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Tmvenaty Microfilms International TMvenaty Microfilm s International MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDS STANDARD REFERENCE MATERIAL 1010a (ANSI and ISO TEST CHART No. 2) i. i i t University Microfilms Inc. 300 N. Zecb Road, Ann Arbor, MI 48106 INFORMATION TO USERS This reproduction was made from a copy of a manuscript sent to us for publication and microfilming. While the most advanced technology has been used to pho­ tograph and reproduce this manuscript, the quality of the reproduction is heavily dependent upon the quality of the material submitted. Pages in any manuscript may have indistinct print. In all cases the best available copy has been filmed. The following explanation of techniques is provided to help clarify notations which may appear on this reproduction. 1. Manuscripts may not always be complete. When it is not possible to obtain missing pages, a note appears to indicate this. 2. When copyrighted materials are removed from the manuscript, a note ap­ pears to indicate this. 3. Oversize materials (maps, drawings, and charts) are photographed by sec­ tioning the original, beginning at the upper left hand corner and continu­ ing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or in black and white paper format.* 4. Most photographs reproduce acceptably on positive microfilm or micro­ fiche but lack clarity on xerographic copies made from the microfilm. For an additional charge, all photographs are available in black and white standard 35mm slide format.* ♦For more information about black and white slides or enlarged paper reproductions, please contact the Dissertations Customer Services Department. T T-A/T-T D'ssertation vJ l V l l Information Service University Microfilms International A Bell & Howell Information Company 300 N. Zeeb Road, Ann Arbor, Michigan 48106 8618788 Jackson, Kelly Michael GANGLIOSIDES SUPPRESS THE PROLIFERATION OF AUTOREACTIVE CELLS IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: THE EFFECTS OF GANGLIOSIDES ON INTERLEUKIN 2 ACTIVITY The Ohio Stale University Ph.D. 1986 University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106 Copyright 1986 by Jackson, Kelly Michael All Rights Reserved GANGLIOSIDES SUPPRESS THE PROLIFERATION OF AUTOREACTIVE CELLS IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: THE EFFECTS OF GANGLIOSIDES ON INTERLEUKIN 2 ACTIVITY DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In the Graduate School of the Ohio State University By Kelly Michael Jackson, B.S. * * * * * The Ohio State University 1986 Dissertation Committee: Approved by Caroline C. Whitacre, Ph.D. Raymond Lang, Ph.D. Frank Kapral, Ph.D. P auli. 'nJJ d WA l& o U' Dennis Pollack, Ph.D. Advisor Department of Medical Microbiology and ImmunologyAllan Yates, M.D., Ph.D. and ImmunologyAllan Copyright by Kelly Michael Jackson 1986 DEDICATION To the memory of my mother, Mrs. Laura T. Jackson - i i - ACKNOWLEDGMENTS I would like to express my sincere appreciation and gratitude to my research advisor, Dr. Caroline Whitacre, for her guidance, support, and especially for her endurance throughout the course of this study. I also thank the other members of Dr. Whitacre’s laboratory, Ingrid Gienapp, Clara Pelfry, Kathy Fuller, and Dina Bitar, for their moral support and friendship. I offer my sincere thanks to my advisory committee, Dr. Raymond Lang, Dr. Dennis Pollack, Dr. Frank Kapral, and Dr. Allan Yates, for their helpful suggestions. Dr. Yates and the members of his laboratory deserve special thanks for training that I received, and for the use of their laboratory space and equipment. I also wish to extend special thanks to Nancy Zinn and Dr. Charles Orosz for their assistance and ideas. I would also like to express my deepest appreciation to my father, Dr. Aubrey Jackson, and to my brothers, Aubrey and Carl, for their support and encouragement throughout this experience. - i i i - VITA February 21, 1958............................... Born - Fairmont, West Virginia 198 0 ...................................................... B.5., Marshall University, Huntington, West Virginia 1 9 8 1 -1 9 8 5 .........................................C.I.C. Minority Student Fellow, The Ohio State University, Columbus, Ohio 1981 - 1986 .........................................Graduate Research Associate, The Ohio State University, Columbus, Ohio BIBLIOGRAPHY Jackson, K. M., A. J. Yates, N. Zinn, C. Orosz, C. C. Whitacre. Experimental Allergic Encephalomyelitis (EAE): The Suppressive Effect of Gangliosides on In Vitro Proliferative Responses. Fed. Proc., 44; 3176, 1985. (Abstract) FIELD OF STUDY Major fields of study: Medical Microbiology and Immunology TABLE OF CONTENTS Page DEDICATION.................................................................. ii ACKNOWLEDGMENTS..................................................... iii VITA .............................................................................. iv LIST OF TABLES........................................................... vi LIST OF FIGURES vii, viti LIST OF PLATES........................................................... ix ABBREVIATIONS............................................................ x, xi CELL LINES..................................................................... xii CHAPTER I. INTRODUCTION.................................................. I II. MATERIALS AND METHODS............................ 36 III. RESULTS............................................................ 48 IV. DISCUSSION..................................................... 91 V. REFERENCES..................................................... 112 -V- LIST OF TABLES Table Page 1 Effects of gangliosides on antigen and mitogen-induced LNC proliferation ............................................................... 53 2 Effects of gangliosides on Con A-induced proliferation of normal unsensitized rat L N C ..................................... 56 3 Effects of gangliosides, asialo GMi, and sulfatides on MBP and Con A-induced LNC proliferation ...................... 57 A Assessment of ganglioside-mediated suppression of MBP-induced LNC proliferation, in the presence of supraoptimal MBP concentrations ............................... 61 5 Proliferative responses of an MBP-reactive rat T-cell l i n e .................................................................................. 65 6 Proliferative responses of an MBP-reative encephalito- genic T-cell line: Effects of gangliosides on MBP- induced proliferation ........................................................ 67 7 Effects of gangliosides on the in vitro activation of LNC to transfer E A E .................................................................. 68 8 Assessment of LNC transfer of EAE in recipients injected with gangliosides ............................................................... 70 9 Direct CTLL-20 indicator cell addition assay for the detection of IL 2 in MBP-activated LNC cultures .... 79 10 Effects of gangliosides on the expression of IL 2 receptors on Con A-activated L N C ..................................................... 87 11 Effects of excess IL 2 on ganglioside-mediated suppression of Con A-induced LNC proliferative responses ................. 89 -v i- LIST OF FIGURES Figure Page 1 Effects of rat and bovine brain gangliosides on the viability of rat and human lymphoid c e l l s ................................................ 50 2 Effects of rat brain gangliosides on the viability of rat lymphoid cells and human PBM C ................................................. 52 3 Effects of gangliosides on the antigen and mitogen-induced proliferation of rat L N C .............................................................. 55 4 Kinetics of ganglioside-mediated suppression of MBP- induced LNC proliferative responses ........................................ 59 5 Effects of supraoptimal MBP concentrations on ganglioside- mediated suppression of LNC proliferative responses . 62 6 Differential suppressive effects of individual gangliosides on the MBP-induced proliferative responses of LNC .... 64 7 Sialic acid concentrations in the sera from unsensitized rats and in the sera from rats with E A E .................................. 72 8 Detection of IL 2 activity in supernatants derived from Con A or MBP-activated cells: Effects of gangliosides on IL 2 ac tivity ............................................................................ 76 9 Effects of gangliosides on IL 2-induced CTLL-20 cell proliferation ............................................................................... 80 10 Effects of various concentrations of gangliosides on CTLL-20 cell proliferation and viability ................................. 83 -v ii- 11 Assessment of IL 2-induced CTLL-20 cell proliferation: Effects of ganglioside-IL 2 preincubation ...................... 84 12 Effects of gangliosides on the cel 1-surface binding of a monoclonal antibody directed against the rat IL 2 receptor ..................................................................... 86 - v ii i- LIST OF PLATES P late Page I TLC pattern of purified rat brain gangliosides .......................... 40 II TLC pattern of gangliosides extracted from the sera of unsensitized and MBP-CFA-sensitlzed rats .... 73 -ix - ABBREVIATIONS AER Active E-rosette ALS Anti-lymphocyte serum ATS Anti-thymocyte serum CFA Complete Freund's adjuvant CNS Central nervous system Con A Concanavalin A DTH Delayed-type
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