Final publication is available at http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=26018088 / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19635号 / 医科博第73号 / 新制||医科||6(附属図書館) / 32671 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妹尾 浩, 教授 渡邊 直樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Prostaglandin F2a (PGF2a), oxytocin and gonadotropin releasing hormone (GnRH) have been used in bulls, rams, boars, stallions or rodents to increase sperm numbers in the ejaculate. Improving sperm quantity in the canine ejaculate would benefit all assisted reproductive techniques used in this species. The purpose of the present study was to evaluate the effects of PGF2a, oxytocin and GnRH on canine ejaculate characteristics. Eight, mature, medium size (25-30 kg), mixed breed dogs were randomly assigned to one of four treatment groups (N=2 dogs each); each group received one treatment per week for four weeks. Treatments were assigned based on a Latin Square design. A two-week training period was used to acclimate the dogs to manual semen collection. Treatments were 0.1 mg/kg PGF2a 15 minutes prior to collection, 2.5 units/dog oxytocin 10 minutes prior to collection, 50 mg/dog GnRH 60 minutes prior to collection, or 1.0 ml of saline 30 minutes prior to collection. An evaluator that was blinded to treatment analyzed ejaculate characteristics. Samples were evaluated for semen volume, concentration of spermatozoa per milliliter, motility, morphology, total sperm number and total morphologically normal motile sperm number (TNMS). In addition, a subjective ease of collection score was assigned following each collection (Scale 1-9, 1 being easiest to manually ejaculate). Semen concentration, motility and morphology were not different between treatments. Semen volume was greater for dogs treated with PGF2a or oxytocin compared to saline. Total sperm number and TNMS were greater when dogs were treated with PGF2a compared to oxytocin, GnRH and saline (p<0.05). The subjective ease of collection score was lower for dogs receiving PGF2a compared to GnRH or saline (p<0.05). In summary, administration of PGF2a or oxytocin prior to semen collection increased semen volume and PGF2a increased total sperm number in the ejaculate of the dog. It did not appear that treatment with GnRH had an effect on semen parameters evaluated in this study. / Master of Science

Yes / Urinary prostaglandin (PG) E metabolite (PGE-M) and 11-dehydro (d)-thromboxane (TX) B2 are biomarkers of cyclooxygenase-dependent prostanoid synthesis. We investigated (1) the effect of aspirin 300 mg daily and eicosapentaenoic acid (EPA) 2000 mg daily, alone and in combination, on urinary biomarker levels and, (2) whether urinary biomarker levels predicted colorectal polyp risk, during participation in the seAFOod polyp prevention trial. Urinary PGE-M and 11-d-TXB2 were measured by liquid chromatography-tandem mass spectrometry. The relationship between urinary biomarker levels and colorectal polyp outcomes was investigated using negative binomial (polyp number) and logistic (% with one or more polyps) regression models. Despite wide temporal variability in PGE-M and 11-d-TXB2 levels within individuals, both aspirin and, to a lesser extent, EPA decreased levels of both biomarkers (74% [P ≤ .001] and 8% [P ≤ .05] reduction in median 11-d-TXB2 values, respectively). In the placebo group, a high (quartile [Q] 2-4) baseline 11-d-TXB2 level predicted increased polyp number (incidence rate ratio [IRR] [95% CI] 2.26 [1.11,4.58]) and risk (odds ratio [95% CI] 3.56 [1.09,11.63]). A low (Q1) on-treatment 11-d-TXB2 level predicted reduced colorectal polyp number compared to placebo (IRR 0.34 [0.12,0.93] for combination aspirin and EPA treatment) compared to high on-treatment 11-d-TXB2 values (0.61 [0.34,1.11]). Aspirin and EPA both inhibit PGE-M and 11-d-TXB2 synthesis in keeping with shared in vivo cyclooxygenase inhibition. Colorectal polyp risk and treatment response prediction by 11-d-TXB2 is consistent with a role for platelet activation during early colorectal carcinogenesis. The use of urinary 11-d-TXB2 measurement for a precision approach to colorectal cancer risk prediction and chemoprevention requires prospective evaluation. / Efficacy and Mechanism Evaluation Programme. Grant Number: NIHR128210. Cancer Research UK. Grant Number: C23434/A24939

Prostaglandins (PGs) are important signalling factors for bone mechanotransduction. The inhibition of cyclooxygenase, responsible for the synthesis of PGs, with non-steroidal anti-inflammatory drugs (NSAIDs) has been shown to influence bone formation induced by mechanical stimulation. The purpose of this study was to examine the timing effects of NSAID administration on: 1) bone formation induced by multiple mechanical loading events in a rat model and 2) the PGE2 response of MLO-Y4 osteocyte like cells stimulated by fluid shear stress. The rat forelimb compression model was used to induce bone formation in male and female rats using a 1-month loading protocol (12 loading sessions). The right forelimbs were loaded and the left forelimbs served as non-loaded controls. NSAIDs were administered orally either before or after loading. Fluorochrome labels were administered to the rats to determine mineral apposition rate (MAR). The NSAIDs examined (indomethacin, NS-398 and ibuprofen) did not significantly affect periosteal MAR, administered either before or after loading, suggesting NSAIDs do not affect bone adaptation to multiple mechanical loading events. To examine in vitro effects of NSAIDs on PGE2 production, an orbital shaker was used to apply fluid shear stress to MLO-Y4 cells seeded in 6-well culture plates. Indomethacin was added to the culture media either before or after loading and media PGE2 concentrations were determined at various time points by enzyme immunoassay. Fluid shear stress increased PGE2 production of MLO-Y4 cells and indomethacin administration inhibited that response when administered both before and after fluid flow. However, PGE2 production was influenced by the media changes that occurred in the in vitro experiments, making it difficult to differentiate between indomethacin effects and media change effects. The in vitro experiments revealed the difficulties of modeling the timing effects of NSAID administration on MLO-Y4 PGE2 production in response to fluid flow. / Thesis / Doctor of Philosophy (PhD) / Bone is a dynamic tissue that can adapt to mechanical loading. Prostaglandins (PGs) are important signalling factors produced by osteocytes, the bone mechanosensing cells, that help to activate various cells and cell processes leading to changes in bone structure. Blocking PG signalling with non-steroidal anti-inflammatory drugs (NSAIDs) has been shown to influence bone formation induced by mechanical stimulation in animals and humans. The purpose of this study was to examine the timing effects of NSAID administration on: 1) bone formation induced by multiple mechanical loading events in rats and 2) the PG production of osteocyte like cells in response to fluid flow stimulation. The results of this study suggest that NSAIDs, administered either before or after loading, do not affect bone responses to multiple mechanical loading events. Further investigation is needed to determine the translatability of these findings to NSAID use around the time of exercise in humans.

Dendritische Zellen (DC) spielen eine Schlüsselrolle im Immunsystem. Sie dienen als professionelle antigenpräsentierende Zellen und können eine antigenspezifische Immunantwort initiieren, indem sie naive T-Zellen primen. DC können auch verwendet werden, um T-Zellen im Kontext der onkologischen Immuntherapie zu stimulieren. In vitro können sie leicht aus Monozyten differenziert werden. Die daraus resultierenden unreifen DC können bereits Antigene phagozytieren und präsentieren, sie aktivieren jedoch noch keine Immunantwort solange keines der aufgenommenen Antigene als pathogen erkannt wird. Die Ausreifung einer unreifen, tolerogenen DC zu einer immunogenen reifen DC kann, neben anderen Methoden, durch einen Cocktail aus TLR-Liganden oder Zytokinen erreicht werden. Die Auswahl der Substanzen in diesem Cocktail bestimmt den Phänotyp und die funktionellen Eigenschaften der resultierenden reifen DC. Einige der benötigten Fähigkeiten der DC in der Tumorimmuntherapie, wo sie aus Patientenmonozyten generiert, mit Tumorantigen beladen und dem Patienten wieder zugeführt werden sollen, umfassen die Migration zu den T-Zell-Zonen der Lymphknoten, Antigenpräsentation auf sowohl MHC-I- als auch MHC-II-Molekülen, Zytokinproduktion für die Direktion der T-Zell-Antwort wie IL-12p70, und die Expression von Oberflächenmarkern wie der kostimulatorischen Moleküle CD80 und CD86. In der Vergangenheit wurde gezeigt, dass durch Zugabe von Prostaglandin E2 (PGE2) zu einem Cocktail mit dem synthetischen TLR3-Liganden poly-I:C und dem TLR7/8-Liganden R848 (Resiquimod) sowohl eine gute migratorische Fähigkeit als auch eine erhöhte IL-12p70-Produktion erreicht werden kann, während etwa die Fähigkeit zur Antigen-Kreuzpräsentation reduziert erschien. Anhand von Monozyten anonymer gesunder Spender beleuchtet diese Arbeit daher den Effekt von PGE2 auf monozytenderivierte DC näher, indem seine konzentrationsabhängige Wirkung auf deren Phänotyp untersucht wird. In den durchgeführten Versuchen wurde dabei die Expressionsdichte der Oberflächenmarker CD83, CD80 und CD86, HLA-DR und CCR7 sowie der monozytäre Marker CD14 durchflusszytometrisch analysiert. Die Ergebnisse zeigen bei Exposition mit PGE2 dosisabhängig eine Heraufregulation von CD80, CD83, CD86 und CCR7 in der Population reifer DC, deren Maximum in unteren mikromolaren Konzentrationen erreicht wird. Gleichzeitig induzierte PGE2 dosisabhängig auch die Entstehung einer zweiten Zellpopulation mit anderen Eigenschaften, die stattdessen den monozytären Marker CD14 re-exprimierte. Dies ist für künftige Studien eine interessante Beobachtung, da sie eine differenzierte Betrachtung beider resultierender Subpopulationen anregt. / Dendritic cells (DC) play a key role in the immune system. They serve as professional antigen presenting cells and can initiate an antigen-specific immune response by priming naive T cells. DC can also be used to stimulate T cells in the context of tumor immunotherapy. In vitro, they can easily be differentiated from monocytes. The resulting immature DC are capable of antigen phagocytosis and presentation, but do not yet activate an immune response as long as none of the uptaken antigens is recognized as pathogenic. The process of converting an immature, tolerogenic DC to an immunogenic mature DC can, among other methods, be achieved by using a cocktail of toll-like receptor (TLR) ligands and cytokines. The choice of the substances included into this cocktail later determines the phenotype and capabilities of the resulting mature dendritic cells. Some of the required DCs' capabilities in the field of cancer immunotherapy, where they are to be generated from patient monocytes, loaded with tumor antigen and re-transferred into the patient, include migration to the T cell areas of lymph nodes, antigen presentation on both MHC-I and MHC-II molecules, cytokine production for shaping the T cell response such as IL-12p70, and the expression of surface markers such as the costimulatory molecules CD80 and CD86. Adding Prostaglandin E2 (PGE2) to a cocktail of the TLR3 ligand poly(I:C) and the TLR7/8 ligand R848 (Resiquimod) has been shown to result in a good migratory capacity as well as an elevated IL-12p70 production. In earlier research, the capability of antigen cross-presentation however appeared to be reduced when PGE2 was added. Hence, using anonymous healthy donor monocytes, this work was designed to further investigate the effects of PGE2 on DC dose-dependently by studying their phenotype. Particularly, the density of the cell surface markers CD83, CD80 and CD86, HLA-DR and CCR7 as well as the monocyte marker CD14 have been studied in flow cytometry. The results suggest a dose-dependent up-regulation by PGE2 of CD80, CD83, CD86 and CCR7 in the population of mature DC reaching its maximum at low µM concentrations. Simultaneously, PGE2 also dose-dependently induced the generation of a second cell population, which instead re-expressed the monocyte marker CD14. This is an interesting finding as well as it encourages a differential look at both resulting subpopulations in future analyses.

Flexor tendonitis is a common cause of lameness and wastage in the equine athlete. Current techniques for tendonitis therapy provide limited success, and horses that do recover tend to return at a decreased level of performance. Current treatment techniques have begun to focus on regenerative medicine to improve tissue healing. Investigations of new treatments are made difficult by the lack of reliable in vitro models that allow for accurate comparison of treatment protocols. New techniques are often implemented into the clinical setting prior to thorough investigation for safety and efficacy. In vitro testing is an important step in the development of new therapeutic agents. However, results of in vitro tests should only be deemed as useful if the model used is one that is reliable and mimics the clinical situation that the reseachers are attempting to investigate. Equine flexor tendonitis is believed to be the result of microdamage caused by cyclic loading of tendons. Cyclic loading of fibroblasts results in increased production of the inflammatory cytokine prostaglandin E2 (PGE2). Thus the exposure of tendon fibroblasts to exogenous PGE2 may induce metabolic changes in the cells similar to what is seen in clinically affected animals making this a useful model for the investigation of therapeutic techniques. Currently a variety of techniques exist for treatment of flexor tendonitis; however, no single treatment has separated itself as superior. A new technique using autogenous conditioned serum (ACS) in humans for treatment of muscle injury has been shown to speed tissue regeneration. ACS produced from human blood has been shown to contain significantly increased levels of III growth factors that may improve tendon fibril formation and strength. We propose to investigate the effect of ACS on cellular metabolism in equine tendon fibroblast monolayers. This will involve cell culture, PGE2-induced cellular injury, and analysis of the cellular response to injury when treated with ACS. Controls will include fetal bovine serum, normal equine serum, and ACS without PGE2-induced cellular injury. The cellular response will be investigated biochemically by quantification of DNA, glycosaminoglycan, and soluble collagen levels and by real time PCR to assess gene expression for matrix metalloproteinases (MMP)-1, MMP-3, and MMP-13, collagen types I and III, and the non-collagenous proteins cartilage oligomeric matrix protein (COMP) and decorin. Data will be analyzed by analysis of variance and post-hoc comparisons. Significance will be set at p<0.05. We hypothesize that the addition of exogenous PGE2 to culture media for monolayers of equine tendon fibroblasts will insight alterations in cellular metabolism that will generate a suitable model for the in vitro study of fibroblast response to novel therapies. We then hypothesize that the addition of ACS to PGE2-treated fibroblasts will result in increased gene expression for collagen types I and III, cartilage oligomeric matrix protein, and decorin. ACS will also stimulate increased protein production of collagen and glycosaminoglycans, and stimulate increased cell proliferation. The use of ACS will decrease gene expression of inflammatory molecules important in tendon degradation, namely matrix metalloproteinases -1, -3, and -13. / Master of Science

The objective of this study was to determine the effects of dietary supplementation of eicosapentanoic (EPA) and docosahexanoic acids (DHA) on reproduction in beef females. In experiment 1, cows (n = 31) were individually fed rumen protected fish meal (FM) or no fish meal (C) supplements. Estrus was synchronized and ovulation induced on d 37. Ovarian follicular growth and diameter were determined by ultrasound on d 35 and d 37. Serum progesterone (P4) profiles were analyzed on d 37 through d 52. On d 52 cows were cannulated, primed with estradiol-17&#946; at -240 min, and stimulated to release PGF2&#945; by oxytocin injection at 0 min with blood sampled every 15 min from -30 min to 240 min. Supplement type did not affect (P > 0.05) follicular diameter, follicular growth or P4 concentrations. In cows fed FM, prostaglandin metabolite (PGFM) concentrations tended (P &#8804; 0.10) to be reduced at 0, 30, and 60 min. In experiment 2, crossbred heifers (n = 214) received FM or C concentrates with corn silage from 30 d before estrous synchronization until 14 d after artificial insemination (AI). Serum fatty acid profiles were determined in five heifers from each group . Estrus detection and AI were conducted from d 37 through d 39. Dietary treatment increased (P < 0.05) EPA and DHA concentrations. Dietary treatment did not affect estrus response or AI conception rates and pregnancy rate. Supplementation of FM increased EPA and DHA concentrations but did not affect reproductive factors. / Master of Science

Prostaglandin F2α (PGF2α) is commonly given to female cattle intramuscularly (IM) for the synchronization of estrus. A novel site for administration of PGF2α that improves beef quality assurance is the ischiorectal fossa (IRF). The objective of this study was to determine whether administration of PGF2α in the IRF results in a similar physiologic response to administration of PGF2α given IM. Yearling angus-cross heifers (n=112) were blocked by weight and randomly assigned within blocks to be injected with 5 mL PGF2α either IM in the neck or in the IRF. Blood samples were taken at 0, 8, 16, 24, 36, and 48 h post-injection. Serum samples were analyzed for progesterone concentration using a radioimmunoassay. Progesterone concentration curves for each heifer were plotted to determine luteolysis. The median times to luteolysis for neck and IRF injections were 18.1 hrs and 20.0 hrs, respectively (p=0.06). Angus cross commercial beef cows (n=1471) at least 30 days post-partum were blocked by age and randomly assigned to be injected with 5 mL PGF2α either IM in the neck muscle or in IRF as part of a 7-Day CO-Synch + CIDR ovulation protocol. Pregnancy diagnosis was performed via ultrasound at 60 days post insemination. Results were analyzed with Proc Glimmix (SAS). Pregnancy rates for neck and IRF injections were 52.6% and 57.2%, respectively (p=0.06). In summary, injection of PGF2α in the IRF for estrus synchronization and lysis of the corpus luteum did not differ from injection in the neck muscle. Utilizing the ischiorectal fossa as an injection site for PGF2α may be considered as an alternative that more closely aligns with beef quality assurance objectives. / Master of Science

by Ho Wai Chan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. [254]-271). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

<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>