Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Bellamkonda, Ravi; Committee Member: LaPlaca, Michelle; Committee Member: McKeon, Robert

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Ravi V. Bellamkonda; Committee Member: Andreas Bommarius; Committee Member: Andrés J. García; Committee Member: Niren Murthy; Committee Member: Robert J. McKeon. Part of the SMARTech Electronic Thesis and Dissertation Collection.

L’arthrose est la plus fréquente des maladies articulaires pour laquelle aucun traitement efficace n’est aujourd’hui disponible. Elle est caractérisée par une destruction du cartilage et de ses composants, dont font partie les protéoglycanes. Les principaux protéoglycanes cartilagineux sont les sulfates de chondroïtines, qui sont de longs polysaccharides linéaires hétérogènes composés d’unités disaccharidiques répétitives constituées d’un acide-D-glucuronique et d’une N-acétyl-D-galactosamine. Dans le but d’étudier la biosynthèse de ces composés et ainsi de mieux comprendre le mécanisme d’action des enzymes qui y sont impliquées, la synthèse chimique d’oligosaccharides de chondroïtines et de sulfates de chondroïtines biotinylés a été réalisée. Grâce à une méthodologie efficace et innovante basée sur une hydrolyse acide d’un polymère de sulfates de chondroïtines, des intermédiaires clés ont rapidement été obtenus. Une stratégie d’élongation les utilisant a ensuite été appliquée pour conduire à une première famille de sept oligosaccharides non sulfatés (du disaccharide à l’octasaccharide). Deux nouvelles familles d’oligosaccharides de sulfates de chondroïtines A et C, sulfatés de façon homogène ont été préparées, à partir d’un précurseur commun par stratégie divergente. Pour la première fois, deux oligosaccharides de sulfate de chondroïtine A, présentant une sulfatation hétérogène, ont également été synthétisés. Ces travaux ont mené à la préparation de treize oligosaccharides biotinylés finaux. / Osteoarthritis is the most frequent articular disease but until now no treatment exists. It is characterized by a destruction of cartilage and its components as proteoglycans. Major cartilage’s proteoglycans are chondroitin sulfate, which are linear and heterogeneous polysaccharides composed of disaccharidic repeating units constituted of a D-glucuronic acid and an N-acetyl-D-galactosamine. In order to study biosynthesis of those compounds and so to better understand working mechanism of the implicated enzymes, chemical syntheses of biotinylated chondroitin and chondroitin sulfate oligomers have been achieved. Thanks to an efficient hydrolysis of a starting chondroitin sulfate polymer, key building blocks were quickly obtained. An elongation strategy using them has been then applied to give a first family of seven non sulfated oligosaccharides (from disaccharide to octasaccharide). Two new families of chondroitin sulfate A and C oligosaccharides, with homogeneous sulfation have been prepared from a common precursor by divergent strategy. For the first time, two chondroitin sulfate A oligosaccharides with an heterogeneous sulfation have been synthetized. This work has led to thirteen final biotinylated oligosaccharides.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13386号 / 論医博第2218号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The use of dietary supplements occurs in the majority of American adults >20 years old and has been reported to be on the rise by the National Health and Nutrition Examination Survey. People are not only taking more dietary supplements, but taking them without the advice of a health care provider. With the lack of education time and focus on this topic, physicians need to know some evidence regarding the most common supplements used, how to understand dietary supplement labels, and where to find reputable information about dietary supplements. Vitamin D, Omega-3, and Glucosamine/ Chondroitin are reviewed here. Also, a description of the various components of a dietary supplement label is explained. Finally, a brief description of independent companies (Consumerlabs.com®, US Pharmacopedial Convention, Natural Medicine Comprehensive Database, and Natural Standard) as well as the federally run Office of Dietary Supplements are presented.

Clinical Question: Does glucosamine or chondroitin reduce pain, improve functional status, or improve prognosis in patients with osteoarthritis?

This diploma thesis deals with experimental study of interaction based on polymer-lipid system. The main goal was to investigate interaction leading to phase separation. Three anionic polyelectrolytes were selected for this purpose: sodium hyaluronate, sodium chondroitin-6-sulfate and sodium poly(4-styrenesulfonate). The liposomes were formulated by sonication of lipid – cationic 1,2-dipalmitoyl-3-trimethylamonium-propane (chloride salt) and zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphocholine. It was found that the phase separation occured at specific ratio of DPTAP and selected polymer. It was also explored that the strong electrostatic interaction which leads to the phase separation can be weaken by increasing the ionic strength in the sample. As we aspected the systems contain the hydrophobic domain and therefore these are able to incorporate hydrophobic substances in their structure.

The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury (SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal regeneration and promote functional recovery when delivered at the site of injury. However, chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity. Maintaining these infusion systems is invasive and clinically problematic. In this dissertation, three studies are reported that demonstrate our strategy to overcome current limitations of using chABC and develop a delivery system for facilitating chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo. Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational change of the enzyme was measured via circular dichroism (CD) with and without trehalose. When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4 weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a sustained release and showed that chABC released from the delivery system is still functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-microtube system was used to locally deliver chABC over two weeks at the lesion site following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and chABC loaded lipid microtubes was implanted at the top of the lesion site immediately following injury. To determine effectiveness of topical delivery of thermostabilized chABC, animal groups treated with single injection or gel scaffold implantation of chABC and penicillinase (P'ase) were included as controls. Two weeks after surgery, the functionality of released chABC and the cellular responses were examined by immunohistological analysis with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results demonstrated that thermostabilized chABC was successfully delivered slowly and locally without the need for an indwelling catheter by using the hydrogel-microtube delivery system in vivo. The results demonstrated that released chABC from the gel scaffold effectively digested CSPGs, and therefore, there were significant differences in CSPG digestion at the lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and controls. Third, a long term in vivo study (45 days) was conducted to examine axonal sprouting/regeneration and functional recovery with both a single treatment each of microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the hydrogel-microtube delivery system. Over the long term study period, the treated animals showed significant improvement in locomotor function and more sprouting of cholera toxin B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers around the lesion site. We demonstrated that this significant improvement of chABC thermostability facilitates the development of a minimally invasive method for sustained, local delivery of chABC that is potentially a useful and effective approach for treating SCI. In addition to that, we demonstrated that combinatorial therapy with chABC and neurotrophic factors could provide a synergistic effect on axonal regrowth and functional recovery after SCI.