Durante a resposta inflamatória alguns mediadores lipídicos, destacando-se o Leucotrieno B4 (LTB4) e a Prostaglandina E2 (PGE2), são liberados no meio e desencadeiam uma série de eventos moleculares e celulares. Não diferentemente do que ocorre em outros tecidos, eventos inflamatórios na polpa também geram a produção destes mediadores lipídicos. Na polpa, entretanto, há presença de células-tronco que persistiram e permanecem indiferenciadas, mas com potencial capacidade de diferenciação em células odontoblast-like. O objetivo do presente estudo foi avaliar a expressão de genes codificadores da síntese e mineralização da matriz dentinária, bem como avaliar a viabilidade celular diante de células indiferenciadas da polpa de camundongos (linhagem OD-21) após estimulação com microesferas de LTB4 e PGE2. Foram preparadas microesferas contendo os mediadores lipídicos (0,01 μM e 0,1 μM) pelo método de simples emulsão óleo-água seguido do processo de evaporação do solvente. Células OD-21 foram mantidas em cultura com os diferentes tratamentos por um período de estimulação de 24 horas para realização de teste de viabilidade celular (Ensaio Colorimétrico MTT). A seguir foi realizada avaliação da expressão gênica relativa dos genes Ibsp, Bmp2, Runx2, Alpl, Msx1 e Bglap pelo método de transcrição reversa e reação em cadeia de polimerase em tempo real (qRT-PCR), utilizando o sistema TaqMan® após estimulação por períodos de 3, 6, 24, 48 e 72 horas. Foi observado aumento significativo no número de células viáveis após um período de 24 horas de estimulação com microesferas contendo PGE2 a 0,1 μM. A estimulação com microesferas, porém, não induziu a expressão de Alpl, Msx1 e Bglap, mas o fez para os genes Ibsp, Bmp2 e Runx2, em períodos mais curtos de estimulação. A PGE2 encapsulada em microesferas foi capaz de modificar o padrão de expressão gênica de Bmp2 e Runx2 em cultura de células OD-21, sendo que o LTB4 mostrou um papel inibidor da expressão gênica de Ibsp. Estes resultados indicam que estes mediadores podem ser importantes no processo de proliferação e diferenciação de células da polpa dental. / During the inflammatory response some lipid mediators, especially Leukotriene B4 (LTB4) and Prostaglandin E2 (PGE2), are released into the environment and trigger a series of molecular and cellular events. Inflammatory events in the pulp also generate the production of these lipid mediators. However in the pulp there is the presence of stem cells that persisted and remain undifferentiated, but with ability to differentiate into odontoblast-like cells. The aim of to this study was to evaluate of gene expression encoding to the synthesis and mineralization of dentin matrix and to assess cell viability in undifferentiated cells of mice pulp (OD-21 strain) after stimulation with PGE2 and LTB4 microspheres. Microspheres containing lipid mediators were prepared (0.01 μM and 0.1 μM) using an oil-in water emulsion solvent extraction-evaporation process. OD-21 cells were maintained in culture with the different treatments during 24 hours for cell viability test (MTT colorimetric assay). After was made the evaluation of the relative gene expression of genes Ibsp, Bmp2, Runx2, Alpl, Msx1 and Bglap by reverse transcription method and real-time polymerase chain reaction (qRT-PCR), using the TaqMan® system after stimulation for 3, 6, 24, 48 and 72 hours. There was a significant increase in the number of viable cells following a 24 hours stimulation with microspheres containing PGE2 0.1 μM. The microspheres stimulation did not induce the expression of Alpl, Msx1 and Bglap, but did in genes Ibsp, Runx2 and Bmp2, in shorter periods of stimulation. PGE2 microespheres modified the pattern of Bmp2 and Runx2 gene expression in OD-21 cell culture whereas LTB4 revealed an inhibitory effect on Ibsp expression. These findings indicate that lipid mediators might be important for dental pulp cell proliferation and differentiation.

Em estudo anterior, observamos que o Met-RANTES, antagonista de receptores CCR1 e CCR5 para quimiocinas, injetado pela via endovenosa (i.v.) reduziu a resposta febril induzida pelo lipopolissacarídeo (LPS) de E. coli, demonstrando o envolvimento da quimiocina RANTES (Regulada sob ativação, expressa e secretada por células T normais) nesta resposta. Além disso, a injeção intrahipotalâmica (i.h.) da RANTES dose-dependentemente aumentou a temperatura corporal de ratos, o qual foi caracterizado como febre, pois foi acompanhada de redução da temperatura da cauda, uma resposta termorregulatória para retenção de calor. Observamos também, que a RANTES aumenta a concentração de prostaglandinas no fluido cerebroespinhal (CSF) e que a febre por ela induzida é sensível aos inibidores não-seletivos para as ciclooxigenases e seletivo para COX-2 (Machado et al., 2007). No presente estudo, aprofundamos a investigação sobre os mediadores, incluindo as prostaglandinas, envolvidos na resposta febril induzida pela RANTES. Verificamos que o paracetamol reduziu, enquanto o diclofenaco de sódio aboliu a resposta febril induzida pela RANTES. Ainda, a injeção i.h. da RANTES promoveu significativa expressão do RNAm para COX-2 no hipotálamo, confirmando ser a COX-2 a enzima responsável pela síntese de prostaglandinas envolvidas no efeito pirogênico desta quimiocina. Através da administração de corante in situ e de cortes histológicos, pode-se averiguar o trajeto da cânula bem como a profundidade alcançada pela agulha durante a injeção na área pré-óptica hipotalâmica anterior (AH/POA). Padronizamos a dose do Met-RANTES (i.h.) que não seria capaz de alterar a temperatura retal dos animais. Posteriormente, avaliou-se o efeito de diferentes doses do Met-RANTES, administrado via intrahipotalâmica, na resposta febril induzida pelo LPS ou pela RANTES. Entretanto, nas doses administradas o pré-tratamento com o antagonista não foi capaz de reduzir a febre induzida por ambos os estímulos. Contudo, o Met-RANTES (i.v.) reduziu a febre induzida pelo TNF-alfa (i.h.), reproduzindo resultados anteriores. O pré-tratamento com Met-RANTES (i.v.) não modificou a febre induzida pela injeção central de interleucina (IL)-6, fator liberador de corticotropina (CRF) e bradicinina (BK). Adicionalmente, a injeção de LPS (i.v.) ou TNF-alfa (i.h.) elevou a concentração da RANTES no tecido hipotalâmico. Antalarmina (antagonista de receptores CRF1) e alfa-helical CRF9-41 (antagonista de receptores CRF1 e CRF2) que reduziram a febre induzida pelo CRF, não alteraram a febre induzida pela administração i.h. da RANTES. O antagonista de receptores B1 (DALBK) que reduziu a segunda fase da resposta febril induzida pela BK, não foi capaz de modificar a febre induzida pela RANTES. Da mesma forma, o antagonista de receptores B2 (Hoe-140) que reduziu a resposta febril induzida pela BK durante todo o período de experimentação, não modificou a febre promovida pela RANTES. Por outro lado, verificamos que o anticorpo anti-IL-6 administrado i.h. reduziu a febre induzida pela IL-6 e pela RANTES. Ainda, a injeção de LPS (i.v.) ou RANTES (i.h.) elevou a concentração de IL-6 no CSF, mas não de IL-1 e TNF-. A RANTES promoveu ativação do fator nuclear-kB (NF-kB) e aumentou a expressão do RNAm para as citocinas IL-1beta, TNF-alfa e IL-6 no hipotálamo dos animais. O pré-tratamento com Met-RANTES reduziu, na 2,5 e 6 h, a neutrofilia induzida pelo LPS. Em síntese, nossos resultados demonstram que durante a resposta febril induzida pelo LPS, este induz a síntese de TNF-alfa o qual promove a síntese da quimiocina RANTES que, ativando os receptores CCR1 e CCR5 promove a transmigração do NF-kB do citoplasma para o núcleo e a subseqüente síntese de IL-6 e de COX-2, esta última, a responsável pela síntese de prostaglandina E2 (PGE2), um dos mediadores finais da resposta febril induzida pelo LPS. Além disso, a RANTES parece ser um mediador da resposta de fase aguda, uma vez que, promove dois sinais importantes desta resposta, febre e neutrofilia. / We showed before that Met-RANTES, CCR1 and CCR5 receptor antagonist, intravenously injected (i.v.) reduced fever induced by lipopolysaccharide (LPS, E. coli), demonstrating the involvement of RANTES (Regulated on activation, normal T cells expressed and secreted) in this response. Also, intrahypothalamic (i.h.) injection of RANTES dose-dependently increased body temperature of rats, this increase was characterized as fever, because it was accompanied of a reduction in the tail skin temperature, a thermoregulatory response for heat retention. We also verified that RANTES increased the concentration of prostaglandin (PG)E2 in the cerebrospinal fluid (CSF), which was sensible to non-selective and selective blockers to cyclooxygenase (COX)-2 (Machado et al., 2007). In the present study, it was investigated which others mediators, including prostaglandins, are involved in the RANTES-induced fever. The effect of paracetamol and sodium diclofenac on fever induced by RANTES was also investigated. Paracetamol reduced, while sodium diclofenac abolished the RANTES-induced fever. The intrahypothalamic (i.h.) RANTES injection promoted a significant COX-2 mRNA expression in the hypothalamus, confirming the role of the COX-2 enzyme in the synthesis of prostaglandin involved in the pyrogenic effect of this chemokine. Through administration of dye in situ and histological analyses, we confirmed that the injection in the preoptic area of the anterior hypothalamus (AH/POA) was correct. Subsequently, we evaluated the effect of different doses of Met-RANTES (i.h.) in the fever induced by both LPS and RANTES. Centrally injected, Met-RANTES did not modify the fever induced by LPS or RANTES. On the other hand, Met-RANTES (i.v.) reduced TNF-alpha-induced fever, but did not modify the fever induced by interleukin (IL)-6, corticotrophin releasing factor (CRF) and bradykinin (BK). Additionally, the injection of LPS (i.v.) or TNF-alpha (i.h.) increased RANTES concentration in the hypothalamus. Antalarmin (a CRF receptor 1 antagonist) and alpha-helical CRF9-41 (CRF 1 and 2 receptor antagonist) that reduced CRF-induced fever did not modify the fever induced by RANTES (i.h.). DALBK (bradykinin B1 receptor antagonist) that reduced the second phase of BK-induced fever did not modify RANTES-induced fever. In the same way, Hoe-140 (bradykinin B2 receptor antagonist) that reduced the fever induced by BK during the whole period of observation, did not modify RANTES-induced fever. On the other hand, we verified that anti-rat IL-6 antibody (i.h.) reduced the fever induced by both IL-6 and RANTES. In addition, the administration of LPS (i.v.) or RANTES (i.h.) increased the CSF IL-6 concentration, but not of IL-1 and TNF-. RANTES promoted nuclear factor-kB (NF-kB) activation and increased IL-1beta, TNF-alpha and IL-6 mRNA expression in the hypothalamus. Pretreatment of the animals with Met-RANTES reduced the LPS-induced neutrophilia. In synthesis, our results suggest that in the fever induced by LPS, RANTES induces TNF- synthesis, which promotes the synthesis of RANTES that, activating CCR1/CCR5 receptors, promotes NF-kB transmigration of cytoplasm to the nucleus and subsequent synthesis of IL-6 and COX-2. The latter, in turn, is responsible by (PGE2) synthesis, one of the final mediators of the febrile response induced by LPS. Moreover, RANTES seem to be a mediator of the acute phase response since it promoted two important signs of this response, fever and neutrophilia.

O glioblastoma (GBM) é o tumor mais frequente do sistema nervoso central com um alto grau de malignidade e um prognóstico desfavorável. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e/ou quimioterapia, não há tratamento eficiente disponível para o GBM. Os prostanoide são derivados do ácido araquidônico e estão envolvidas com vários processos do desenvolvimento e progressão do câncer. O objetivo deste estudo foi analisar in vitro o perfil de diferentes prostanoides nas linhagens de GBM. Além de analisar o papel dos prostanoides e dos receptores na migração celular de GBM. Os resultados demostraram um perfil dos prostanoides da série 2 diferente entre as linhagens, além da expressão dos genes envolvidos na biossíntese de PGE2. Nos ensaios de migração os dados demostraram que os tratamentos realizados com os prostanoides exógenos aumentaram a migração celular e os tratamentos com os antagonistas de EP2 e EP4 diminuiram a migração. Em conjunto esses resultados, demonstram o papel importante dos prostanoides, especialmente PGE2, no processo de migração das células de GBM. / Glioblastoma (GBM) is the most common tumor of the central nervous system with a high degree of malignancy and poor prognosis. Despite advances in surgical techniques and radiation therapy and/or chemotherapy, there is no effective treatment available for GBM. The prostanoid are derived from arachidonic acid and are involved in many processes of development and progression of cancer. The aim of this study was to analyze in vitro profile of different prostanoids in the lines of GBM. In addition to analyzing the role of prostanoids and receptors on the cell migration of GBM. The results showed a profile of series 2 prostanoids the different between the cell lines, in addition to expression of genes involved in the biosynthesis of PGE2. In migration testing data showed that the treatments performed with exogenous prostanoids increased cell migration and treatment with antagonists of EP2 and EP4 decreased migration. Together these results demonstrate the important role of prostanoids, especially PGE2, in the migration process of the GBM cells.

A modulação das respostas imunológicas em modelos experimentais infectados por Trypanosoma cruzi tem contribuído de maneira importante nas investigações de novas terapias contra a doença de Chagas. Neste estudo, avaliamos a produção de citocinas Th1 e Th2 relevantes na fase aguda da doença, assim como, níveis de prostaglandina E2, nitrito, parasitemia sanguínea e parasitemia tecidual cardíaca em ratos Wistar machos infectados pela cepa Y de T. cruzi. Foram investigados nos 7°, 14° e 21° dias de infecção, as citocinas padrão Th1: IL-2, IFN-, TNF- e padrão Th2: IL-4, IL-10. Os parâmetros foram dosados e interpretados após a administração, ou não, de meloxicam, melatonina ou ambos. A administração de melatonina contribuiu na proteção do hospedeiro submetido à infecção experimental por T. cruzi devido às ações imunomodulatórias previamente atribuídas a esta substância. O bloqueio da síntese de PGE2 atribuído à administração de meloxicam e/ou melatonina durante a fase aguda da infecção foi seguido por uma modulação de citocinas pertencentes aos padrões Th-1 e Th-2. Houve um aumento da síntese de citocinas importantes na proteção do hospedeiro durante a fase aguda da infecção, tais como IFN-, IL-2 e NO. Estes efeitos demonstrados no estudo foram benéficos, sendo evidenciados pela diminuição da carga parasitária dos animais experimentais. Os resultados poderão auxiliar a estabelecer mecanismos alternativos de tratamento na infecção chagásica aguda através de um melhor entendimento das respostas imunológicas anti-T cruzi. / The modulation of the immune responses in experimental models infected with Trypanosoma cruzi has effectively contributed for the investigations of new therapies used to treat Chagas disease. In this study, it was evaluated the production of Th1 and Th2 cytokines, which play a role during the acute phase of the disease, as well as prostaglandin E2 and nitrite, number of blood parasites and cardiac tissue parasitism in male Wistar rats infected with the Y strain of T. cruzi. Experiments were performed on 7, 14 and 21 days after infection in which Th1 cytokines such IL-2, IFN-, TNF- were done and Th2 cytokines as IL-4 and IL-10. The parameters were evaluated with/without the administration of meloxicam, melatonin or both. Melatonin contributed for the hosts protection in animals experimentally infected with T. cruzi through its immunomodulator actions. The blockage of PGE2 synthesis was attributed to the administration of meloxicam and/or melatonin during the acute phase of infection, followed by a modulation of the Th1 and Th2 cytokines. In this work enhanced levels of IFN-, IL-2 and NO were observed. The analysis of these data was beneficial for the hosts protection through a reduced number of amastigote nests in heart tissue. These results permit to establish alternative mechanisms to treat the acute chagasic infection through a better understanding of the immune responses against T cruzi.

Durante a resposta inflamatória alguns mediadores lipídicos, destacando-se o Leucotrieno B4 (LTB4) e a Prostaglandina E2 (PGE2), são liberados no meio e desencadeiam uma série de eventos moleculares e celulares. Não diferentemente do que ocorre em outros tecidos, eventos inflamatórios na polpa também geram a produção destes mediadores lipídicos. Na polpa, entretanto, há presença de células-tronco que persistiram e permanecem indiferenciadas, mas com potencial capacidade de diferenciação em células odontoblast-like. O objetivo do presente estudo foi avaliar a expressão de genes codificadores da síntese e mineralização da matriz dentinária, bem como avaliar a viabilidade celular diante de células indiferenciadas da polpa de camundongos (linhagem OD-21) após estimulação com microesferas de LTB4 e PGE2. Foram preparadas microesferas contendo os mediadores lipídicos (0,01 μM e 0,1 μM) pelo método de simples emulsão óleo-água seguido do processo de evaporação do solvente. Células OD-21 foram mantidas em cultura com os diferentes tratamentos por um período de estimulação de 24 horas para realização de teste de viabilidade celular (Ensaio Colorimétrico MTT). A seguir foi realizada avaliação da expressão gênica relativa dos genes Ibsp, Bmp2, Runx2, Alpl, Msx1 e Bglap pelo método de transcrição reversa e reação em cadeia de polimerase em tempo real (qRT-PCR), utilizando o sistema TaqMan® após estimulação por períodos de 3, 6, 24, 48 e 72 horas. Foi observado aumento significativo no número de células viáveis após um período de 24 horas de estimulação com microesferas contendo PGE2 a 0,1 μM. A estimulação com microesferas, porém, não induziu a expressão de Alpl, Msx1 e Bglap, mas o fez para os genes Ibsp, Bmp2 e Runx2, em períodos mais curtos de estimulação. A PGE2 encapsulada em microesferas foi capaz de modificar o padrão de expressão gênica de Bmp2 e Runx2 em cultura de células OD-21, sendo que o LTB4 mostrou um papel inibidor da expressão gênica de Ibsp. Estes resultados indicam que estes mediadores podem ser importantes no processo de proliferação e diferenciação de células da polpa dental. / During the inflammatory response some lipid mediators, especially Leukotriene B4 (LTB4) and Prostaglandin E2 (PGE2), are released into the environment and trigger a series of molecular and cellular events. Inflammatory events in the pulp also generate the production of these lipid mediators. However in the pulp there is the presence of stem cells that persisted and remain undifferentiated, but with ability to differentiate into odontoblast-like cells. The aim of to this study was to evaluate of gene expression encoding to the synthesis and mineralization of dentin matrix and to assess cell viability in undifferentiated cells of mice pulp (OD-21 strain) after stimulation with PGE2 and LTB4 microspheres. Microspheres containing lipid mediators were prepared (0.01 μM and 0.1 μM) using an oil-in water emulsion solvent extraction-evaporation process. OD-21 cells were maintained in culture with the different treatments during 24 hours for cell viability test (MTT colorimetric assay). After was made the evaluation of the relative gene expression of genes Ibsp, Bmp2, Runx2, Alpl, Msx1 and Bglap by reverse transcription method and real-time polymerase chain reaction (qRT-PCR), using the TaqMan® system after stimulation for 3, 6, 24, 48 and 72 hours. There was a significant increase in the number of viable cells following a 24 hours stimulation with microspheres containing PGE2 0.1 μM. The microspheres stimulation did not induce the expression of Alpl, Msx1 and Bglap, but did in genes Ibsp, Runx2 and Bmp2, in shorter periods of stimulation. PGE2 microespheres modified the pattern of Bmp2 and Runx2 gene expression in OD-21 cell culture whereas LTB4 revealed an inhibitory effect on Ibsp expression. These findings indicate that lipid mediators might be important for dental pulp cell proliferation and differentiation.

Orientador: José Luiz Laus / Banca: Carlos Augusto Araújo Valadão / Banca: Paula Diniz Galera / Resumo: Estudou-se a eficácia do meloxicam e do carprofeno, aplicados por diferentes vias, em uveítes experimentais em cães. Realizou paracentese de câmara anterior em dois momentos (M0 e M1), com intervalo de cinco horas entre si. Em M0 e M1, colheram-se 0,2 ml de humor aquoso e determinou-se a concentração de proteína total e de prostaglandina E2 (PGE2). Em um primeiro período, constituíram-se quatro grupos (n = 5), que receberam meloxicam ao final de M0 pelas vias subcutânea (GIm), subconjuntival (GIIm) e tópica (GIIIm). Um quarto grupo não recebeu tratamento (Controle). Decorridos sete dias, os animais foram submetidos aos mesmos procedimentos adotados previamente e receberam carprofeno. Avaliação clínica foi também realizada, assim como histopatologia da conjuntiva dos animais dos grupos GIIm e GIIc. Os resultados foram avaliados estatisticamente (p LÜ 0,05). Em todos os grupos, encontrou-se aumento significativo dos níveis protéicos e de PGE2 em M1 (p < 0,001). Não se observou diferença significativa entre os grupos para os valores de proteína total e de PGE2 em M1 (p > 0,05). Observou-se correlação positiva entre proteína total e PGE2 (p < 0,05) apenas no GIm, GIc, GIIIm, GIIIc e GIIm. Exsudado inflamatório de caráter agudo e hemorragia discreta foram vistos à histopatologia após a aplicação de ambos os fármacos (p > 0,05). O meloxicam e o carprofeno foram ineficazes em inibir a síntese de PGE2 e o influxo de proteínas para a câmara anterior, por qualquer uma das vias testadas. A redução nos níveis de 44% proteínas, quando o carprofeno foi utilizado pela via tópica, sugere que por esta via, ele pode ser utilizado como adjuvante no controle da uveíte em cães. / Abstract: Efficacy of meloxican and carprofen, administered by different routes, in experimental uveitis in dogs were studied. Anterior chamber paracenteses was accomplished at two different moments (M0 and M1), with a five hour interval among them. At M0 and M1, 0,2 ml of aqueous humor were collected and total protein and prostaglandin E2 (PGE2) concentration was determined. Four groups were formed in a first period (n = 5), which received meloxican at the end of M0, by the following routes: subcutaneous (GIm), subconjunctival (GIIm), and topical (GIIIm). A fourth group that received no treatment was instituted (Control). Seven days after, animals underwent the same procedures described previously and received carprofen. Clinical evaluation was also performed, as well as conjunctival histopathology of the conjunctiva of the animals of GIIm and GIIc. Results were evaluated statistically (p LÜ 0,05). In all groups, protein and PGE2 values enhanced significantly in M1 (p < 0,001). Protein and PGE2 values, did not change significantly between groups at M1 (p > 0,05). Positive correlation among total protein and PGE2 (p < 0,05) was only noted in GIm, GIc, GIIIm, GIIIc and GIIm. Inflammatory exudate of acute character and mild hemorrhage were seen at histopathology, after both agents were administered. Meloxican and carpofen were unable to inhibit PGE2 synthesis and the protein influx to the anterior chamber by any of the tested routes. The lowering of 44% in protein levels, when carprofen was used by the topical route, suggests that by this route, it can be used as an adjuvant to control uveitis in dogs. / Mestre

O glioblastoma (GBM) é o tumor mais frequente do sistema nervoso central com um alto grau de malignidade e um prognóstico desfavorável. Apesar dos avanços nas técnicas cirúrgicas e de radioterapia e/ou quimioterapia, não há tratamento eficiente disponível para o GBM. Os prostanoide são derivados do ácido araquidônico e estão envolvidas com vários processos do desenvolvimento e progressão do câncer. O objetivo deste estudo foi analisar in vitro o perfil de diferentes prostanoides nas linhagens de GBM. Além de analisar o papel dos prostanoides e dos receptores na migração celular de GBM. Os resultados demostraram um perfil dos prostanoides da série 2 diferente entre as linhagens, além da expressão dos genes envolvidos na biossíntese de PGE2. Nos ensaios de migração os dados demostraram que os tratamentos realizados com os prostanoides exógenos aumentaram a migração celular e os tratamentos com os antagonistas de EP2 e EP4 diminuiram a migração. Em conjunto esses resultados, demonstram o papel importante dos prostanoides, especialmente PGE2, no processo de migração das células de GBM. / Glioblastoma (GBM) is the most common tumor of the central nervous system with a high degree of malignancy and poor prognosis. Despite advances in surgical techniques and radiation therapy and/or chemotherapy, there is no effective treatment available for GBM. The prostanoid are derived from arachidonic acid and are involved in many processes of development and progression of cancer. The aim of this study was to analyze in vitro profile of different prostanoids in the lines of GBM. In addition to analyzing the role of prostanoids and receptors on the cell migration of GBM. The results showed a profile of series 2 prostanoids the different between the cell lines, in addition to expression of genes involved in the biosynthesis of PGE2. In migration testing data showed that the treatments performed with exogenous prostanoids increased cell migration and treatment with antagonists of EP2 and EP4 decreased migration. Together these results demonstrate the important role of prostanoids, especially PGE2, in the migration process of the GBM cells.

Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.

<p>Prostaglandin (PG) E₂ is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE₂ has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE₂ is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE₂ synthesising enzymes are not completely established.</p><p>PGE₂ was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA₂a (cPLA₂a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE₂. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH₂. The conversion of PGH₂ to the final product PGE₂ was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA₂α was markedly advantageous for the formation of PGE₂.</p><p>Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE₂ in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA₂a.</p>

Low-load repetitive work (LLRW) and mental load are important risk factors for the development of workrelated muscle pain. The link between these risk factors and the development of pain is still not understood, but stimulation of chemo-sensitive receptors in the muscle probably plays an important role. It has been suggested that sensitising substances may accumulate in the muscle during LLRW, especially when combined with mental load. The overall purpose of this thesis was to try to shed some light on the effects of LLRW on the concentration of sensitising substances (glutamate, prostaglandin E2 (PGE2), norepinephrine (NE)) and on metabolism (lactate, pyruvate and oxygenation) in the trapezius muscle of healthy controls (CON) and subjects with trapezius myalgia (TM). A first step was to investigate whether females with TM exhibit higher absolute concentrations of glutamate and PGE2 in the affected muscle during rest. Using Microdialysis (MD) females with TM and asymptomatic controls were studied during four hours of rest. [Glutamate] and [PGE2] during rest did not differ between groups. A second step was to investigate, in a simulated occupational setting, the effects of LLRW on the concentration of sensitising substances and metabolism in the trapezius muscle of TM and CON, and whether increased work duration resulted in a progressive effect. Asymptomatic females were studied during baseline rest, 30 versus 60 min work and recovery, using MD and near infrared spectroscopy (NIRS). Subjects with TM were studied during baseline rest, 30 min work and recovery. [Glutamate] and [lactate] increased in response to work, but not progressively with increased work duration. [Glutamate] was at all time points significantly lower in TM. [PGE2]and oxygenation remained unchanged during work for CON, while for TM oxygenation decreased significantly during work. In TM [pyruvate] increased during both work and recovery, and a significant interaction between groups was found for [pyruvate] during recovery; while moderately increased in CON it increased progressively in TM. The effects of LLRW with and without superimposed mental load on intramuscular [NE], muscle activity and oxygen saturation in the trapezius were also investigated and compared. Using MD, electromyography and NIRS, healthy females were studied on two occasions; during 30 min LLRW and during 30 min LLRW with superimposed mental load. During work [NE], and muscle activity, were increased, while oxygenation decreased, but no differences between occasions. However, recovery of [NE] to baseline was slower after LLRW with superimposed mental load. The findings of the present thesis suggest: (i) no inflammation, or increased interstitial [glutamate] in TM; (ii) LLRW causes an increased anaerobic metabolism in both TM and CON; (iii) no effect of work duration was found; (iv) a significant difference in the effects of LLRW on the interstitial milieu of the trapezius muscle in TM as compared to CON; (v) LLRW causes a significant increase in [NE], but superimposed mental load does not cause a further increase; (vi) LLRW with a superimposed mental load may result in a slower recovery to baseline [NE] as compared with LLRW alone.