INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I 75-26,685 WHITACRE, Caroline Clement, 1949- TRANSPLANTATION STUDIES IN A CANINE MODEL: CHARACTERIZATION OF A CANINE ANTI-LYMPHOCYTE SERUM. Hie Ohio State University, Ph.D., 1975 Health Sciences, imnunology Xerox University Microfilms,Ann Arbor, Michigan 48106 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. TRANSPLANTATION STUDIES IN A CANINE MODEL: CHARACTERIZATION OF A CANINE ANTI-LYMPHOCYTE SERUM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Caroline C. Whitacre, B.A. ******** The Ohio State University 1975 Reading Committee: Raymond W. Lang, Ph.D. Vincent V. Hamparian, Ph.D. Abramo Ottolenghi, Ph.D. Advisor/ Norman L. Somerson, Ph.D. Department cS$r Medica Microbiology ACKNOWLEDGMENTS My respect and sincere thanks go to my advisor, Dr. Raymond Lang, who was always available for consultation. His dedication to his field provides an example for any beginning investigator. Many thanks also go to the members of my graduate advisory committee for their time and con­ structive criticism: Dr. Norman L. Somerson, Dr. Vincent V. Hamparian, and Dr. Abramo C. Ottolenghi. I would also like to acknowledge Dr. James A. Madura who was responsible for all coberman immunizations and bleedings. I wish to express my thanks to my mother, Mrs. Rosalyn Whitacre, for her understanding and encouragement toward graduate education. To my future husband, Michael Para, goes much gratitude for his unending moral support. VITA November 4, 1949 Born - Cincinnati, Ohio 1971 B.A./ The Ohio State University, Columbus Ohio 1971-1975 Graduate Research Assoc­ iate, Department of Medical Microbiology, The Ohio State Univer­ sity, Columbus, Ohio Publications Whitacre, C., Wilson, G.P., Madura, J.A., St. Pierre, R. L., and Lang, R.W., "Specificity of DEAE Fractions of Canine Anti-Lymphocyte Serum." Abstract in Tissue Antigens, 3 (1973), 167. Whitacre, C., and Lang, R.W., "Method for Isolation of Canine Lymphocytes." Abstract in Am. Soc. Microbiol., (1973),98. Whitacre, C., and Lang, R.W., "Characterization of IgG- Containing DEAE Fractions of Canine Serum." Transplanta­ tion Proceedings, (1975), in press. Whitacre, C., and Lang, R.W., "A Technique for Separation of Canine Lymphocytes and Their Use in the Lymphocytotoxic, Blastogenic, and Rosette Assays." Transfusion (1975), in press. Fields of Study Major Field: Medical Microbiology Major Area of Study: Immunochemistry iii TABLE OP CONTENTS Page ACKNOWLEDGMENTS ....................................... ii VITA ................................................. iii LIST OF TABLES ............................... vi LIST OF FIGURES ............................... vii INTRODUCTION ......................................... 1 PART I. CHARACTERIZATION OF IgG- CONTAINING DEAE FRACTIONS OF CANINE SERUM ........................... 10 Introduction .............................. 11 Materials and Methods .................... 12 Results ................................... 14 D i s c u s s i o n ................................ 23 PART II. DEAE CHROMATOGRAPHIC CHARACTERIZA­ TION OF CANINE SERUM AND ISOLATION OF AN IgG SUBCLASS ........................ 27 Introduction .............................. 28 Materials and Methods .................... 29 Results ................................... 32 Discussion ................................ 47 PART III. A TECHNIQUE FOR SEPARATION OF CANINE LYMPHOCYTES AND THEIR USE IN THE LYMPHOCYTOTOXIC, BLASTOGENIC, AND ROSETTE ASSAYS ............................ 50. Introduction .............................. 51 Materials and Methods ..................... 52 Results ................................... 55 iv Page D i s c u s s i o n .............................. 62 PART IV. CHARACTERIZATION OF A DL-B TISSUE TYPING ANTISERUM ................ 64 BIBLIOGRAPHY ........................................ 73 V LIST OF TABLES Table Page 1. Localization of cytotoxic activity in DEAE column fractions .............. 24 2. Analysis of leukocyte preparations ....... 56 3. Analysis of lymphocyte preparations ..... 57 4. PHA and PWM stimulation of lympho­ cytes following separation from canine blood ................... 59 5. E rosette formation of lymphocytes following separation from canine b l o o d ........i ......................... 60 6. Comparison of cytotoxic titers with lymphocyte preparations ............... 51 vi LIST OP FIGURES Figure Page 1. The family history of the "coberman" dogs .... 13 2. DE-52 chromatograph of canine anti-lympho­ cyte serum SI4113 and chromatograph of pooled and concentrated pre-hemoglobin fractions............................ 15 3. Immunoelectrophoresis of DE-52 fractions 1, 2, 3, 4, 5, commercial canine IgG, and commercial canine transferrin ............. 17 4. Osserman Immunoelectrophoresis test for identification of DE-52 column frac­ tions ..................................... 20 5. Analytical DE-52 chromatographs of canine serum and serum fractions : A. Reconstituted lyophilized commercial dog serum ................. 33 B. Pooled normal mongrel serum ......... 34 C. Normal coberman serum ............ .. .. 35 D. Coberman anti-lymphocyte serum ...... 36 . E. Commercial canine Cohn fraction II ... 37 F. Human serum, canine serum, human s e r u m ................................. 39 6. Direct immunoelectrophoresis of fraction 1, fraction 2, and commercial canine IgG..... 41 7. Osserman immunoelectrophoretic technique for identification of fraction 3, and 2 components ...................... 43 8. Polyacrylamide gel electrophoresis of fractions 1-5, whole serum S14113 and canine IgG .................. 44 vii Figure Page 9. Characterization of anti-fraction 1 and anti-fraction 2 antisera ................ 46 10. The immunizing dose and cytotoxic anti­ body response of dog 7 1 ............ 67 11. Percentage of dogs from populations react­ ing with whole ALS and the IgM frac­ tion of whole ALS and the level of response ................................. 69 viii INTRODUCTION Studies concerning the immunoglobulins of the canine were begun in the early 19 60's by Patterson and co-workers at Northwestern University. The immunologic response of dogs was investigated with the finding that dogs were poor producers of precipitating antibodies in response to solu­ ble antigens (Patterson et ad., 1963a). However, the incor­ poration of Freund's adjuvant with the antigen resulted in high antibody levels as detected by tanned red blood cell hemagglutination and precipitation reactions. The demon­ stration of both canine precipitating and non-precipitating antisera against bovine serum albumin prompted further study into the canine immune response (Patterson et al., 1964a; Patterson et al., 1964b). The precipitating antisera showed incomplete precipitation of antigen in the region of anti­ body excess as measured by coprecipitation and quantitative isotope precipitation. The non-precipitating antisera con­ tained only one active component, a $ 2 globulin, while the precipitating antisera demonstrated a $ 2 as well as a y globulin, the combined effect supposedly producing the characteristic canine precipitin curve. The first completely comprehensive study of dog immuno­ globulins was carried cut by Jphnson and Vaughan (1967a) when 1 they described six immunoglobulin classes: y2a, y2b, y2c, 7Sy^, intermediate Sy^, and yM. y2a and y2b were found to be inseparable on agar gel electrophoresis, ultracentrifuga­ tion both sedimenting at 6.7 S, starch zone electrophoresis, and DEAE cellulose chromatography. y2c was designated