京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21375号 / 農博第2299号 / 新制||農||1067(附属図書館) / 学位論文||H30||N5148(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Colorectal carcinoma is an important, feasible and attractive target for chemoprevention because a) it is a major cause of mortality in the United States and in other developed countries worldwide, b) there is a high mortality associated with advanced disease, c) there is a well described molecular carcinogenesis pathway and d) recent advances in molecular genetics will improve the ability to identify high-risk subjects. Epidemiological data, colonoscopic screening and advances in molecular genetics has made possible the identification and selection of subjects at increased risk of developing colorectal cancer. Due to this new information it may be possible to impede malignant cellular transformation with drugs. Such intervention with relatively simple maneuvers, such as a low daily dose of aspirin, can potentially reduce mortality from colorectal cancer. Prospective trials need to confirm experimental and epidemiological data supporting the efficacy of aspirin and other NSAID as chemopreventive agents before they can be used in the general population at risk. To use cancer chemopreventives effectively and safely in an asymptomatic population, the risks should be minimized and the benefits maximized by determination of optimal dose, schedule and chemopreventive mechanism of the NSAID. By linking the putative mechanism of drug action to effect endpoints, we expect to know whether the chemopreventive intervention is likely to be effective in a given individual.

Purpose: Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. The purpose of this project is to quantify ACh release from isolated murine splenocytes when challenged with different mediators that stimulate T cells in this pathway. Methods:Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.01 - 5 micrograms/ml (0.068 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine (0.5 mM) to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours. Splenocytes were challenged with saline (control) or 1 mM (-) isoproterenol for 4 and 24 hours in the next set of experiments, and ACh in the medium was quantified. We also evaluated separate and combined effects of isoproterenol and activation of T cells with CD3 and CD28 antibodies on ACh release. Results:Correlation coefficients (R2) indicate linearity for ACh and Ch in culture media in the calibration range. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated in cell culture media for 4 and 24 hours, with and without splenocytes, showed a small but statistically significant loss of ACh after 24 h compared to 0 time media controls. However, the average loss of ACh was less than 10% and was not affected by the presence of splenocytes, suggesting that it was due to chemical hydrolysis. Incubation for 4 hr with and without splenocytes did not affect recovery of ACh. Treatment of splenocytes with isoproterenol for 4 hours did not cause significant release of ACh. However, significant release of ACh was detected after 24 hours exposure to isoproterenol or T cell activation. Media from untreated splenocytes had an ACh concentration of 0.14 +/- 0.07 mcg/mL. Isoproterenol treated had 0.28 +/- 0.14 mcg/mL, T-cell activated had 0.32 +/- 0.17 mg/mL, and isoproterenol + T-cell activation had 0.47 +/- 0.16 mcg/mL. Using a 1-way analysis of variance, statistically significant differences were detected between each of these groups. Conclusion: The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway in isolated splenocytes. Statistically significant differences in ACh release between control splenocytes and those treated with isoproterenol and T-cell activation can be detected. Quantitative investigation of this pathway helps provide an improved understanding of ACh dynamics as a mediator released from leukocytes. Further studies using this model and methodology will provide novel insights into cholinergic anti-inflammatory mechanisms and other immunomodulatory actions of non-neuronal ACh.

Leaves from Combretum collinum Fresen (Combretaceae) are commonly used for the treatment of inflammatory conditions, wound healing and bacterial infections in traditional West African medicine. This research focuses on the characterization of the phenolic profile and lipophilic compounds of leaves extracts of C. collinum. Studies of the in vitro anti-inflammatory activity were performed in TNFα stimulated HaCaT cells and antibacterial activity was evaluated with agar well diffusion and microdilution assays. Antioxidant activity was determined by DPPH and ABTS assays and compared to standards. The phytochemical studies confirmed myricetin-3-O-rhamnoside and myricetin-3-O-glucoside as major components of the leaves extracts, each contributing significantly to the antioxidant activity of the hydrophilic extracts. GC-MS analysis identified 19 substances that were confirmed by comparison with spectral library data and authentic standards. Combretum collinum aqueous leaves extract decreased pro-inflammatory mediators in TNFα stimulated HaCaT cells. Further investigations showed that myricetin-3-O-rhamnoside has an anti-inflammatory effect on IL-8 secretion. In the antimicrobial screening, the largest inhibition zones were found against S. epidermidis, MRSA and S. aureus. MIC values resulted in 275.0 µg/mL for S. epidermidis and 385.5 µg/mL for MRSA. The in vitro anti-inflammatory, antibacterial and antioxidant activity supports topical use of C. collinum leaves extracts in traditional West African medicine.

Inflammatory diseases such as PCOS, autoimmune diseases, irritable bowel syndrome, etc. are all highly uncomfortable diseases with several negative side effects. By adding antioxidants and omega-3 fatty acids to patients with inflammatory diseases diets, studies show that symptoms of these diseases will lessen. The objective of this study is to create a banana bread with increased omega-3 fatty acids and increased antioxidants to be served on trays of patients with inflammatory diseases and for patients to make at home to decrease symptoms related to inflammation. The experimental food should be an equal substitute for the control flavor, aroma, and texture wise. The control banana bread was substituted for an anti-inflammatory banana bread with the addition of cinnamon, dark chocolate, extra eggs, and pecans. The banana bread was made without dairy and grain for celiac patients and lactose intolerant patients. Both variations were equally accepted according to the hedonic scale, completed by 9 participants. Research was continued to confirm the of increased omega-3 fatty acids within the anti-inflammatory bread. Furthermore, walnuts were switched for pecans to test the antioxidant and fatty acid composition of both variations. Overall, we found that the walnut variation had more fatty acids, but pecans had more antioxidants. Our research suggests that both variations can be used to accommodate patients with inflammatory diseases. Further research can be done for long-term research for inflammatory disease patients that swapped the control for the variations.

No description available.

Red seaweeds are reported to represent the largest group of algae, with more species accounted for than the combination of brown and green seaweeds. Due to the high amount of polysaccharides in red seaweeds, they are mainly utilized for commercial agar and carrageenan production in industry. However, increasing studies indicate other valuable compounds such as lipids and polyphenols could be potential utilized for multiple human needs (e.g., drug development) (1, 2). With increasing studies demonstrating the potential health benefits of seaweed components, two red seaweed species commonly consumed in Asia, hong qı´ lı´n c a`i (HQL), Eucheuma sp and zhe` gu¯ ca`i (ZGC), Caloglossa leprieurii, were investigated on to determine the anti-inflammatory effects of their extractable lipophilic bioactives (ELB) and bound lipophilic bioactives (BLB) in lipopolysaccharide( LPS)-treated RAW 264.7 macrophages. The chemical composition of ELB and BLB was characterized in terms of total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), oxygen radical absorbance capacity (ORAC), and etc. Six phenolic compounds were identified in ZGC extracts and one was detected in HQL. All extracts inhibited the nitric oxide (NO) production in LPS-induced macrophages, which was associated with downregulation of iNOS and COX-2 protein expression and up-regulation of HQ-1 and NQO1 protein expression. Overall, our results showed that both ELB and BLB in HQL and ZGC seaweeds presented potential anti-inflammatory activities. These results warrant future investigations to determine the mode of actions of red seaweed bioactives and their efficacy in humans.

This thesis presents the design and optimization of a dendrimer-based cationic nanoparticle system tailored for versatile applications, ranging from anti-inflammatory scavenging to targeted pain relief through endosomal delivery. By harnessing the unique attributes of this platform, various strategies were devised to overcome hurdles in drug delivery, offering promising avenues for nanomedicine in anti-inflammatory and nociceptive treatments. In our scavenging screening project, we rigorously screened various materials to find the best universal anti-inflammatory carrier. We started by exploring the potential of dendrimer-based materials as scavengers of inflammatory signals and studied how they could be used to develop therapeutic carriers. With intrinsic therapeutic properties and the ability to create tunable nanocarriers, dendrimer-based delivery systems are powerful multimodal delivery systems. The dendrimer base of our delivery system, cationic PAMAM Generation 3 dendrimer (PAMAM-G3), was selected due to its efficient scavenging ability and low biotoxicity. Conjugation with cholesterol facilitated the formation of polymeric micelles, exhibiting a cationic and hydrophilic exterior coupled with a hydrophobic interior, resulting in a high drug-loading capacity. Among the developed scavengers, PAMAM-Cholesterol (PAMAM-Chol) nanoparticles demonstrated a potent reduction in toll-like receptor activation with minimal toxicity and extended endosomal retention. We then exploited the endosomal retention of PAMAM-Chol nanoparticles to target the activated PAR2 receptor within endosomes of relevant cancer cells, aiming to alleviate oral cancer-induced nociception. Extensive characterization confirmed the platform's stability, physical attributes, and ability to encapsulate PAR2 inhibitor, AZ3451. The platform exhibited high drug loading capacity and sustained release profiles across various pHs. Cellular uptake studies demonstrated efficient endosomal targeting, with subsequent modulation of PAR2 signaling pathways. Preclinical studies in oral cancer pain models revealed a significant and prolonged reduction in nociception for over 24 hours, surpassing the efficacy of free drugs. Further diversification of the PAMAM-Chol platform explored its potential as a "Push" chemotherapy carrier and a "Pull" cfDNA scavenger against chemotherapy-induced neurological and neuropathic side effects. Evaluation in wild-type mice demonstrated the platform's effectiveness in mitigating chemobrain and chemotherapy-induced peripheral neuropathy, highlighting its translational potential for multimodal cancer therapy. We found that NPs loaded with chemotherapy significantly reduced the painful effects of chemotherapy-induced peripheral neuropathy and decreased recovery times. Collectively, this body of work underscores the potential of PAMAM-Chol as a versatile tool in drug delivery and endosome-localized pain therapeutics. It contributes to the evolving landscape of precision medicine through tailored therapeutic approaches for minimizing side effects and enhancing patient well-being. The innate therapeutic properties coupled with efficient and sustained drug delivery mechanisms position the PAMAM-Chol platform as a foundational element for the development and delivery of next-generation therapeutics.

Lanthionine synthetase C-like protein 2 (LANCL2) is a member of the LANCL protein family, which is broadly expressed throughout the body. LANCL2 is the molecular target of abscisic acid (ABA), a compound with insulin-sensitizing and immune modulatory actions. LANCL2 is required for membrane binding and signaling of ABA in immune cells. Direct binding of ABA to LANCL2 was predicted in silico using molecular modeling approaches and validated experimentally using ligand-binding assays and kinetic surface plasmon resonance studies. The therapeutic potential of the LANCL2 pathway ranges from increasing cellular sensitivity to anticancer drugs, insulin-sensitizing effects and modulating immune and inflammatory responses in the context of immune-mediated and infectious diseases. A case for LANCL2-based drug discovery and development is also illustrated by the anti-inflammatory activity of novel LANCL2 ligands such as NSC61610 against inflammatory bowel disease in mice. This dissertation discusses the value of LANCL2 as a novel therapeutic target for the discovery and development of new classes of orally active drugs against chronic metabolic, immune-mediated and infectious diseases and as a validated target that can be used in precision medicine. Specifically, in Chapter 2 of the dissertation, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of LANCL1 as a template. Our molecular docking studies predicted that ABA and other PPAR - agonists share a binding site on the surface of LANCL2. In Chapter 3 of the dissertation, structure-based virtual screening was performed. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. In Chapter 4 of the dissertation, we developed a novel integrated approach for creating a synthetic patient population and testing the efficacy of the novel pre-clinical stage LANCL2 therapeutic for Crohn's disease in large clinical cohorts in silico. Efficacy of treatments on Crohn's disease was evaluated by analyzing predicted changes of Crohn's disease activity index (CDAI) scores and correlations with immunological variables were evaluated. The results from our placebo-controlled, randomized, Phase III in silico clinical trial at 6 weeks following the treatment shows a positive correlation between the initial disease activity score and the drop in CDAI score. This observation highlights the need for precision medicine strategies for IBD. / Ph. D.