The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

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.

Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Chronic inflammation has been linked to cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Although both COX isoforms have similar structure and function, they are encoded by different genes and show distinct expression patterns. COX-1 is expressed in most cells and tissues and remains constant under most physiologic conditions to play a housekeeping role whereas the COX-2 form is inducible and usually only expressed in response to various inflammatory stimuli. COX enzymes may be involved in both tumor establishment and maintenance of existing tumors. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. Prevention may be the best approach to reduce ovarian cancer. Ovarian cancer is the fifth leading cause of cancer death among women and the most lethal gynecological malignancy. There are at least 3 well established risk factors for ovarian cancer: age, family history and environmental factors. Ovarian cancer is mainly seen in older women when their ovaries are not reproductively functional. Close to half of the women with ovarian cancer (48%) are in the age group of 65 or older. Epidemiological and preclinical studies indicate that increased dietary intake of omega-3 fatty acids (OM-3FAs) reduces the incidence and growth of various cancers. Thus, increasing the consumption of OM-3FAs may be a nontoxic way to prevent or suppress ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Fish oil is a source of OM-3FAs which may be effective in prevention of ovarian cancer. The main OM-6FA, Linoleic Acid (LA), is a direct precursor of the Arachidonic Acid (AA). Alpha-linolenic acid (ALA) is the main OM-3FA found in flax oil, whereas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the OM-3FAs in fish oil. ALA is elongated to form EPA and DHA in the intestine. Celecoxib is a non-steroidal anti-inflammatory (NSAID) drug that selectively inhibits COX-2. There are evidences showing that Celecoxib has some anti-cancer properties. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. Our first objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by Immunohistochemistry, western blot, quantitative real-time PCR and LC-MS-MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. Since no effect on ovarian cancer incidence was detected after feeding the 2. 5 year old hens with 10% flaxseed for 1 year, we designed a long-term study using 6 month old hens. Our objectives were: 1) to examine the expression of COX enzymes and PGE2 levels in ovaries and correlate them to ovarian cancer and aging 2) to determine if long-term consumption of a flaxseed enriched diet decreases ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE2 concentration. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4 years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured by western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE2 concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE2 concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE2. Furthermore, elevated PGE2 in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. The results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE2 and expression of COX-2 were diminished in ovaries of hens fed flaxseed. Our findings suggest that the lower levels of COX-2 and PGE2 are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer. Based on our previous findings, targeting COX expression and prostaglandin biosynthesis by dietary intervention using OM-3FAs and selective COX inhibitor can be an effective approach to prevent or suppress ovarian cancer. Thus, we conducted a series of studies to assess effect of fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined or flax oil and Celecoxib combined on COX-1 and COX-2 expression, PGE2 concentrations, proliferation and apoptosis in normal and cancerous ovaries of laying hens. This study had not been performed in hens before, thus the first step was to find the optimum doses. In order to do so, we utilized one year old hens, divided them to groups of 6 hens, and fed them different doses of fish oil (50, 100, 175, 375 and 700 mg/kg), flax oil (100, 250, 500, 1000 and 1500 mg/kg) or Celecoxib (35, 65 and 100 mg/kg) for three weeks. The OM3-FAs andomega-6 fatty acids contents of egg yolks were determined by gas chromatography. Proliferation, apoptosis,COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, western blot, quantitative real-time qPCR and ELISA, respectively. The results indicated that 100 mg/kg fish oil was the most effective dose in reducing COX enzymes and PGE2, and increased apoptosis and reduced proliferation in ovaries. The lower doses of fish oil incorporated more OM-3FAs into yolks, reduced OM-6FAs and increased the egg laying frequency but did not affect EP4 expression. Unlike fish oil, the highest dose of flax oil (1500 mg/kg) caused the most significant reduction in COX expression and PGE2 concentration. Celecoxib was not perfectly selective in targeting COX-2, however, treating the hens with 65 mg/kg Celecoxib resulted in the most significant amelioration of PGE2 levels in ovaries. Using the optimum doses of fish oil, flax oil and Celecoxib, we aimed to investigate if these components can alter ovarian cancer end-points in normal and cancerous hen ovaries. There is an adverse relation between ovulation and health of ovaries. Thus, 3-4 year old hens were monitored for egg laying frequency and the hens with the least ovulation rate were selected for health assessment. The hens presenting poor health were scanned using ultrasound and if tumor mass and/or ascites were detected, they were chosen for this study. The hens with normal and cancerous ovaries were divided to groups and were fed fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined, or flax oil and Celecoxib combined for 42 days. The results showed that fish oil and flax oil increased the incorporation of OM-3FAs into egg yolks in both normal and cancerous ovaries of hens. Fish oil reduced COX-1 and COX-2 in normal and cancerous ovaries. Fish oil, flax oil and Celecoxib reduced the COX-2 expression in ovaries. Combination of fish oil and Celecoxib and flax oil and Celecoxib decreased COX and PGE2 more than each of these treatments alone. The cancerous ovaries of hens treated with fish oil, flax oil, Celecoxib, and flax oil and Celecoxib combined increased the percentage of apoptotic cells compared to cancerous ovaries of control hens. The cancerous ovaries of hens treated with fish oil and Celecoxib had the highest number of apoptotic cells indicating that the combination of fish oil and Celecoxib is more effective than fish oil or Celecoxib alone. To our knowledge the present study provides the first insight into the efficacy of fish oil, flax oil, Celecoxib, alone or combined on the reduction of COX enzyme expression, PGE2 concentration and apoptosis in the normal and cancerous ovaries and further demonstrates the utility of the hen model for ovarian cancer. Our studies provided new insight into the potential mechanism of action of flaxseed, fish oil, flax oil and Celecoxib in the reduction of ovarian cancer and will establish the foundation for clinical trials to test the efficacy of dietary intervention for the prevention and suppression of ovarian cancer in women.

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-03-26Bitstream added on 2014-06-13T20:45:02Z : No. of bitstreams: 1 vergani_sa_dr_arafo.pdf: 639289 bytes, checksum: 6483b0519cf455414c9e0881668e277f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi o de avaliar os efeitos do controle de placa supragengival, em pacientes com doença periodontal crônica generalizada, por meio dos parâmetros clínicos microbiológicos e imunológicos. Foram selecionados 30 pacientes, com idade entre 32 e 59 anos, apresentando 4 bolsas entre 3 e 5mm e 4 bolsas entre 6 e 10mm e sem envolvimento sistêmico. Após o exame inicial, os pacientes foram submetidos a raspagem supragengival e receberam instruções de higiene oral, sendo acompanhados semanalmente por um período de 30 dias. Os parâmetros clínicos avaliados foram profundidade de sondagem, recessão gengival, índice de placa, índice gengival e sangramento à sondagem. Amostras de fluido crevicular das bolsas foram coletadas para análise da concentração dos mediadores de inflamação interleucina-1b e prostaglandina E2 e identificação pela reação de polimerase em cadeia dos microrganismos Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia e Prevotella nigrescens. Os resultados demonstraram a ocorrência de melhoras evidenciadas pelos parâmetros clínicos avaliados. Observou-se ainda uma redução significante no número de Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia e Prevotella nigrescens. O microrganismo A. actinomycetemcomitans não apresentou diferença significante. Para o exame imunológico não foram encontradas diferenças nos níveis de interleucina-1b, sendo constatado ainda um aumento nas concentrações de prostaglandina E2 nas bolsas de 6 a 10mm que apresentavam sangramento no final do tratamento. Concluiu-se que decisão da necessidade de tratamento periodontal em sítios profundos, após a realização do controle de placa supragengival, baseada somente na ausência de sangramento à sondagem pode subestimar... . / The aim of the present study was to evaluate the effect of supragingival plaque control, in patients with generalized chronic periodontal disease using clinical, microbiological and immunological parameters. 30 patients were selected, aged 32 to 59 years, presenting 4 periodontal pockets between 3 and 5mm and 4 pockets between 6 and 10mm. After initial exam patients received supragingival scaling and oral hygiene instructions, which were reinforced once a week for 30 days. Clinical parameters were probing depth, gingival resection, plaque index, gingival index and bleeding upon probing. Samples of crevicular fluid were taken for analyses of interleukin-1b and prostaglandin E2 and for identification by polymerase chain reaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia and Prevotella nigrescens. The results demonstrated an improvement of the clinical parameters. The microbiological findings indicate reduction of Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, and Prevotella nigrescens. A. actinomycetemcomitans did not show significant difference. In regard to the immunological exam, no differences were observed in interleukin-1b concentration. Prostaglandin E2 concentrations were elevated in pockets between 6 and 10mm with persistent bleeding upon probing after treatment. In conclusion the decision for treatment needs after supragingival plaque control only based on the absence of bleeding upon probing might underestimate the periodontal disease activity.

Made available in DSpace on 2014-06-11T19:23:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-27Bitstream added on 2014-06-13T20:48:19Z : No. of bitstreams: 1 ribeiro_ap_me_jab.pdf: 821460 bytes, checksum: 9540c58da44188e3ae8a41593d305a9c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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. / 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.

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.