The objective of this proposal is to develop new carbohydrate-based therapeutics for selectin-mediated inflammation. Infiltration of leukocytes into the synovium and joint fluids represents a significant pathological reaction in rheumatoid arthritis. The leukocytes release proteases and reactive oxygen species which results in degradation of cartilage and enhanced inflammation. Thus, methods to block leukocyte infiltration should ameliorate some of the symptoms of rheumatoid arthritis and may prevent exacerbation of the disease. The following specific aims should accomplish this goal: 1: Synthesize glycosides that resemble constituents of sialyl Lewis X. A series of disaccharides will be made based on the structure of sialyl Lewis X (sLex) that act as glycosyltransferase substrates in vitro. Attaching them to a hydrophobic aglycone, such as naphthalenemethanol, and modifying the hydroxyl groups should permit the glycosides to permeate cells and enter the Golgi where the glycosyltransferases reside. Preliminary studies indicate that acetylated Galbeta1-4GlcNAcbeta-O- naphthalenemethanol (AcLacNAc-NM) will prime oligosaccharide chains. Other blocking groups will be tested to increase the efficiency of uptake and priming. 2. Examine glycosides for inhibition of sLeX assembly and priming. Established cell lines and human monocytes and neutrophils will be grown in the presence of various amounts of acetylated disaccharides to test if they inhibit the formation of sLex. Preliminary results show that AcLacNAc-NM decreases sLex on HL-60 cells. Analytical studies will establish if the compounds prime oligosaccharide chains and alter sLex synthesis on endogenous glycoconjugates. 3. Test if glycosides inhibit adhesion of leukocytes to selections and stimulated endothelial cells. Adhesion assays will employ HL-60, U-937 and human neutrophils and monocytes binding to plates coated with selectin chimeras or TNF-alpha stimulated human umbilical vein endothelial cells. Both static and dynamic assays will be tried so that the potency of the disaccharides can be evaluated prior to testing in animals. Inhibition of selectin-mediated adhesion would suggest that the compounds might have potentially useful therapeutic activities. 4. Examine if compounds active in vitro have anti-inflammatory activity in animal models of rheumatoid arthritis. Mice immunized with bovine Type II collagen or bovine cartilage proteoglycan (aggrecan) develop symptoms resembling rheumatoid arthritis. The anti-arthritic effects of the disaccharides can be judged by measuring the number of affected paws and paw width, and by histological analysis of affected joints. We also plan to examine leukocytes from treated animals to detect differences in expression of sLex and their ability to adhere.
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