This dissertation has been microfilmed exactly as received 69-4926 LARKIN, Gary Freeman, 1941- SEROLOGIC SPECIFICITY OF ANTIBODIES TO RIBONUCLEIC ACID IN NORMAL AND RHEUMATOID ARTHRITIS SERA. The Ohio State University, Ph.D., 1968 Health Sciences, immunology University Microfilms, Inc., Ann Arbor, Michigan SEROLOGIC SPECIFICITY OF ANTIBODIES TO RIBONUCLEIC ACID IN NORMAL AND RHEUMATOID ARTHRITIS SERA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Gary Freeman Larkin, B»S<,, MoS„ The Ohio State University 1968 Approved by Adviser Microbiology ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation and thanks to Dr. Matthew C. Dodd whose sound advice and encouragement were invaluable during the course of graduate studies at this Uni­ v e rsity . The writer is also deeply grateful to Dr. Nancy J. Bigley for her enthusiasm, interest, and most valuable criticism during the present investigation. Appreciation is also extended to James C, Darnel’ for his sug­ gestions and interest in this study. VITA July 16, 19;+1 Bom — Massillon, Ohio 1963• B.S., The Ohio State U niversity, Columbus, Ohio 196^ 1966.... Teaching A ssistant, Department, of Microbiology, The Ohio State U niversity, Columbus, Ohio 1966...c.e.o. M.S., The Ohio State U niversity, Columbus, Ohio 1966-196?.... Graduate Research Assistant, Department of Micro­ biology, The Ohio State U niversity, Columbus, Ohio 1967-1968.... Graduate Research Associate, Department of Micro­ biology, The Ohio State U niversity, Columbus, Ohio FIELDS OF STUDY Major Field: Microbiology Studies in Immunology. Professor Matthew C, Dodd, Profes­ sor Nancy J. Bigley, Professor Frank V/, Chorpenning Studies in Pathogenic Microbiology. Professor Melvin S. Rheins Studies in Bacterial Physiology. Professor Chester I. Randles , Studies in Food Microbiology. Professor Harry H. Weiser Studies in Virology. Professor David A. Wolff i i i CONTENTS Page INTRODUCTION.. ___ ......................... 1 REVIEW OF THE LITERATURE. ......... 3 MATERIALS AND METHODS.. « . « . 2 3 RESULTS OF EXPERIMENTATION. 37 DISCUdSION. .. .. .. ..6oe..o.ffoe..o.....0oe*effeooee.ee«eoo..o. 72 SUMMARY . .o. o. .9 0 . oe. .......... «.o.o. 87 LITERATURE CITED..........o o o ............................... xv TABLES Table Page 1. Percentage of complement fix atio n in h ibition of anti-RNA a c tiv ity in normal human serum by- purine and pyrimidine bases.......................... 39 2. Percentage of complement fix atio n in h ib itio n of anti-RNA a c tiv ity in normal human serum by purine and pyrimidine ribonucleosides................ 39 3. Percentage of complement fix atio n inh ib itio n of anti-RNA a c tiv ity in normal human serum by purine and pyrimidine ribonucleotides». .... .......... 40 4. Percentage of complement fix atio n in h ibition of anti-RNA a c tiv ity in normal human serum by adenine-containing compounds. • . •». 41 5. Percentage of complement fix atio n inh ib itio n of • anti-RNA a c tiv ity in normal human serum by guanine-containing compounds. 41 6. Percentage of complement fix atio n in h ib itio n of anti-RNA a c tiv ity in normal human serum by cytosine-containing compounds. 42 7. Percentage of complement fix atio n inh ib itio n of anti-RNA a c tiv ity in normal human serum by thymine-containing compounds. 43 8. Percentage of complement fix atio n inh ib itio n of anti-RNA a c tiv ity in normal human serum by uracil-containing compounds .......... 43 9. Results of whole serum electrophoresis of rheumatoid arthritis sera and normal sera............ 46 10. Conglutination reactivity of rheumatoid arthritis sera and normal sera ......... 48 v Table Page 11. Passive hemagglutination reactivity of rheumatoid arthritis sera with nucleic acids and their derivatives...............p . 31 15. Results of passive hemagglutination absorptions with various antigens upon rheumatoid az tin x tis seia.......oooo........... 5^* 13. Results of passive hemagglutination absorptions with base pairs upon rheumatoid a r th r itis s e ia . .......... 63 v i INTRODUCTION Complement fixation techniques as well as precipitation reactions have demonstrated that antibodies exist in serum with specificities directed toward cellular as well as subcellular portions of tissues and organs. Furthermore, different specificities can be shown in comparisons between normal and malignant cells and subcellular components. With the discovery of, and subsequent investigations into, the area of nucleic acid antigenicity, work in this laboratory and others has been carried out in order to determine the exact im­ munological. specificity of these-antigenic substances. The work of Friou, Finch and Detre (30) and the subsequent identification of the lupus erythematosis factor as a gamma globulin with deoxyribonucleo- protein specificity was a major achievement in the increased body of knowledge regarding the immunologically associated autoimmune disease processes. Previous work in this laboratory has shown that ribonucleic acid (RNA) linked to bentonite particles can detect antibodies to RNA in sera of both diseased and normal individuals. Furthermore, there are differences in the specificity of the antibody response, to this antigen. It is the purpose of 'Hie present study to further elucidate these differences and to study in greater depth the iimn.unocheiin.cal relationship between the antigen and its specific antibody. 1 By utilizing the quantitative micro-comploment fixation and micro-complement fixation inhibition techniques of Wasserman and Levin© (92), normal serum anti-RNA antibody sp e c ific ity was studied. Since rheumatoid arthritis sera were anti-complementary, attempts were made to identify these factors. Passive hemagglutination titrations and absorptions were performed upon rheumatoid arthritis sera using nucleic acids, nucleotides, nucleosides and bases as anti­ gens. Base pair absorptions were also carried out. Direct comparisons between the bentonite flocculation technique, the passive hemagglu­ tin a tio n technique and the complement fix atio n technique were made. By using various nucleotides, nucleosides, and purine and pyrimidine bases, the immunologic specificity of such antibodies was also com­ pared. The nature of the antigenic determinant was investigated. REVIEW OF THE LITERATURE Previous reports have indicated a wide range of immunolog- . ically active substances present in tissue and organ extracts. Duran-Reynals (27) reported that sera from chickens and other fowls flocculated crude salin e extracts of huiuan tissu e s. Using complement fixation, Kidd and Friedewald (45) demonstrated the reactivity of normal rabbit serum with saline extracts of rabbit kidney,.liver, lung, brain, spleen, and heart tissue. Further investigations have shorn th a t animals immunised with wholo brain tissu e react with alco- . holic extracts of brain (.96), both of homologous and heterologous origin. Thyroid tissue appears to contain both organ and species specific factors. Isoantibodies may be produced in the rabbit causing changes in the organ of the immunised animal (69, 97). Sim ilarly, many patients with chronic thyroiditis possess antibody reactivity toward the microsomal fraction of the extracted gland (84). Furthermore, lens (37), uveal (22), and pancreatic tissue (98) can serve as auto­ antigens as well as iso- and hotero-genetic antigens. Gajdusek (34) used complement fixing antibody techniques to demonstrate autoanti­ bodies to human tissue antigens in the sera of patients known to have acute or chronic diseases as well as for the detection of autoantibody to normal human tissue (33)» In addition to normal tissue and organ antigens, there are many specific antigens which are produced as the 3 kr result of experimentally induced tumors (55) and naturally occurring tumors (85t 99) • Wiederman (95) has shown that antibodies with specifieity directed toward the cytoplasmic constituents of the liver, namely the mitochondria, lysosomos, microsomos and the soluble fractio n appear in sera of patients with lupus erythematosis and infectious hepatitis. Asherson (1) demonstrated furthermore complement fixation reactions with the cytoplasmic fraction of cells as well as with nuclear compo­ nents in the sera of patients with systemic lupus erythematosis, lupoid hepatitis, syphilis, and macroglobulinemia. Deicher, eh al. (2h) demonstrated that sera of patients with SLE and other diseases when mixed with subeellular fractions (human liv e r, kidney or calf liv e r, and r a t liv e r microsomes and mitochondria) bound complement. Treatment of these preparations with DNase, RNase, or trypsin did not change the titer of the complement fixation reac­ tions indicating that nucleic acids were probably not the cause of antibody production. As a logical consequence of these studies, Macks.y and Gajdusek (52) stated that cell proteins, particulate compo­ nents, polysaccharides, and nucleoproteins could become antigenic and act as autoantigens if they were released from the cell and came into contact with cells of the retieulo-endothelial system. When, in 19h8, Hargraves, Richmond and Morton (hO) discovered the LE serum factor which is responsible for LE cell formation and when Haserick, et al. (hi) determined it to be a gamma globulin, interest was turned toward discovering the specificity of the LE fac­ tor in serum. Friou, Finch, and Detre (30) showed that this serum facto r combined with iso lated n u c le i, and Friou (31)