Os linfócitos T CD4+ orquestram a resposta imune adaptativa, auxiliando os macrófagos, os linfócitos T CD8+ e os linfócitos B na resposta mais eficiente frente a um antígeno. As etapas que caracterizam uma resposta imune adaptativa são: apresentação do antígeno, ativação e diferenciação dos linfócitos T, expansão clonal e morte celular para retorno à homeostasia via AICD. Este tipo de morte ocorre via FAS/FASL. Tendo em vista que o mecanismo pelo qual a PGE2 inibe a expressão de fasl ainda não é conhecido, o presente trabalho tem como objetivo compreender o mecanismo molecular de atuação da PGE2 sobre os linfócitos T CD4+ na inibição gênica do FASL, tendo como hipótese o envolvimento do repressor transcricional ICER. Foi verificado que PGE2 10-8 M é capaz de proteger as DO11.10 da AICD e que, nesta concentração, ela induz a expressão de icer. Este repressor apresenta uma expressão transiente e observa-se seu aumento concomitantemente à inibição da expressão de fasl. Sendo assim, sugere-se a participação de ICER na via de inibição do fasl pela PGE2. / CD4+ T lymphocytes orchestrate the adaptive immune response, helping the macrophages, CD8+ T lymphocytes and B lymphocytes to reach an efficient antigen-specific immune response. The adaptive immune response is characterized by different phases: antigen, T lymphocyte activation and differentiation, clonal expansion and, finally, clonal cell death to return to homeostasis via AICD. This cell death occurs via FAS/FASL. Since the mechanism by which PGE2 inhibits fasl expression is not known, our aim is to understand the molecular mechanism responsible for PGE2 inhibition of fasl in CD4+ lymphocytes. Our hypothesis is that the transcriptional repressor ICER, which binds to CRE sites on gene promoters, is involved in this process. We verified that PGE2 10-8 M protects DO11.10 cells from AICD, and in that concentration, PGE2 increases icer expression. This repressor has a transient expression and we observed that its expression is increased in the same time that fasl expression is inhibited. Thus, we suggest the involvement of ICER in fasl inhibition pathway by PGE2.

Os linfócitos T CD4+ orquestram a resposta imune adaptativa, auxiliando os macrófagos, os linfócitos T CD8+ e os linfócitos B na resposta mais eficiente frente a um antígeno. As etapas que caracterizam uma resposta imune adaptativa são: apresentação do antígeno, ativação e diferenciação dos linfócitos T, expansão clonal e morte celular para retorno à homeostasia via AICD. Este tipo de morte ocorre via FAS/FASL. Tendo em vista que o mecanismo pelo qual a PGE2 inibe a expressão de fasl ainda não é conhecido, o presente trabalho tem como objetivo compreender o mecanismo molecular de atuação da PGE2 sobre os linfócitos T CD4+ na inibição gênica do FASL, tendo como hipótese o envolvimento do repressor transcricional ICER. Foi verificado que PGE2 10-8 M é capaz de proteger as DO11.10 da AICD e que, nesta concentração, ela induz a expressão de icer. Este repressor apresenta uma expressão transiente e observa-se seu aumento concomitantemente à inibição da expressão de fasl. Sendo assim, sugere-se a participação de ICER na via de inibição do fasl pela PGE2. / CD4+ T lymphocytes orchestrate the adaptive immune response, helping the macrophages, CD8+ T lymphocytes and B lymphocytes to reach an efficient antigen-specific immune response. The adaptive immune response is characterized by different phases: antigen, T lymphocyte activation and differentiation, clonal expansion and, finally, clonal cell death to return to homeostasis via AICD. This cell death occurs via FAS/FASL. Since the mechanism by which PGE2 inhibits fasl expression is not known, our aim is to understand the molecular mechanism responsible for PGE2 inhibition of fasl in CD4+ lymphocytes. Our hypothesis is that the transcriptional repressor ICER, which binds to CRE sites on gene promoters, is involved in this process. We verified that PGE2 10-8 M protects DO11.10 cells from AICD, and in that concentration, PGE2 increases icer expression. This repressor has a transient expression and we observed that its expression is increased in the same time that fasl expression is inhibited. Thus, we suggest the involvement of ICER in fasl inhibition pathway by PGE2.

Orientador: Alexandra Ivo de Medeiros / Resumo: A prostaglandina E2 (PGE2) é um mediador lipídico que participa tanto na diferenciação como na expansão de linfócitos T helper (Th) Th1 e Th17. Esse prostanoide vem sendo descrito como um importante mediador envolvido no agravamento da Encefalomielite Autoimune Experimental (EAE). A EAE é uma doença mediada por células Th1/Th17 autorreativas, responsáveis pela intensa resposta inflamatória contra antígenos do sistema nervoso central (SNC). Alguns estudos descrevem que a inibição da síntese desse prostanoide, ou o bloqueio de seus receptores EP, reduzem os níveis de IL-17A e IFN- e atenuam drasticamente o desenvolvimento da doença. A coexistência de linfócitos Th1 e Th17 na EAE, assim como a presença de células Th17 produtoras de IFN-γ (Th1-like) no SNC sugerem uma possível plasticidade destas subpopulações de linfócitos. No entanto, até o momento, não há relatos na literatura se a presença de PGE2, presente no SNC, estaria envolvida na plasticidade de linfócitos Th17 em Th1 nessa autoimunidade. Portanto, a hipótese desse estudo é que as células Th17 migrariam para o SNC e desencadeariam o recrutamento de células inflamatórias e o aparecimento dos primeiros sinais clínicos da doença. A presença de PGE2, associado esse microambiente inflamatório, favoreceria a plasticidade das células Th17 para um padrão Th1, resultando na diferenciação de células T CD4+ patogênicas (IL17+IFN+) e células Th1-like. Os resultados obtidos demonstram que as células Th17, quando cultivadas em cond... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

PGE2 plays an important role in liver inflammation and carcinogenesis. Its metabolism is regulated by a cascade of reactions catalyzed by enzymes including COX-1/2, mPGES-1/2, 15-PGDH. Among these regulators, mPGES-1 is a cytokine-inducible enzyme mainly responsible for catalyzing terminal synthesis of PGE2, 15-PGDH catalyzes the oxidation of PGE2 to 15-keto-PGE2. In this context, we exogenously expressed mPGES-1 or 15-PGDH genes in mice hepatocytes to constitute a physiological condition ideal for evaluating PGE2 and its metabolites function in liver pathogenesis. In the first part, we developed transgenic mice with targeted expression of mPGES-1 in the liver and assessed the response of the transgenic mice to Fas-induced hepatocyte apoptosis and acute liver injury. Compared to wild type mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase and aspartate transaminase levels, less hepatic necrosis/apoptosis, and lower levels of caspase activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analyses revealed increased expression and activation of the serine/threonine kinase Akt and associated anti-apoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling. This finding is consistent with previous reports of the anti-apoptotic and pro-proliferative role of PGE2. Our results suggest that induction of mPGES-1 or treatment with PGE2 may represent a potential therapeutic strategy for the prevention and treatment of Fas-associated liver injuries. In the second part, we generated transgenic mice with targeted expression of 15-PGDH in the liver and the animals were subjected to LPS/GalN-induced acute liver inflammation and injury. Compared to the wild type mice, the 15-PGDH Tg mice showed lower levels of alanine aminotransferase and aspartate aminotransferase, less liver tissue damage, less hepatic apoptosis/necrosis, less macrophage activation, and lower inflammatory cytokine production. In Kupffer cell cultures, treatment with 15-keto-PGE2 or the conditioned medium (CM) from 15-PGDH Tg hepatocyes inhibited LPS-induced cytokine production. Both 15-keto-PGE2 and the CM from15-PGDH Tg hepatocyes also up-regulated the expression of PPAR-γ downstream genes in Kupffer cells. In cultured hepatocytes, 15-keto-PGE2 treatment or 15-PGDH overexpression did not influence TNF-α-induced hepatocyte apoptosis. These findings suggest that 15-PGDH protects against LPS/GalN-induced liver injury and the effect is mediated via 15-keto-PGE2, which activates PPAR-γ in Kupffer cells and thus inhibits their ability to produce inflammatory cytokines. Accordingly, we observed that the PPAR-γ antagonist, GW9662, reversed the effect of 15-keto-PGE2 in Kupffer cell in vitro and restored the susceptibility of 15-PGDH Tg mice to LPS/GalN-induced acute liver injury in vivo. Our findings not only support the pro-inflammatory role of PGE2, but also reveal a novel anti-inflammatory role of 15-keto-PGE2. The data suggest that induction of 15-PGDH expression or utilization of a 15-keto-PGE2 analog may be therapeutic for treatment of endotoxin-associated liver inflammation/injury. Consistent with a pro-carcinogenic role for PGE2, overexpression mPGES-1 enhances growth of either HCC or cholangiocarcinoma cells, while overexpression 15-PGDH inhibits tumor cell growth in vitro. In the third part, we use a pharmacological method to induce 15-PGDH in cholangiocarcinoma tumor cells to inhibit PGE2 production. Our results indicated that treatment of human cholangiocarcinoma cells (CCLP1 and TFK-1) with ω-3 PUFA (DHA) or transfection of these cells with the Fat-1 gene (encoding Caenorhabditis elegans desaturase which converts ω-6 PUFA to ω-3 PUFA) significantly increased 15-PGDH protein level in cholangiocarcinoma cell lines. Human cholangiocarcinoma cells treated with DHA or transfected with a Fat-1 expression vector showed reduction of miRNA26a and miRNA26b (both miRNAs target 15-PGDH mRNA thus inhibiting 15-PGDH translation). Consistent with these findings, we observed that overexpression of miR26a or miR26b decreased 15-PGDH protein, reversed ω-3 PUFA-induced accumulation of 15-PGDH protein, and prevented ω-3 PUFA-induced inhibition of cholangiocarcinoma cell growth. Knockdown of 15-PGDH also attenuated ω-3 PUFA-induced inhibition of tumor cell growth. We observed that ω-3 PUFA suppressed miRNA26a and miRNA26b by inhibiting c-myc, a transcription factor that co-regulates a gene cluster comprised of miR-26a/b and carboxy-terminal domain RNA polymerase II polypeptide A small phosphatases (CTDSPs). Accordingly, overexpression of c-myc enhanced the expression of miRNA26a/b and prevented ω-3 PUFA-induced inhibition of tumor cell growth. Taken together, our results support a pro-tumorigenic role for PGE2, and suggest induction of 15-PGDH as potential way for the prevention and treatment of human cholangiocarcinoma. / 1 / LU YAO

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Intra-articular bupivacaine is a common analgesic used in dogs with orthopedic disease. Bupivacaine has been linked to chondrotoxicity. The mechanism for bupivacaine's chondrotoxicity is unknown, but may involve inflammation. Prostaglandin E2 (PGE2) is an inflammatory mediator and a marker of joint inflammation. The aim of this study was to compare synovial fluid PGE2 concentrations after a single intra-articular injection of bupivacaine with a saline control in normal canine stifles. We hypothesized that bupivacaine stifles would have a significantly elevated PGE2 concentration compared to controls. Stifles from eight healthy, adult Beagles were randomly selected as the treated stifle and infused with bupivacaine. The contralateral stifle was injected with saline. Synovial fluid was collected before and after injection. PGE2 was quantified using a commercial ELISA. Data were transformed and mixed model ANOVA was performed with significance set at p<0.05. There were no significant differences in PGE2 concentration between treatment groups or times. Samples acquired with one or two aspiration attempts had significantly lower PGE2 concentrations than samples with =3 aspiration attempts (p=0.001). When adjusted for number of attempts, PGE2 concentrations were significantly higher 24 (p=0.003) and 48 (p=0.041) hours after injection compared to baseline in the bupivacaine group, but not in the saline group. Intra-articular bupivacaine injection did not result in increased synovial fluid PGE2 concentrations compared to controls; however, multiple aspiration attempts did, suggesting that synovial fluid PGE2 concentration is sensitive to multiple fluid collection attempts. Future studies investigating synovial fluid inflammatory mediators should consider methods to minimize aspiration attempts. / M. S. / Intra-articular bupivacaine is a popular pain relief medication commonly used in joint surgery. Despite its historically wide use, bupivacaine has been scrutinized due to its potentially toxic effects on joint cartilage. Currently, the mechanism of this toxicity has not been identified, though it may be associated with inflammation. Prostaglandin E₂ (PGE₂) is considered an indicator of joint inflammation. The purpose of this study was to quantify the concentration of PGE₂ within the joint fluid following a single injection of bupivacaine in normal canine stifles as compared to a saline control. Eight healthy, adult Beagles were used for this study. Stifles were randomized into treatment (bupivacaine) or control (saline) groups such that each dog had one stifle infused with bupivacaine and the opposite stifle with saline. Joint fluid was collected at the following time points: before injection (T0), 30 minutes, 60 minutes, 24 hours and 48 hours. Samples were analyzed in duplicate for PGE₂ concentration. There was no significant effect of treatment group (bupivacaine vs. saline) or time on joint fluid PGE₂ concentration. The number of sampling attempts did have an effect. Samples acquired with only one or two attempts had significantly lower PGE₂ concentrations than samples that required 3 or more sampling attempts. When adjusted for number of attempts, PGE₂ concentrations were significantly higher 24 and 48 hours after injection compared to baseline within the bupivacaine group, but not the saline group. Intra-articular bupivacaine injection did not result in increased joint fluid PGE₂ concentration compared to saline control. The data indicates that joint fluid PGE₂ concentration is highly sensitive to fluid collection attempts.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19887号 / 医博第4136号 / 新制||医||1016(附属図書館) / 32964 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 横出 正之, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25167号 / 医博第5053号 / 新制||医||1071(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 生田 宏一, 教授 椛島 健治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25498号 / 医博第5098号 / 新制||医||1074(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 渡邊 直樹, 教授 寺田 智祐, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Os antiinflamatórios não esteroidais (AINEs) constituem um grupo de medicamentos de uso rotineiro na terapêutica clínica, principalmente no tratamento do processo inflamatório e doloroso. O efeito colateral mais comum constatado pelo seu uso é a irritação da mucosa gástrica. A atividade ulcerogênica dos AINEs tem variabilidade diária, uma vez que quando administrados à noite, são melhor tolerados que durante o dia. Tal observação chama a atenção para a possível ação do hormônio pineal, melatonina, sintetizado no período de escuro, tanto por animais de hábitos noturnos como diurnos. No presente estudo foram investigados os efeitos da administração de melatonina sobre a atividade antiinflamatória e lesão gástrica induzida pelo piroxicam e o efeito da melatonina e do piroxicam sobre a síntese de PGE2 pela mucosa gástrica, testando esse hormônio nos seguintes modelos: edema de pata de rato induzido por carragenina, lesão da mucosa gástrica induzida pelo piroxicam e determinação da síntese de PGE2 por enzima-imunoensaio (EIA). A administração intragástrica de melatonina não alterou o efeito aniinflamatório do piroxicam, mas inibiu o efeito colateral ulcerogênico. O efeito antiulcerogênico da melatonina foi observado após administração intragástrica, mas não após a administração subcutânea. As diferenças observadas entre as duas vias de de administração sugere um efeito local desse hormônio sobre a mucosa gástrica. O efeito antiulcerogênico da mealtonina intragástrica esta relacionada à prevenção da inibição da produção der PGE2 da mucosa gástrica pelo piroxicam. A pinealectomia aumentou o efeito ulcerogênico da adminstração noturna de piroxicam. Mesmo utilizando fenilbutazona, que é um AINEs ulcerogenicamente mais potente, no período da manhã, quando o efeito ulcerogênico dos AINEs parece ser acentuado, a melatonina administrada intragastricamente promoveu proteção da mucosa gástrica. Por outro lado, a melatonina administrada intragastricamente não interferiu com ulceração induzida pelo estresse, sugerindo que o efeito antiulcerogênico da melatonina está relacionado mais especificamente com fatores importantes na patogênese da lesão gástrica induzida pelos AINEs (piroxicam e fenilbutazona) em ratos, prevenindo a inibição da síntese de PGE2 da mucosa gástrica induzida pelo piroxicam, sem afetar a ação antiinflamatória. / Non-steroidal antiinflammatory drugs are, the most frequently consumed drugs worlwide. They also cause gastrointestinal adverse effects, like gastric ulceration and bleedig. Several reports indicate that, both antiinflammatory effect and the susceptibility to mucosal damage produced by NSAIDs in rats and humans show a circadian variation. Nighttime administration of NSAIDs is better tolerated than morning administration in human and rats. Melatonin, the principal hormone of the pineal gland is secreted at night both in nocturnal and diurnal animals. The purpose of this experiment was to study the effect of melatonin on the antiinflammatory action and gastric mucosal damage induced by piroxicam and the effect of melatonin and piroxicam on the gastric mucosal prostaglandin E2 (PGE2) synthesis. Intragastric administration of melatonin significantly attenuated the gastric lesions induced by piroxicam, but, did not entiinflammatory action accessed by measuring paw edema after carrageenin adminstration. Melatonin anti-ulcerogenic effect was observed after intragastric, but not subcutaneus administration. The differences observed between the two administration routes point to a local effect of the hormone on the stomach mucosa. Pinealectomy increases the ulcerogenic effect of nocturnal administred piroxicam. The anti-ulcerogenic effect of intragastric melatonin was related to prevention of the inhibition of gastric mucosal PGE2 production induced by piroxicam. Indeed, intragastric administration of melatonin protect the gastric mucosa damage induced by effect on stress-induced gastric ulceration. These data suggest that melatonin may attenuate the severity of NSAID-induced gastric mucosal lesions in rats by preventing NSAID-induced inhibition of mucosal PGE2 production whithout affecting th anti-inflamamtory action.