Die Mastzellen der Haut sind bedeutende Immuneffektorzellen in der Pathogenese der Caninen Atopischen Dermatitis (CAD; OLIVRY et al. 1997). Diese Zellen schütten in der Sofort- und in der Spätphase der Überempfindlichkeitsreaktion des Typs I Entzündungsmediatoren aus. Diätetisch verabreichte Fettsäuren werden in zelluläre Membranen eingebaut und sind somit in der Lage, die Produktion und Freisetzung dieser Entzündungsmediatoren zu beeinflussen. In der Praxis konnte gezeigt werden, dass eine diätetische Ergänzung von n6- und n3-Fettsäuren im Verhältnis von 5 zu 1 eine Linderung der klinischen Symptomatik bei 40% der an CAD leidenden Hunde herbeiführte (SCOTT et al. 1997). Das Ziel der vorliegenden Arbeit war es, zu überprüfen, welche Auswirkungen der Einbau supplementierter n6- und n3-Fettsäuren auf die Fettsäurenzusammensetzung und die Prostaglandinfreisetzung caniner Mastocytomzellen (C2) hat und ob diese Zellen in Bezug auf ihren Fettsäurenstoffwechsel als Modell für die CAD geeignet sind. Die Kultivierung der Zellen erfolgte in einem Grundmedium (DEH) oder in mit 14 µM Linol- (C18:2n6, DEH-LA), Gammalinolen- (C18:3n6, DEH-GLA), Arachidon- (C20:4n6, DEH-AA), a-Linolen- (C18:3n3, DEH-LnA), Eicosapentaen- (C20:5n3, DEH-EPA) oder Docosahexaensäure (C22:6n3, DEH-DHA) angereichertem Medium. Das Wachstum der C2 wurde in allen Kulturmedien über 11 Tage kontrolliert. Für die weiteren Untersuchungen wurden die Zellen am 4. bzw. 8. Tag geerntet, zweimal mit phosphatgepufferter Kochsalzlösung gewaschen und anschließend unter Stickstoff getrocknet. Die Ermittlung der Fettsäurenzusammensetzung der C2 erfolgte mittels Gaschromatographie nach Extraktion und Umesterung der Phospholipide. Dabei wurde L-a-Phosphatidylcholin-C17:0 als Interner Standard genutzt. Für die Bestimmung der Prostaglandine (PG) D2 und E2 wurden die Zellen mit dem Wespengift Mastoparan stimuliert. PGD2 wurde mittels eines PGD2-Methoxim-Enzym-Immunoassay (EIA) und PGE2 wurde mit Hilfe eines Radio-Immunassays (RIA) bestimmt. Die C2 zeigten in allen Kulturmedien eine Vermehrung lebender Zellen bis zum 8. Kultivierungstag, danach nahm die Zahl der abgestorbenen Zellen deutlich zu. Die Fettsäurensupplementierung beeinflusste das Zellwachstum nicht. Die erhöhte Zufuhr der Fettsäuren bewirkte eine Konzentrationserhöhung der entsprechenden Fettsäuren in den C2 (LA 4,9-fach, GLA 6,9-fach, AA 6-fach, LnA 9,3-fach, EPA 6,5-fach, DHA 8,4-fach). Weiterhin wurden signifikante Erhöhungen von Fettsäurenmetaboliten, die über die Elongasen und die D6-Desaturase aus den zugegebenen Fettsäuren gebildet werden, in den C2 gefunden. Produkte der D5-Desaturase waren dagegen nur in geringen Mengen nachweisbar. Ein zeitabhängiger Effekt des Einbaus der geprüften supplementierten Fettsäuren konnte nur für LA festgestellt werden, welche nach 8 Tagen in DEH-LA kultivierten C2 signifikant stärker eingebaut wurde als nach 4 Tagen. Die vorliegenden Ergebnisse lassen die Schlussfolgerung zu, dass in den C2 eine geringe Aktivität der D5-Desaturase vorliegt. Da eine niedrige Aktivität dieser Desaturase als möglicher Pathogenesemechanismus für das Auftreten der CAD verantwortlich gemacht wird, erscheinen die C2 als Modell für weitere Untersuchungen der CAD geeignet. Die durch Mastoparan stimulierte Freisetzung von PGE2 der C2 war bei der Kultivierung der Zellen im DEH-LnA und DEH-DHA signifikant erniedrigt und im DEH-AA und DEH-EPA signifikant erhöht. Die Ursache für die unterschiedlichen PGE2-Konzentrationen in C2 nach dem Zusatz der verschiedenen n3-Fettsäuren (LnA, EPA, DHA) ist bisher unklar. Verschiedene Möglichkeiten der Beeinflussung des Prostaglandinstoffwechsels durch diese Fettsäuren werden diskutiert. Auf Grund der erhaltenen Ergebnisse können die C2 als Modell genutzt werden, um die Mechanismen der Produktion von Prostaglandinen oder anderen Entzündungsmediatoren näher zu untersuchen und somit zur Erforschung der Pathogenesemechanismen der atopischen Dermatitis des Hundes sowie des Menschen beizutragen. / Cutaneous mast cells are considered as key immune effector cells in the pathogenesis of canine atopic dermatitis (CAD; OLIVRY et al. 1997). These cells release immediate-phase and late-phase mediators of inflammation. Dietary fatty acids are incorporated in cellular membranes and seem to influence mediator production and release. A dietary intervention with n6- and n3-fatty acids with a ratio from 5 to 1 alleviated clinical symptoms in 40% of atopic dogs (SCOTT et al. 1997). The purpose of this study was to examine the effects of n6- and n3-fatty acids on the fatty acid composition and the production of prostaglandins in canine mastocytoma cells (C2) as a possible model for CAD. Cells were cultured in a basic medium (DEH) or with additional 14 µM linoleic (C18:2n6, DEH-LA), gammalinolenic (C18:3n6, DEH-GLA), arachidonic (C20:4n6, DEH-AA), a-linolenic (C18:3n3, DEH-LnA), eicosapentaenoic (C20:5n3) or docosahexaenoic acid (C22:6n3, DEH-DHA). Cell growth was examined for 11 days in all media. The cells were harvested after 4 or 8 days, washed twice with phosphated buffered saline and dried under nitrogen for fatty acid analysis. The fatty acid composition was determined by gas chromatography after extraction and transesterification of the phospholipids using di-C17-phosphatidylcholin as internal standard. For measurment of prostaglandin (PG) D2 and E2 the C2 were stimulated with the wasp venom peptide mastoparan. PGD2 was measured by PGD2-methoxim-enzymimmunoassay (EIA) and PGE2 was determined by radioimmunoassay (RIA). Cell growth increased from day 1 to 8 and decreased thereafter in all media conditions. The supplied fatty acid did not influence the cell growth. Added fatty acids increased the concentration of these fatty acids in C2 (LA 4.9-fold, GLA 6.9-fold, AA 6-fold, LnA 9.3-fold, EPA 6.5-fold, DHA 8.4-fold). Futhermore elongated and D6-desaturated products of the corresponding fatty acids were significantly elevated, however D5-desaturated products were not measurable. An increased time dependent incorporation was only detectable for LA after culturing C2 in DEH-LA. The results let us assume that C2 has no activity of the D5-desaturase. If the assumed low activity of these desaturase is one of the mechanisms underlying the pathogenesis of CAD, C2 seems to be an adequate model for CAD. The production of PGE2 after stimulation with mastoparan was significantly reduced when C2 were cultured in DEH-LnA and DEH-DHA and was significantly increased when C2 were cultured in DEH-AA and DEH-EPA. The reason for the different PGE2-production in C2 after the treatment with the n3-fatty acids (LnA, EPA or DHA) being unsettled. The observed results suggest, that C2 could be used to investigate the mechanisms of production and release of prostaglandins or other mediators as a model to improve our understanding of the pathogenesis of canine or human atopic dermatitis.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21403号 / 生博第404号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 HEJNA,James, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Capsaicin, prostaglandin E2 (PGE2) and polydopamine (PDA) were used to target macrophage and myofibroblast activity to reduce biomaterial-induced fibrosis. The lifetime and efficacy of implantable biomedical devices are determined by the foreign body response. Immediately after implantation, proteins nonspecifically adsorb onto the material and initiate inflammation. Macrophages recruited to the site can differentiate into M1 and M2 phenotypes and upregulate inflammation and fibrosis which interferes with the intended function. M1 macrophages secrete pro-inflammatory mediators that induce chronic inflammation and promote myofibroblast differentiation while M2 macrophages are wound healing cells that suppress inflammation and regulate fibroblast activity. The fibrotic tissue is developed by myofibroblasts which produce collagen in an unregulated fashion. Collagen thickening and biomaterial encapsulation decreases efficacy and sensitive of biomedical devices. We investigated the in vitro and in vivo effects of capsaicin, PGE2 and polydopamine surface modification on macrophages and myofibroblasts. Capsaicin and PGE2 reduced poly(lactic-co-glycolic) acid (PLGA)-induced fibrosis by promoting M2 macrophage phenotype to secrete anti-inflammatory IL-10 and suppressing myofibroblast marker α-smooth muscle actin (α-SMA). Capsaicin decreased collagen by 40% and upregulated IL-10 secretion by 35% while PGE2 reduced collagen by 55% after 14 days of implantation and 40% less collagen after 42 days. PDA was used to bind an anti-fibrotic compound to the surface of a poly(dimethyl siloxane) (PDMS-PDA) to reduce fibrosis. However, PDMS-PDA controls gave an unexpected result by reducing fibrosis to the same extent as anti-fibrotic compound bound PDMS- v PDA. PDA modification reduced cellularity by 50% and significantly decreased collagen thickness by 30%. Overall, our results showed that biomaterial-induced fibrosis can be reduced by promoting M2 macrophage activity and inhibiting myofibroblast differentiation. This research demonstrates three compounds that have potential to reduce fibrosis and extend the lifetime and efficacy of implantable biomedical devices. / Thesis / Master of Applied Science (MASc) / Capsaicin, prostaglandin E2 (PGE2) and polydopamine were used to reduce scar tissue development around implanted polymers. Biomedical devices implanted in the body can undergo severe scar tissue formation, or fibrosis, and fail. Fibrosis is described by the accumulation of collagen and encapsulation of an implanted polymer. Macrophages regulate fibrosis by secreting pro-fibrotic compounds and myofibroblasts produce unregulated amounts of collagen. In this thesis, capsaicin, PGE2 and polydopamine were incorporated into implants to target macrophage and myofibroblast activity and reduce fibrosis in mice. Capsaicin and PGE2, released from a degradable polymer, altered macrophages to secrete anti-fibrotic compounds and decreased collagen by 40% and 55%, respectively. Polydopamine surface modified implants gave an unexpected result and suppressed overall cell activity to reduce fibrosis by 30%. The research conducted shows the potential of these compounds to reduce fibrosis and extend the lifetime of implantable devices.

Erythema is a hallmark skin response to excessive ultraviolet radiation (UVR) and is associated with cutaneous inflammation. Both are mediated by inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2) and chemoattractants such as 12-hydroxyeicosatetraenoic acid (12-HETE) leading to vasodilation and increased leukocyte infiltration. The erythematous response is more pronounced in individuals with low basal melanin levels or who fail to respond to UVR with a robust up-regulation of melanogenesis. While melanin production is a key function of melanocytes, these cells can also produce NO and PGE2, and are located in close proximity to the dermal vasculature. It has been hypothesized that melanocytes with poor melanogenic capacity may participate in the inflammatory response to UVR. The aim of this project was to investigate the inflammatory response in the skin of individuals with either skin phototype (SPT) 1 or 4 to UVR. Sixteen normal healthy individuals were selected for study (8 SPT-1 & 8 SPT-4). Buttock skin was investigated by immunohistochemistry for leukocyte subtypes, eicosanoid producing enzymes and NO synthases under basal and UVR-stimulated conditions. In addition primary cultures of epidermal melanocytes (EM) were established from 16 individuals (8 SPT-1 & 8 SPT-4) and assessed for the presence of eicosanoid-producing enzymes, melanogenic enzymes and NO synthases, by immunocytochemistry, Polymerase Chain Reaction and Western Blotting and for the production of the main pro-inflammatory eicosanoid PGE2 by ELISA and Mass Spectrometry. Moreover, the fatty acid composition of cultured melanocytes was assessed by Gas Chromatography. Results showed that individuals with SPT-1 had significantly greater neutrophil infiltration into the epidermis than those with SPT-4 at 24 hrs post-UVR. Moreover, CD3+ lymphocyte infiltration into the dermis was significantly greater in individuals with SPT-4 than those with SPT-1 at 24 and 72 hrs post-UVR. NOS-1, NOS-3, 12-LOX and COX-2 expression were significantly increased in SPT-1 skin, while NOS-2 and 15-LOX were significantly increased in SPT-4 skin. As 12-LOX and COX-2 products are chemoattractive (for neutrophils) and pro-inflammatory respectively these data could explain the greater observed neutrophil infiltration in SPT-1. The 15-LOX product (15-HETE) is anti-inflammatory and may suggest that 15-LOX up-regulation in SPT-4 skin may aid resolution of the sunburn response, which in part may be mediated by CD3+ lymphocytes and a class-switch in eicosanoid production from COX to LOX products. Melanocyte primary cultures surprisingly showed that SPT was not correlated with melanin content or melanogenic enzyme expression/activity suggesting that all melanocytes in vitro contained the necessary cellular machinery to produce melanin. This finding may reflect also their equal treatment under these enriched culture conditions, which may or may not be available to these cells in situ. Moreover, all melanocytes expressed the necessary machinery (PLA2, COX-1, cPGES) to produce PGE2. However, only some cultures did so at baseline and in response to UVR, and this was not correlated with SPT. A positive correlation was found however between expression level of dopachrome tautomerase (DCT) and protection against PGE2 production in response to UVR, which may suggest a novel role for DCT unrelated to melanogenesis. In summary this research project has generated data that highlights differences between the skin of individuals with SPT-1 and those with SPT-4, and may provide evidence that the keratinocyte partner contributes significantly to the SPT-associated response. This research may also suggest DCT as a novel therapeutic target to protect EM from participation in the UVR-associated inflammatory response in skin.

En dépit du nombre croissant d’études cliniques sur le canal artériel (CA), des failles méthodologiques entretiennent plusieurs incertitudes concernant l’efficacité et la sécurité des traitements chez les bébés nés prématurés. L’objectif de cette recherche était de comparer les concentrations de prostaglandine E2 (PGE2) et les mesures du flux mésentérique par échographie Doppler chez les enfants nés prématurément et ayant un canal artériel traité à l’ibuprofène par voie intraveineuse ou entérale, en utilisant la méthodologie randomisée contrôlée et à double insu. Dans notre étude pilote, 20 nouveau-nés prématurés de moins de 34 semaines ayant un CA symptomatique confirmé par échocardiographie, furent randomisés au traitement à l’ibuprofène par voie intraveineuse ou entérale. La voie d’administration fut maintenue à l’insu de l’équipe traitante, des cardiologues et des investigateurs. Des dosages des prostaglandines plasmatiques ont été mesurés avant le début du traitement ainsi que 3, 24 et 48 h après le début du traitement. Les mesures du flux mésentérique ont été effectuées avant le traitement à l’ibuprofène ainsi que 1 h et 3 h après le traitement. Nous avons démontré à partir de nos observations que les niveaux plasmatiques de prostaglandines E2 diminuent chez les patients ayant répondu au traitement à l’ibuprofène, indépendamment de la voie d’administration. Nous n’avons pas observé de changement dans l’évolution des dosages de PGE2 chez les patients qui n’ont pas répondu au traitement. Les paramètres mesurés par échographie Doppler au niveau de l’artère mésentérique supérieure n’étaient pas affectés par la voie d’administration du traitement à l’ibuprofène, intraveineuse ou entérale. La présente étude suggère ainsi que le traitement du CA par ibuprofène intraveineux ou entéral n’influe pas sur le flux sanguin mesuré par échographie Doppler. La baisse de la prostaglandine E2 coïncide avec la fermeture du CA, et son dosage pourrait jouer un rôle dans la gestion du traitement. Nous avons démontré la faisabilité d’une étude clinique randomisée à double insu dans le traitement du canal artériel; une méthodologie qui devrait désormait être employé dans la recherche clinique sur les traitements de la persistance du CA. / Despite the growing body of research on the patent ductus arteriosus (PDA), issues with clinical research methodology impairs much of our understanding regarding treatment efficacy and safety in the preterm population. The purpose of this study was to determine plasma prostaglandin E2 (PGE2) concentrations in preterm infants with symptomatic persistence of the ductus arteriosus treated with IV and oral ibuprofen, and measure Doppler flow parameters in the superior mesenteric artery, utilizing randomized controlled and double-blind methodology. Twenty patients age < 34 wks with a symptomatic PDA confirmed by echocardiography randomized to oral vs intravenous ibuprofen regimen. Treating physician, cardiologists and study investigators were blinded to treatment allocation. Plasma PGE2 levels were measured prior to ibuprofen treatment and at 3, 24 and 48 h after treatment. Mesenteric Doppler measurements were taken prior to ibuprofen treatment, and 1 h and 3 h after treatment. Our results showed that plasma PGE2 levels decreased over time in patients that exhibited ductal closure after IV or oral ibuprofen treatment; no time-dependent changes in PGE2 were seen in subjects that failed to respond to ibuprofen. Superior mesenteric artery Doppler flow measurements were not affected by ibuprofen treatment (IV or oral), regardless of efficacy on ductal closure and of PGE2 changes. We conclude that treatment with oral or intravenous ibuprofen does not impact on superior mesenteric artery blood flow measured by Doppler ultrasound. Decreases in plasma PGE2 concentrations coincide with ibuprofen efficacy, and may be more cost-effective to monitor than ultrasound. This study also demonstrated the successful use of double blinded randomized controlled research methodology, which should be more strictly applied in future clinical research on PDA treatment.

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.

O carcinoma epidermóide de cabeça e pescoço (CECP) representa 6ª malignidade mais comum no mundo. Para melhor entender os mecanismos envolvidos na iniciação tumoral, progressão e metástase, é necessária a elucidação dos eventos moleculares que guiam esses processos. É também importante a investigação da interação e modulação das células tumorais e seu microambiente. A participação de agentes inflamatórios no desenvolvimento e manutenção do CECP pode ser resumida na superexpressão da cicloxigenase 2 (COX-2) e na secreção de prostaglandina E2 (PGE2) pelas células tumorais. A PGE2 ativa seus receptores EP1-4 que são ligados a proteínas G. As proteínas G ativam outras vias de sinalização responsáveis por processos celulares como proliferação e angiogênese. Embora a participação do EP2 no câncer de cólon seja bem estabelecida, o papel dos receptores de PGE2 no CECP ainda permanece incerto. Este trabalho teve como objetivo avaliar o papel da PGE2 e de seus receptores na proliferação celular em linhagens celulares de CECP, bem como a expressão dos receptores em tissue microarrays de CECP. Inicialmente as linhagens de CECP foram utilizadas para analisar o padrão de expressão da COX-2 e dos receptores EP1-4 por meio da técnica de western blotting. A inibição da secreção da PGE2 pelos inibidores de COX-2 foi mensurada por meio da técnica de ELISA. A expressão dos receptores EP1-3 e da COX-2 foi também avaliada por meio da imuno-histoquímica em dois diferentes tissue microarray. A fim de esclarecer a indução da proliferação celular pela PGE2 e de apontar um de seus receptores como responsável pelo processo, duas PGE2 sintéticas, um antagonista do EP2 e um antagonista do EP3 foram utilizados para estimular a proliferação celular. Foi realizado o bloqueio do receptor EP2 por meio da interferência de RNA. Seus efeitos sobre a proliferação foram avaliados por meio do ensaio de incorporação de timidina. Os resultados mostraram que o CECP expressa constitutivamente a COX-2, o EP1, o EP2 e o EP3; e que é capaz de secretar PGE2. Os inibidores de COX-2 inibiram a secreção de PGE2 em baixas concentrações, mas não foram capazes de inibir a proliferação. A COX-2 e os receptores EP1-3 foram amplamente expressos nos tissue microarrays. Foi observada correlação entre EP1 e EP2; EP1 e EP3; e EP2 e EP3 (p<0,05). Somente o EP1 mostrou correlação com a COX-2 (p<0,05). A PGE2 induziu a proliferação por meio da indução da síntese de DNA nas linhagens celulares de CECP. O agonista de EP3 também induziu a síntese de DNA, sugerindo sua participação na proliferação dos CECPs. Os efeitos do siRNA para EP2 sobre a síntese de DNA não foram conclusivos. As proteínas ativadas por segundos mensageiros do EP2 também não foram afetadas pelo bloqueio do mesmo. Este estudo indica três importantes achados: 1. a PGE2 é secretada por linhagens de CECP; 2. a COX-2 é superexpressa nos CECPs; 3. os receptores de PGE2 são constitutivamente expressos nos CECPs. No entanto, esse trabalho mostra que esta via inflamatória parece ser independente aos mecanismos indutores da proliferação nos CECPs. / Head and neck squamous cell carcinoma (HNSCC) is the 6th most common malignant lesion worldwide. To better understand the mechanisms of tumor initiation, progression, and metastasis a better understanding of the molecular networks that guides these process is needed. Towards this goal, it is important to investigate the interaction and modulation of cancer cells over its surrounding microenvironment. The involvement of inflammatory agents in HNSCC development and maintenance can be resumed in the overexpression of cycloxygenase 2 (COX-2) and secretion of prostaglandin E2 (PGE2) by tumor cells. Prostaglandin E2 activates its receptors EP1-4 which are coupled to G proteins. G protein activates other pathways responsible for cellular processes such as proliferation and angiogenesis. The participation of EP2 in colon cancer is well established however the role of PGE2 receptors in HNSCC is still poorly understood. This work aims to investigate the role of PGE2 and its receptors in cellular proliferation in HNSCC cell lines and the clinical relevant expression pattern in HNSCC tissue microarrays. HNSCC cell lines were initially used to access the expression pattern of COX-2 and EP1-4 by using western blotting technique. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA antibody specific assay. Also, EP1, EP2, EP3 and COX-2 expression were evaluated by immuno-histochemistry in two different sets of HNSCC tissue microarrays. To address the question about PGE2 inducted cell proliferation and which PGE2 receptor are involved in the process, two synthetic PGE2, an EP2 agonist and an EP3 agonist were used to stimulate cell proliferation. Finally, the knockdown of EP2 receptor was performed by siRNA transfection assay and its effect was evaluated in cell proliferation by radioactive thymidine incorporation assay. The results presented here shows that HNSCC constitutively express COX-2, EP1, EP2 and EP3 and that they are able to secret PGE2. COX-2 selective inhibitors are able to suppress PGE2 secretion in lower concentrations but not to inhibit cell proliferation. Also, COX-2, EP1, E2 and EP3 are widely expressed in HNSCC tissue microarrays. A correlation between EP1 and EP2; EP1 and EP3; and EP2 and EP3 (p<0.05) was observed. Only EP1 showed correlation with COX-2 in tissue microarrays (p<0,05). PGE2 was able to induce cell proliferation as it induces DNA synthesis in HNSCC cell lines. EP3 agonist also induced DNA synthesis addressing its role in cell proliferation induction in HNSCC. The siRNA for EP2 effects in DNA synthesis was not conclusive and the downstream proteins activated by EP2 second messenger were not affected following its expression knockdown. This study indicates three important findings. First, PGE2 is secreted by HNSCC. Second, COX-2 is found to be overexpressed in HNSCC; and third, PGE2 receptors are found to be constitutively expressed in HNSCC. Most interesting, we show here that this inflammatory pathway seems to be independent of the mechanisms that induce HNSCC proliferation.

O carcinoma epidermóide de cabeça e pescoço (CECP) representa 6ª malignidade mais comum no mundo. Para melhor entender os mecanismos envolvidos na iniciação tumoral, progressão e metástase, é necessária a elucidação dos eventos moleculares que guiam esses processos. É também importante a investigação da interação e modulação das células tumorais e seu microambiente. A participação de agentes inflamatórios no desenvolvimento e manutenção do CECP pode ser resumida na superexpressão da cicloxigenase 2 (COX-2) e na secreção de prostaglandina E2 (PGE2) pelas células tumorais. A PGE2 ativa seus receptores EP1-4 que são ligados a proteínas G. As proteínas G ativam outras vias de sinalização responsáveis por processos celulares como proliferação e angiogênese. Embora a participação do EP2 no câncer de cólon seja bem estabelecida, o papel dos receptores de PGE2 no CECP ainda permanece incerto. Este trabalho teve como objetivo avaliar o papel da PGE2 e de seus receptores na proliferação celular em linhagens celulares de CECP, bem como a expressão dos receptores em tissue microarrays de CECP. Inicialmente as linhagens de CECP foram utilizadas para analisar o padrão de expressão da COX-2 e dos receptores EP1-4 por meio da técnica de western blotting. A inibição da secreção da PGE2 pelos inibidores de COX-2 foi mensurada por meio da técnica de ELISA. A expressão dos receptores EP1-3 e da COX-2 foi também avaliada por meio da imuno-histoquímica em dois diferentes tissue microarray. A fim de esclarecer a indução da proliferação celular pela PGE2 e de apontar um de seus receptores como responsável pelo processo, duas PGE2 sintéticas, um antagonista do EP2 e um antagonista do EP3 foram utilizados para estimular a proliferação celular. Foi realizado o bloqueio do receptor EP2 por meio da interferência de RNA. Seus efeitos sobre a proliferação foram avaliados por meio do ensaio de incorporação de timidina. Os resultados mostraram que o CECP expressa constitutivamente a COX-2, o EP1, o EP2 e o EP3; e que é capaz de secretar PGE2. Os inibidores de COX-2 inibiram a secreção de PGE2 em baixas concentrações, mas não foram capazes de inibir a proliferação. A COX-2 e os receptores EP1-3 foram amplamente expressos nos tissue microarrays. Foi observada correlação entre EP1 e EP2; EP1 e EP3; e EP2 e EP3 (p<0,05). Somente o EP1 mostrou correlação com a COX-2 (p<0,05). A PGE2 induziu a proliferação por meio da indução da síntese de DNA nas linhagens celulares de CECP. O agonista de EP3 também induziu a síntese de DNA, sugerindo sua participação na proliferação dos CECPs. Os efeitos do siRNA para EP2 sobre a síntese de DNA não foram conclusivos. As proteínas ativadas por segundos mensageiros do EP2 também não foram afetadas pelo bloqueio do mesmo. Este estudo indica três importantes achados: 1. a PGE2 é secretada por linhagens de CECP; 2. a COX-2 é superexpressa nos CECPs; 3. os receptores de PGE2 são constitutivamente expressos nos CECPs. No entanto, esse trabalho mostra que esta via inflamatória parece ser independente aos mecanismos indutores da proliferação nos CECPs. / Head and neck squamous cell carcinoma (HNSCC) is the 6th most common malignant lesion worldwide. To better understand the mechanisms of tumor initiation, progression, and metastasis a better understanding of the molecular networks that guides these process is needed. Towards this goal, it is important to investigate the interaction and modulation of cancer cells over its surrounding microenvironment. The involvement of inflammatory agents in HNSCC development and maintenance can be resumed in the overexpression of cycloxygenase 2 (COX-2) and secretion of prostaglandin E2 (PGE2) by tumor cells. Prostaglandin E2 activates its receptors EP1-4 which are coupled to G proteins. G protein activates other pathways responsible for cellular processes such as proliferation and angiogenesis. The participation of EP2 in colon cancer is well established however the role of PGE2 receptors in HNSCC is still poorly understood. This work aims to investigate the role of PGE2 and its receptors in cellular proliferation in HNSCC cell lines and the clinical relevant expression pattern in HNSCC tissue microarrays. HNSCC cell lines were initially used to access the expression pattern of COX-2 and EP1-4 by using western blotting technique. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA antibody specific assay. Also, EP1, EP2, EP3 and COX-2 expression were evaluated by immuno-histochemistry in two different sets of HNSCC tissue microarrays. To address the question about PGE2 inducted cell proliferation and which PGE2 receptor are involved in the process, two synthetic PGE2, an EP2 agonist and an EP3 agonist were used to stimulate cell proliferation. Finally, the knockdown of EP2 receptor was performed by siRNA transfection assay and its effect was evaluated in cell proliferation by radioactive thymidine incorporation assay. The results presented here shows that HNSCC constitutively express COX-2, EP1, EP2 and EP3 and that they are able to secret PGE2. COX-2 selective inhibitors are able to suppress PGE2 secretion in lower concentrations but not to inhibit cell proliferation. Also, COX-2, EP1, E2 and EP3 are widely expressed in HNSCC tissue microarrays. A correlation between EP1 and EP2; EP1 and EP3; and EP2 and EP3 (p<0.05) was observed. Only EP1 showed correlation with COX-2 in tissue microarrays (p<0,05). PGE2 was able to induce cell proliferation as it induces DNA synthesis in HNSCC cell lines. EP3 agonist also induced DNA synthesis addressing its role in cell proliferation induction in HNSCC. The siRNA for EP2 effects in DNA synthesis was not conclusive and the downstream proteins activated by EP2 second messenger were not affected following its expression knockdown. This study indicates three important findings. First, PGE2 is secreted by HNSCC. Second, COX-2 is found to be overexpressed in HNSCC; and third, PGE2 receptors are found to be constitutively expressed in HNSCC. Most interesting, we show here that this inflammatory pathway seems to be independent of the mechanisms that induce HNSCC proliferation.

<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>

The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function. In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin. Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.