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dc.contributor.advisorSiahaan, Teruna J.
dc.contributor.authorManikwar, Prakash
dc.date.accessioned2010-10-03T03:21:14Z
dc.date.available2010-10-03T03:21:14Z
dc.date.issued2010-06-23
dc.date.submitted2010
dc.identifier.otherhttp://dissertations.umi.com/ku:11062
dc.identifier.urihttp://hdl.handle.net/1808/6731
dc.description.abstractThe 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.extent231 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectPharmaceutical chemistry
dc.subjectConjugation
dc.subjectExperimental autoimmune encephalomyelitis
dc.subjectFluorescein isothiocyanate
dc.subjectLeukocyte function associated antigen-1 (lfa-1)
dc.subjectLfa 1 i-domain
dc.subjectProteolipid protein (plp)
dc.titleIN VITRO AND IN VIVO EVALUATION OF NOVEL I-DOMAIN CONJUGATES FOR DRUG TARGETING TO IMMUNE CELLS AND SUPPRESSION OF EAE IN MICE
dc.typeDissertation
dc.contributor.cmtememberKrise, Jeffrey P.
dc.contributor.cmtememberLaurence, Jennifer S.
dc.contributor.cmtememberBerkland, Cory J.
dc.contributor.cmtememberDeGuzman, Roberto
dc.thesis.degreeDisciplinePharmaceutical Chemistry
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
kusw.bibid8085531
dc.rights.accessrightsopenAccess


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