casacata inflamatÃria, como, por exemplo, o anticorpo monoclonal anti-TNF&#945;, infliximabe, que tem um efeito benÃfico comprovado no tratamento da doenÃa de Crohn, artrite reumatÃide e espondilite anquilosante. Hà escassos estudos na literatura de terapia alvo anti-citocinas inflamatÃrias no contexto da mucosite intestinal induzida por quimioterÃpico. Objetivo: Avaliar o papel do Infliximabe sobre as alteraÃÃes inflamatÃrias e disfunÃÃo intestinal associados à MI induzida por Irinotecano. MÃtodos: Camundongos C57BL&#61487;6 foram divididos em grupos (n=5-9) e tratados por 4 dias com salina (5 ml&#61487;kg, i.p) ou irinotecano (75 mg&#61487;kg, i.p) ou prÃ-tratados com infliximabe (5 mg&#61487;kg, e.v, dose Ãnica) 1 hora antes da primeira injeÃÃo de irinotecano. O peso e mortalidade foram avaliados diariamente. No 5o dia avaliou-se a diarrÃia por escores, o leucograma e, apÃs sacrifÃcio, coletou-se o duodeno para dosagem da atividade de mieloperoxidase (MPO) e de IL-1&#946;, expressÃo de Ãxido nÃtrico sintase induzida (iNOS), anÃlise de escores histopatolÃgicos, morfometria e contratilidade in vitro ao carbacol. Resultados: Os animais tratados com irinotecano apresentaram mucosite intestinal, evidenciada por aumento significativo (p<0,05) nos escores de diarrÃia, diminuiÃÃo do comprimento e Ãrea do vilo, aumento da atividade de MPO, aumento no nÃvel tecidual de IL-1&#946; e de expressÃo de iNOS e hipercontratilidade da musculatura lisa duodenal, alÃm de terem apresentado leucopenia, quando comparado com o grupo tratado com salina. O grupo de animais que recebeu infliximabe previamente ao tratamento com irinotecano apresentou melhora significativa (p<0,05) nos parÃmetros inflamatÃrios avaliados (atividade de MPO, nÃvel tecidual de IL-1&#946;, expressÃo de iNOS), comparado com o grupo tratado apenas com irinotecano. Contudo, a perda ponderal, a mortalidade e a disfunÃÃo intestinal (diarrÃia e contratilidade intestinal in vitro) nÃo foram afetadas pelo tratamento com infliximabe (p>0,05). ConclusÃes: Os resultados mostram o proeminente efeito antiinflamatÃrio da terapia anti TNF-&#945; com infliximabe na MI induzida por irinotecano. Tal droga, no entanto, nÃo foi capaz de alterar a sobrevida e de proteger contra a disfunÃÃo na MI experimental por irinotecano. Adicionalmente, acentuou a leucopenia induzida pelo irinotecano, o que pode aumentar o risco de complicaÃÃes infecciosas. / Introduction: Intestinal mucositis (IM) is a common limiting side effect of anticancer therapy with irinotecan. Previous studies have reported the involvement of cytokines, such as TNF-&#945;, IL-1&#946; e IL-18 in the pathogenesis of irinotecan-induced IM, besides demonstrating that nonselective inhibitors of cytokines such as pentoxifylline, attenuate chemotherapy-induced IM. The role of cytokines is well established in the context of inflammatory bowel disease. It is used in clinical practice treatments that target inflammatory cascade components, for example, the anti-TNF monoclonal antibody, infliximab, which has a beneficial effect in the treatment of Crohnâs disease, rheumatoid arthritis and ankylosing spondylitis. There is a lack of information regarding the effect of selective cytokine target therapy on anticancer drug toxicity. Aims: To investigate the protective role of a selective TNF-&#945; inhibitor, infliximab, on irinotecan-induced IM. Methods: C57BL&#61487;6 mice were divided into groups (n=5-9) and treated for 4 days with saline (5 mL/kg, i.p.) or irinotecan (75 mg/kg/4 days, i.p) or were given infliximabe (5 mg/kg, e.v, single dose) 1 hour before the first irinotecan injection. Body weight and mortality were assessed daily. On the 5th day the diarrhea was evaluated using scores and the blood was collected for white blood cell count (WBC). After euthanasia, samples of duodenum was collected for myeloperoxydase assay, tissue IL-1&#946; dosage, western blot (WB) of the inducible nitric oxide synthase, morphometric analysis and in vitro evaluation of duodenal contractility. Results: Irinotecan induced IM demonstrated by the significant (p<0,05) increase in diarrhea, decrease in length and area of the villi increased MPO activity, increased IL-1&#946; dosage and expression of iNOS and intestinal smooth muscle over-contractility when compared with saline treated group. The group treated with irinotecan also presented leucopenia, when compared with control group. The group treated with infliximab previously to irinotecan showed a significant improvement of inflammatory parameters (MPO activity, IL-1&#946;, expression of iNOS), when compared withn irinotecan-treated group. However, animal weight loss, mortality and gut disfunction (diarrhea and intestinal contractility) were not affected by infliximab treatment (p>0,05). Conclusion: The results suggest for the first time the proeminent anti-inflammatory effect of the target therapy anti-TNF-&#945; with infliximab on irinotecan-induced IM. However, such treatment did not alter the survival and did not protect against intestinal dysfunction in irinotecan-induced IM. Additionally accentuated leukopenia induced by irinotecan, which can increase the risk of infections. / Introduction: Intestinal mucositis (IM) is a common limiting side effect of anticancer therapy with irinotecan. Previous studies have reported the involvement of cytokines, such as TNF-&#945;, IL-1&#946; e IL-18 in the pathogenesis of irinotecan-induced IM, besides demonstrating that nonselective inhibitors of cytokines such as pentoxifylline, attenuate chemotherapy-induced IM. The role of cytokines is well established in the context of inflammatory bowel disease. It is used in clinical practice treatments that target inflammatory cascade components, for example, the anti-TNF monoclonal antibody, infliximab, which has a beneficial effect in the treatment of Crohnâs disease, rheumatoid arthritis and ankylosing spondylitis. There is a lack of information regarding the effect of selective cytokine target therapy on anticancer drug toxicity. Aims: To investigate the protective role of a selective TNF-&#945; inhibitor, infliximab, on irinotecan-induced IM. Methods: C57BL&#61487;6 mice were divided into groups (n=5-9) and treated for 4 days with saline (5 mL/kg, i.p.) or irinotecan (75 mg/kg/4 days, i.p) or were given infliximabe (5 mg/kg, e.v, single dose) 1 hour before the first irinotecan injection. Body weight and mortality were assessed daily. On the 5th day the diarrhea was evaluated using scores and the blood was collected for white blood cell count (WBC). After euthanasia, samples of duodenum was collected for myeloperoxydase assay, tissue IL-1&#946; dosage, western blot (WB) of the inducible nitric oxide synthase, morphometric analysis and in vitro evaluation of duodenal contractility. Results: Irinotecan induced IM demonstrated by the significant (p<0,05) increase in diarrhea, decrease in length and area of the villi increased MPO activity, increased IL-1&#946; dosage and expression of iNOS and intestinal smooth muscle over-contractility when compared with saline treated group. The group treated with irinotecan also presented leucopenia, when compared with control group. The group treated with infliximab previously to irinotecan showed a significant improvement of inflammatory parameters (MPO activity, IL-1&#946;, expression of iNOS), when compared withn irinotecan-treated group. However, animal weight loss, mortality and gut disfunction (diarrhea and intestinal contractility) were not affected by infliximab treatment (p>0,05). Conclusion: The results suggest for the first time the proeminent anti-inflammatory effect of the target therapy anti-TNF-&#945; with infliximab on irinotecan-induced IM. However, such treatment did not alter the survival and did not protect against intestinal dysfunction in irinotecan-induced IM. Additionally accentuated leukopenia induced by irinotecan, which can increase the risk of infections.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-03-03T12:08:21Z No. of bitstreams: 1 victorsoarescavalcanticosta.pdf: 3451719 bytes, checksum: 9019e3c3a2f3b904f3e1f38804fb82f8 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T01:42:12Z (GMT) No. of bitstreams: 1 victorsoarescavalcanticosta.pdf: 3451719 bytes, checksum: 9019e3c3a2f3b904f3e1f38804fb82f8 (MD5) / Made available in DSpace on 2016-04-24T01:42:12Z (GMT). No. of bitstreams: 1 victorsoarescavalcanticosta.pdf: 3451719 bytes, checksum: 9019e3c3a2f3b904f3e1f38804fb82f8 (MD5) Previous issue date: 2013-09-18 / A talidomida é conhecida por sua atividade antiinflamatória e imunomoduladora, controlando os sinais clínicos das doenças mais variadas, dentre elas o eritema nodoso leproso, doença de Crohn, artrite reumatóide, câncer, entre outros distúrbios vasculares e inflamatórios. Estudo anterior realizado em nosso laboratório mostrou que análogos da talidomida contendo diaminas e estrutura ftalimídica aberta são capazes de inibir in vitro a produção de citocinas pró-inflamatórias como TNF-a, IL-12, IL-6, IFN-g, CXCL9, CXCL10 e CD80, além de elevar os níveis de IL-10. Neste estudo, a atividade antiinflamatória de dois análogos de talidomida (GI-16 e SC-15) foi avaliada in vivo, utilizando o modelo de inflamação na pata induzido por injeção subplantar de carragenina em camundongos BALB/c, e o modelo de inflamação pulmonar induzida por inoculação intratraqueal com LPS em camundongos C57Bl/6. Além disso, a toxicidade aguda e sub-crónica destes compostos foi avaliada em ratos Wistar. Os resultados mostram que o tratamento com GI-16 (10mg/kg e 50mg/kg) e SC-15 (50mg/kg) reduziu significativamente (53-77%) o edema de pata induzido pela injeção subplantar de carragenina 2% por até 24 horas. No modelo de inflamação pulmonar com LPS (200μg/ml), os compostos GI-16 (20mg/kg e 50mg/kg) e SC-15 (50mg/kg) inibiram significativamente a produção de TNF- a (34%) e IL-6 (66% a 89%) no homogenato pulmonar dos animais testados. O composto SC-15 (50mg/kg) e a talidomida (50mg/kg) causaram aumento dos níveis de IL-10 (p<0,05). Conforme esperado, a talidomida e o fármaco de referência dexametasona provocaram inibição significativa do edema de pata induzido por carragenina, assim como redução dos níveis de TNF-a e IL-6 induzidos pela administração de LPS. Análises histopatológicas revelaram que o tratamento com GI-16 causou redução da inflamação induzida por LPS, havendo discreta congestão capilar. Por outro lado, animais tratados com SC- 15 mostraram um quadro inflamatório de moderado a intenso, A análise histopatológica dos pulmões dos camundongos apenas estimulados com LPS demonstrou quadro inflamatório de moderado a intenso, sendo detectado espessamento da parede alveolar e congestão vascular, na dose de 20mg/kg, com redução do quadro inflamatório quando aplicada a dose de 50mg/kg. 8 Ratos Wistar fêmeas e machos administrados com GI-16 e SC-15 (20mg/kg) não desenvolveram sinais clínicos de toxicidade aguda (dose única) ou de toxicidade sub-crónica (doses em dias alternados por 28 dias), imediatamente ou durante o período pós-tratamento. Não houve mortalidade durante os períodos de tratamento avaliados. O ganho de peso corporal ao longo do tempo foi semelhante entre os grupos sem alteração significativa nos parâmetros bioquímicos ou hematológicos. Os cortes histológicos de tecido cardíaco, hepático e renal dos ratos estudados não mostraram alterações sob a microscopia ótica comum de campo claro. Em conjunto, os resultados indicam uma forte atividade antiinflamatória dos compostos GI-16 e SC-15, não havendo sinais de toxicidade aguda ou sub-crônica, o que os torna drogas promissoras para o tratamento de doenças inflamatórias. / Thalidomide exhibits anti-inflammatory and immunomodulatory effects, improving clinical symptoms in a variety of diseases, including erythema nodosum leprosum, Crohn disease, rheumatoid arthritis, cancer and other vascular and inflammatory diseases. In previous study from our laboratory it has been shown that thalidomide analogues containing diamines and open phthalimide structure, showed high inhibitory in vitro activity on key molecules such as TNF-a, IL-12, IFN-g, IL-6, CXCL9, CXCL10 and CD80. In contrast, some compounds induced an increase in IL-10 production. In this study, the anti-inflammatory activity of two thalidomide analogues (GI-16 and SC-15) were evaluated in vivo using the carrageenan-induced paw inflammation on BALB/c mouse and the LPS-induced lung inflammation in C57Bl/6 mice. In addition, the acute and sub-chronic toxicity of the compounds on Wistar rats were evaluated. The treatment with both GI-16 (10mg/kg and 50mg/kg) and SC-15 (50mg/kg) reduced significantly (53-77%) the paw oedema over 24h evoked by subplantar injection of 2% carrageenan. GI-16 (20mg/kg and 50mg/kg) and SC-15 (50mg/kg) greatly inhibited LPS-induced TNF-a (around 34%) and IL-6 (66- 89%) in lung homogenate. In contrast, thalidomide (50mg/kg) and SC-15 (50mg/kg) enhanced IL-10 (p<0,05). As expected, thalidomide and the reference drug, dexamethasone, caused significant inhibition of both carrageenan-induced oedema and of TNF-a and IL-6 production 24 h after intratracheal administration of LPS (200ug/ml). Histopathological analysis has shown that GI-16 treatment induced reduction in LPS–induced inflammation characterized by discrete capillary congestion. In contrast, animals treated with SC-15, showed moderate to intense inflammatory infiltrate, thickening of alveolar wall and vascular congestion at 20mg/kg, and reduced inflammation at 50mg/kg. Female and male Wistar rats administered with GI-16 and SC-15 (20mg/kg) did not develop any clinical signs of acute toxicity (single dose) or sub-chronic toxicity (every other day for 28 days) either immediately or during the posttreatment period. No mortality occurred in both, control and treated animals either immediately or during the treatment period. Body weight gain over time 10 was similar in all groups. There was not significant alteration in biochemical or hematological parameters. Sections of heart, liver and kidneys tissues of the studied rats showed no pathological alterations under light microscopy. These results strongly reinforce the anti-inflammatory effects of GI-16 and SC-15, which make them very attractive drug candidates to treat a broad range of inflammatory diseases.

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Introduktion: Inflammation är en bidragande orsak till flera av dagens folksjukdomar t ex diabetes, ateroskleros, neurodegenerativa sjukdomar och vissa former av cancer. Inflammation karaktäriseras av en 2-4 gånger förhöjd nivå av anti- och proinflammatoriska cytokiner (framför allt IL-6 och TNF-a) samt ökad halt C-reaktivt protein. Idag behandlas inflammation i första hand med NSAID-preparat. Då träning frisätter antiinflammatoriska cytokiner är syftet med detta arbete att undersöka om det finns vetenskapliga bevis för att träning skulle kunna vara en behandlingsmetod vid låggradig systemisk inflammation. Metod: Studien har genomförts genom att studera och utvärdera vetenskapliga artiklar inom området, framför allt översiktsartiklar och kliniska studier, vilka har erhållits genom sökning i vetenskapliga databaser. Resultat: Resultaten från studierna som mäter den antiinflammatoriska effekten av träning pekar på att träning minskar halten av TNF-a och CRP samt ökar halten av IL-6. NSAID verkar sänka nivån TNF-a, IL-6 och CRP. Vid inflammatoriska tillstånd ses en förhöjd halt IL-6, som produceras från T-celler och makrofager och som effekt av ökande halt TNF-a. I samband med träning ses en ökning av IL-6 från den kontraherande muskeln. IL-6 från muskler verkar ge såväl en metabolisk som en antiinflammatorisk effekt genom att påverka lipidmetabolismen respektive minska utsöndringen av TNF-a. Slutsats: Träning höjer halten IL-6, vilket även leder till en hämning av TNF-a. Den här studien kan dock inte avgöra om NSAID eller träning har bäst effekt för att minska systemisk låggradig inflammation.

Master of Public Health / Department of Diagnostic Medicine/Pathobiology / John A. Pickrell / Beef cattle feed lots produce significant, local point source pollution of the atmosphere. The dusts generated in the CAFOs are complex mixture of fine and ultra fine particles, organic compounds, transition metals, and adsorbed toxic gases. Since each component is toxic in itself, we do not fully understand the relative importance of each component in the dust and their interactions to inducing inflammatory changes in the lung. We did extensive literature searches to understand the mechanism of dust toxicity in respiratory system. This lead to focusing on solubility of dust in lung simulant fluid, and in-vitro study of release of two common biomarkers of inflammatory processes IL-6 and IL-8 from lung epithelial cells. Various concentrations (1 to 50%) of the dust extract induced release of IL-6, and IL-8 from lung epithelial cell as indicators of pro-inflammatory changes (IL-6), and amplification and maintenance of inflammation (IL-8). IL-6 release had dose dependence; peak production was seen with 25% dust extract. IL-8 production went down as the concentration of the dust extract increased from 1% to 25%. However, 50% dust extract was cytotoxic to the cell leading to 10-15% cell viability. At non-cytotoxic concentrations for lung epithelial cells, production of IL-8 was reduced. These findings suggested that higher exposure concentration were required to initiate inflammation as indicated by IL-6 release. Lower exposure concentrations (1 and 5% extracts) were related to optimal release of IL-8 needed to amplify and maintain the inflammatory response. Inhibition of endotoxin didn't significantly change the pattern of IL-6 or IL-8 release from epithelial cells. This finding suggested that at least a portion of the mechanism by which particle induced cytokine release from the lung epithelial cells was not endotoxin dependent. Heating samples at 1200C for 5 minutes modified some of the toxic properties of the dust extracts but didn't completely detoxify it. We observed that longer incubation period was required to peak release for both IL-6 and IL-8. However, the higher concentration of sample (50% extract) found to be cytotoxic in non-heat treated sample was no longer cytotoxic and induced both IL-6 and IL-8 release from the lung epithelial cells. This result suggested that heat treatment could reduce some of the dust extract's cytotoxic properties. However, the extract's potential to induce peak cytokine release increased.

Les cellules du système immunitaire sont programmées pour reconnaître et éliminer les cancéreuses, pourtant les cellules tumorales ont la capacité de mettre en œuvre les divers moyen pour échapper à la mort induite par les effecteurs du système immunitaire. Au cours de cette thèse, nous avons étudié plus particulièrement PD-L1, qui est impliqué dans la protection des cellules tumorales contre une attaque du système immunitaire et induit au cours de la TEM. / EMT foster cancer progression by acting on mechanisms allowing tumors to exide immune surveillance. Recent clinical advances immunotherapy demonstrated that some cancer with established lymphocyte infiltrates, express immune checkpoint inhibitory molecules, such as PD-L1, to allow their progression. During this thesis, another link between EMT and immune escape, through the regulation of PD-L1 in non-smal cell lung caricinoma was established. A new role of IKKƐ in the regulation of PD-L1 during EMT was also been shown.

Painful conditions having the origin within the musculoskeletal system is a common cause for people to seek medical care. Between 20-40% of all visits to the primal care in Sweden are coupled to pain from the musculoskeletal system. Muscle pain and impaired muscle function can be caused by muscles being repetitively overused and/or via heavy load. Skeletal muscle is a dynamic tissue which can undergo changes in order to fulfill what is best for optimal function. However, if the load is too heavy, morphological changes including necrosis, as well as pain can occur. The extension of the skeletal muscle is the tendon. Tendinopathy refers to illness and pain of the tendon. The peritendinous tissue is of importance in the features related to tendon pain. Common tendons/origins being afflicted by tendinopathy/pain are the Achilles tendon and the extensor origin at the elbow region.    Tumor necrosis factor alpha (TNF-alpha) is a cytokine that is involved in several biological processes. It is well-known for its involvement in the immune system and is an important target for inflammatory disorders such as rheumatoid arthritis. It is not known to what extent the TNF-alpha system is involved in the process of muscle inflammation and damage due to overuse.    Studies were conducted on rabbit and human tissue, tissues that either had undergone an excessive loading activity or tissue that was removed with surgery due to painful conditions. The tissues were evaluated via staining for morphology, in situ hybridization and immunofluorescence.    Unilateral experimental overuse of rabbit muscle (soleus muscle) led to morphological changes in the soleus muscle tissue bilaterally. The longer the experiment extended, the more was the tissue affected. This included infiltration of white blood cells in the tissue (myositis) and abnormal muscle fiber appearances. TNF-alpha mRNA was seen in white blood cells, in muscle fibers interpreted to be in a reparative stage and in white blood cells that had infiltrated into necrotic muscle fibers.  There was an upregulation in expressions of TNF receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2) in muscles that were markedly overused, with expressions in white blood cells, fibroblasts, blood vessel walls and muscle fibers. Immunoreactions for the receptors were seen in nerve fascicles of markedly overused muscles but only occasionally in normal muscles. The upregulations were seen for both experimental and contralateral sides. Overall the two receptors showed somewhat different expression patterns. Tendinopathy is associated with an increase in blood flow and infiltration of white blood cells in the tissue adjacent to the tendon. It is called the peritendinous tissue and is also richly innervated. The white blood cells and the blood vessels walls in this tissue were showing immunoreaction for TNFR1 and TNFR2. Two types of nerve fascicles were found in this tissue, one normally appearing when staining for nerve markers and one type with signs of axonal loss. The latter had clearly strong immunoreactions for TNFR1 and TNFR2.    The findings suggest that the TNF-alpha system is involved in both myopathies occurring due to overuse and in features in the peritendinous tissue in the tendinopathy situation. TNF-alpha and its receptors seem to be involved in degeneration but also in regeneration and healing of the tissue. The findings also suggest that TNF-alpha has effects on nerves showing axonal loss. The changes in the TNF-alpha system were seen both on the experimental side and contralaterally. / Smärta och funktionsbortfall från rörelseapparaten är vanligt förekommande. Mellan 20-40% av alla besök i primärvården är kopplade till smärta från rörelseapparaten. Det är också en vanlig orsak till sjukfrånvaro. Överansträngning inklusive repetitivt enformigt muskelarbete kan leda till muskelsmärta och bristande muskelfunktion (ex nedsatt styrka och uthållighet, inskränkt rörlighet). Muskelvävnad är en dynamisk vävnad som kan ändras utefter vilka påfrestningar den utsätts för och därigenom vilka behov den ställs inför. Men om belastningen blir för hård, alternativt återhämtningen blir för kort, kan negativa förändringar i vävnadsstrukturen uppstå, inklusive celldöd och vävnadsskada. Förlängningen av muskeln är senan. Senan är den vävnad som förbinder muskeln med skelettet. Tendinopati innefattar smärtsamma sjukdomstillstånd i senan. När sjukdom i en sena uppstår, exempelvis en smärtande hälsena, har man sett att den lösa bindväven som omger senan är av betydelse. Den genomgår morfologiska förändringar och man tror att det är den som är med och bidrar till smärtan vid tillståndet. Akillessenan och ”tennis-armbåge” är vanliga ställen för tendinopati. Akillessenan förbinder den trehövdade vadmuskeln med hälbenet. Tennis-armbåge omfattar ett område för flera musklers ursprung vid armbågen. Dessa muskler ansvarar framför allt för att sträcka i handleden. TNF-alfa är en signalsubstans som är involverad i flertalet biologiska processer. Den är känd för sin del i immunförsvaret och den är ett viktigt mål för behandling av autoimmuna sjukdomar som exempelvis reumatoid artrit. Det är inte känt om TNF-alfa är inblandad i processen som uppstår vid muskelinflammation/muskelskada efter kraftig överansträngning. TNF-alfa har flera receptorer, i det här arbetet har utbredning av TNFR1 och TNFR2 analyserats. Studier har utförts på djur (kaniner) och människa. Kaniner har genomgått ett träningsexperiment, där de utsatts för repetitiva muskelkontraktioner som lett till överansträngningsskador och muskelinflammation. Den muskel som studerats är soleus-muskeln, en del i den trehövdade vadmuskeln. Vävnadsprover har tagits från patienter med smärta i Akillessenan eller tennisarmbåge. Vävnadsproverna från kanin och människa har analyserats med färgningar för morfologi, immunohistokemi för detektering av TNF-alfa och dess receptorer samt för in situ hybridisering för detektion av mRNA i TNF-alfa systemet. Parallellt med färgningar för faktorerna i TNF-alfa systemet har uttryck för andra faktorer studerats. Ensidig överbelastning hos kaniner ledde till samma morfologiska förändringar på båda sidor, det vill säga även i muskeln i det ben som inte hade genomgått träningsexperimentet. Ju längre experimentet pågick, desto större blev de morfologiska förändringarna. TNF-alfa sågs i vita blodkroppar, TNF-alfa mRNA sågs även i förändrade muskelfibrer. Resultatet av parallella dubbelfärgningar tolkades som att dessa muskelfibrer antingen var i en regenererande process eller i en destruktiv process. TNFR1 och TNFR2 uttrycktes i större utsträckning ju längre experimentet pågick och ju mer muskelvävnaden var påverkad av inflammation. TNF receptorer sågs i vita blodkroppar, fibroblaster, muskelfibrer och nervstrukturer hos experimentdjuren. Det såg lika ut på båda sidor, inklusive det ben som inte ingått i experimentet. De två receptorerna skilde sig åt i uttryck. Vävnad från patienter med smärtande senor/smärta vid muskelursprungs-region genomgick också färgningar för faktorer i TNF-alfa systemet. Man kunde se att den lösa bindväven runt senan (den peritendinösa vävnaden) innehöll mycket blodkärl och nerver. De nerver som sågs i denna vävnad var av två typer, en som såg normal ut och en typ som uppvisade tecken på förlust av axoner. Den senare varianten hade en tydlig uppreglering av båda TNF receptorerna. Dessa resultat tyder på att TNF-alfa systemet är involverat i muskelsjukdomar som rör muskelinflammation till följd av kraftig överansträngning och i processerna i bindväven vid smärtande senor. TNF-alfa och dess receptorer verkar vara inblandade i både nedbrytning och uppbyggnad av muskelvävnad, samt påverka nerver som visar tecken på förlust av axoner. Förändringarna i TNF-alfa systemet sågs både på experimentsidan och kontralateralt.

Introduction: Gout is a painful form of acute inflammatory arthritis associated with elevated uric acid crystal deposition especially in the joints, but also in tendons and the kidney. Between 1 and 2% of Western populations are affected and in severe cases, gout sufferers can be completely incapacitated. Despite the number of gout sufferers, the high number of risk factors and high incidence of adverse drug reactions using the standard treatment regimens, little research involving gout has been done within the highly diverse multiracial and multicultural population of South Africa. Hypothesis: This study was a hypothesis generating observational study to assess whether serum levels of pro-inflammatory cytokines and acute phase protein levels could be used as markers of the gout status of a patient. Method: Thirty gout patients were enrolled onto the study and attended two visits. At the screening visit; medical history, vital signs and demographic details were collected from intercritical gout patients. At both visits, patients completed visual analogue scales; namely: subject’s assessment of pain and subject’s assessment of disease activity. A doctor completed the physician’s assessment of disease activity at both of the visits. At the end visit, patients experiencing an acute gout attack were asked to list various foods and beverages that triggered said attacks. Patients were requested to return for their second visit as soon as they experienced a gout attack, however, those patients that did not experience a gout attack were asked to return to the clinic to complete the follow up visit four months after their baseline visit. Uric acid, IL-1β, TNF-α and CRP were measured for each patient at both visits. Results: Many of the patients displayed risk factors for metabolic syndrome. The mean subject’s assessment of pain score increased from 31mm at the screening visit to 40mm at the end visit (p=0.1947; n=26), while the mean subject’s assessment of disease activity score and the mean physician’s assessment of disease activity increased from 30mm to 37mm (p=0.3196; n=26) and 23mm to 35 mm (p=0.0937; n=26) respectively. Uric acid levels decreased from 1.053mmol/L to 0.871mmol/L between visits (p=0.0926; n=25) while CRP concentrations increased significantly from 10.2mg/L to 26.6mg/L (p=0.0278, n=24). IL-1β concentrations remained similar (12.17pg/ml to 12.54pg/ml) while TNF-α concentrations decreased from 12.63pg/ml to 3.54pg/ml, however neither of these were statistically significant differences. Upon stratifying results into active and non-active patients, both IL-1β and TNF-α concentrations decreased between non-active and active patients, while CRP and urate concentrations increased. However, none of these differences were statistically significant. Conclusion: The visual analogue scales all showed an increase between the screening and final visits, although this was not statistically significant. Uric acid concentrations decreased between visits, however this increase was once again not statistically significant. There appears to be no association between inflammatory markers and the level of gout activity, although this needs to be tested in a larger sample population. Results in South African patients have confirmed results from previous studies where gout patients are at a higher risk of metabolic syndrome than the normal population. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted