dc.contributor.advisor | Siahaan, Teruna J. | |
dc.contributor.author | Manikwar, Prakash | |
dc.date.accessioned | 2010-10-03T03:21:14Z | |
dc.date.available | 2010-10-03T03:21:14Z | |
dc.date.issued | 2010-06-23 | |
dc.date.submitted | 2010 | |
dc.identifier.other | http://dissertations.umi.com/ku:11062 | |
dc.identifier.uri | http://hdl.handle.net/1808/6731 | |
dc.description.abstract | The long-term objective of this project is to utilize the I-domain of leukocyte function associated antigen-1 (LFA-1) to target antigenic peptides and drugs to intercellular adhesion molecule-1 (ICAM-1) expressed on the surface of immune cells. The objectives of the dissertation are: 1) to characterize the binding properties of the I-domain to ICAM- 1 receptors on the surface of lymphocytes (Raji cells), and 2) to evaluate the efficacy of PLP-I-domain conjugates in suppressing experimental autoimmune encephalomyelitis (EAE) in the mouse model, an animal model for multiple sclerosis. To accomplish these objectives, the I-domain protein was labeled with fluorescein isothiocyanate (FITC) at several lysine residues to produce the FITC-I-domain. Along with trypsin digestion and peptide mapping, we utilized a specific fragmentation of the fluorochrome moiety from the modified residues in the electrospray ionization-mass spectrometry (ESI-MS) to identify the conjugation sites more quickly (Chapter 2). The FITC-I-domain binds to ICAM-1 in a calcium-, time- and energy-dependent manner. It enters the Raji cells via receptor-mediated endocytosis. FITC-I-domain binding to ICAM-1 was blocked by anti- I-domain mAb; in contrast, anti-ICAM-1 mAb to D1 and D2-domain enhance FITC-I- domain binding to ICAM-1 on the cell surface (Chapter 3). The I-domain protein was conjugated to an antigenic peptide, PLP139-151, to produce PLP-I-domain-1 and -2 conjugates. We evaluated the biological activities of the conjugates in female SJL/J mice induced with EAE (Chapter 4). The in vivo studies showed that PLP-I-domain-1 has excellent efficacy in suppressing EAE, similar to that of the best positive control (i.e., Ac-PLP-cIBR1-NH2). Although PLP-I-domain-2 could delay the onset of EAE compared to PBS, it was not as potent as PLP-I-domain-1. The chemical structure differences between PLP-I-domain-1 and -2 were determined using tryptic digestion followed by mass spectroscopic analysis. The number of conjugation sites in PLP-I-domain-1 is higher than in PLP-I-domain-2; this suggests that these additional sites in PLP-I-domain- 1 contribute to its biological activity. In conclusion, the I-domain protein binds to and is internalized by ICAM-1 receptors on the surface of immune cells. The proper conjugation of PLP peptides to I-domain (i.e., PLP-I-domain-1) is necessary for suppressing EAE in the animal model. | |
dc.format.extent | 231 pages | |
dc.language.iso | en | |
dc.publisher | University of Kansas | |
dc.rights | This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author. | |
dc.subject | Pharmaceutical chemistry | |
dc.subject | Conjugation | |
dc.subject | Experimental autoimmune encephalomyelitis | |
dc.subject | Fluorescein isothiocyanate | |
dc.subject | Leukocyte function associated antigen-1 (lfa-1) | |
dc.subject | Lfa 1 i-domain | |
dc.subject | Proteolipid protein (plp) | |
dc.title | IN VITRO AND IN VIVO EVALUATION OF NOVEL I-DOMAIN CONJUGATES FOR DRUG TARGETING TO IMMUNE CELLS AND SUPPRESSION OF EAE IN MICE | |
dc.type | Dissertation | |
dc.contributor.cmtemember | Krise, Jeffrey P. | |
dc.contributor.cmtemember | Laurence, Jennifer S. | |
dc.contributor.cmtemember | Berkland, Cory J. | |
dc.contributor.cmtemember | DeGuzman, Roberto | |
dc.thesis.degreeDiscipline | Pharmaceutical Chemistry | |
dc.thesis.degreeLevel | Ph.D. | |
kusw.oastatus | na | |
kusw.oapolicy | This item does not meet KU Open Access policy criteria. | |
kusw.bibid | 8085531 | |
dc.rights.accessrights | openAccess | |