Abstract

Type 1 diabetes mellitus (T1D) is a debilitating chronic inflammatory disease of the insulin-producing pancreatic islet β-cells that results from a combination of genetic and environmental factors. Attempts to suppress Th1-mediated autoimmune diseases such as T1D by mucosal delivery of autoantigens for immunotolerization have yielded only partial success. Attainment of satisfactory levels of sustained immunological tolerance remains to be accomplished. To restore self-tolerance requires delivery of sufficient amounts of autoantigen to stimulate regulatory T helper cells that function to survey the gut and induce tolerance to consumed antigens such as food. Oral delivery of autoantigens has previously been shown to elicit IL-4, IL-10, and TGF-β suppressor cytokine release by regulatory T-cells, but large autoantigen doses are usually required to overcome protease degradation in the gut.

To enhance the effectiveness of oral tolerance therapy, the diabetes autoantigen proinsulin was linked to the non-toxic B chain of ricin—an enterocyte-binding plant lectin that possesses intrinsic cell-delivery and immunostimulatory properties. The human proinsulin (INS) ricin toxin B chain (RTB) fusion protein was expressed in Escherichia coli and purified for use in an animal inoculation experiment to test for the suppression of diabetes symptoms. Non-obese diabetic (NOD) mice were orally inoculated and boosted with purified INS-RTB. Histopathological examination of immunized mouse pancreatic tissue showed significantly less lymphocyte infiltration into pancreatic islets of Langerhans (regions of insulin production by β-cells) than did mice dosed with INS alone or in untreated animals.

The bacterial-produced INS-RTB was synthesized as a denatured aggregate that, despite numerous attempts to dialyze in a variety of refolding buffers, would only yield ~6 - 8% of native, biologically active INS-RTB protein. Therefore, the INS-RTB genes were introduced into the genome of potato plants by Agrobacterium tumefaciens mediated transformation for the generation of a properly folded, post-translationally modified source of INS-RTB fusion protein characteristic of a eukaryotic production system. Synthesis of the INS-RTB protein was confirmed by tissue immunoprint and Western blot using antibodies that bind INS and RTB. Lectin activity of natively folded RTB fusion protein was determined by binding to asialofetuin in an enzyme-linked immunosorbent assay.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate Studies

First Advisor

William H. R. Langridge

Second Advisor

Penelope J. Duerksen-Hughes

Third Advisor

Alan P. Escher

Fourth Advisor

Daila S. Gridley

Fifth Advisor

E. Clifford Herrmann

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2010

Date (Title Page)

6-2010

Language

English

Library of Congress/MESH Subject Headings

Diabetes Mellitus, Type 1 -- immunology; Diabetes Mellitus, Type 1 -- therapy; Ricin -- genetics; Ricin -- administration & dosage; Receptor, Insulin -- metabolism; Insulin -- genetics; Insulin -- administration & dosage; Immune Tolerance; Polymerase Chain Reaction; Protein Subunits -- genetics; Protein Subunits -- immunology; Cell Fusion; Recombinant Fusion Proteins -- genetics; Recombinant Fusion Proteins -- immunology; Mice, Inbred NOD.

Type

Dissertation

Page Count

xix; 152

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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