IN VIVO AND IN VITRO EVALUATION OF CELL~MEDIATED IMMUNITY. IN THE DOG _ Thesis for the Degree of M. S. MICHIGAN STATE. UNIVERSITY ALFRED MAURICE LEGENDRE 1 974 0- fifi~o~nnm THESI' r " ’w gag-v.9.- g2: "‘v-m..- _ . J y jLILllx -t~.,'_ hi'fihgfin SC; 1‘: ”:3- UAVLEN’Y par " I ~ .' 3-1 J’f‘“ M" 'M' A ' BINBING BY :3" mm 5 sons « BOOK ammav mc LIBRAR- BINDERS nnhl - .ns.uln -1/ |\\ ‘l A Nil'ri u\§\\‘ A Fair:- OVERDUE FINES: 25¢ per day per Item gramme LIBRARY MATERIALS: Place In book return to remove charge from circulation records ABSTRACT IN VIVO AND IN VITRO EVALUATION OF CELL-MEDIATED IMMUNITY IN THE DOG BY Alfred Maurice Legendre Methods to evaluate naturally and artificially induced cell- mediated immunity in the dog were tested. Twenty dogs of mixed age, sex and breed were injected intradermally with Monilia, Dermatophyton 0, Trichophyton, Aspergillus and Streptokinase-Streptodornase to determine if a delayed skin reaction occurred regularly to ubiquitous antigens. On the basis of visible reactions and histological examina- tion of biopsied injection sites, it was concluded that only Streptokinase-Streptodornase had promise. To test artificially induced cell mediated immunity, 10 of the dogs were injected subcutaneously with viable BCG in incomplete Freund's adjuvant, and 10 dogs were sensitized with DNCB in DMSO applied to the plantar surface of the foot. Three weeks or more after sensitization the following tests were made: intradermal tuberculin testing, skin challenge with DNCB and in vitro migration inhibition of blood leukocytes by tuberculin. Undiluted tuberculin was unsuitable for intradermal testing in the dog because of false positive reactions. Tuberculin diluted 1:50 was 90% accurate as an intradermal test in differentiating between BCG Alfred Maurice Legendre and DNCB sensitized dogs. Tuberculin diluted 1:50 produced a reaction characterized by a predominantly polymorphonuclear leukocyte infiltra- tion in BCG sensitized dogs. Half of the BCG sensitized dogs also had a distinct mononuclear cell infiltration. The response of sensitized dogs to tuberculin appears to be different than in other species. All DNCB and 30% of the BCG sensitized dogs had microscopic but not gross responses to DNCB challenge. DNCB in DMSO is not a suitable agent for skin sensitization in dogs. The direct radial migration inhibition of peripheral dog leuko- cytes by 1:100 tuberculin was 80% accurate in differentiating between BCG and DNCB sensitized dogs. The indirect method, which measures the migration inhibition effect of the supernates of dog leukocyte cultures on normal guinea pig peritoneal cells, produced an excessive number of false positive reactions. The indirect method was inferior to the direct method in evaluating cell mediated immunity in the dog. Tuberculin 1:100 was more effective than more purified tuberculo- proteins in the migration inhibition test. The average of a number of migration inhibition tests is more accurate than individual tests. The direct radial migration inhibition test of peripheral leukocytes appears to be a good in vitro method to study cell mediated immunity in the dog. IN VIVO AND IN VITRO EVALUATION OF CELL-MEDIATED IMMUNITY IN THE DOG By Alfred Maurice Legendre A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Small Animal Surgery and Medicine 1974 Dedicated to my parents and my wife ii ACKNOWLEDGEMENTS I wish to express my thanks to Dr. Leonard Gideon and Dr. Robert Michel for their suggestions and help, and to Dr. Robert Schirmer, my major professor, for his counsel, guidance and support in this study and during the past three years. A special thanks to Dr. Virginia Mallmann for the many hours she spent on the planning and development of this project. I also want to thank Mrs. Jane Walsh for performing the in vitro studies, Ms. Roberta Milar for preparation of the tissue sections, and Ms. Janice Fuller for the skillful editing and typing of the manuscript. Lastly, to the students of the immunology research group, my sincere appreciation for the many hours devoted to the project. Without their help, the successful completion of this study would not have been possible. iii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 17 Animals. . . . . . . . . . . . . . . . . . . . . . . . . . 17 Experimental Groups. . . . . . . . . . . . . . . . . . . . l7 Intradermal Testing. . . . . . . . . . . . . . . . . . . . 18 Bacillus of Calmette—Guerin (BCG) Sensitization. . . . . . 20 Dinitrochlorobenzene (DNCB) Sensitization. . . . . . . . . 21 In vivo and in vitro Evaluation of BCG Sensitized Dogs . . 22 In vitro Test of Migration Inhibition of Dog Leukocytes with Tuberculin by the Radial Migration Method . . . . . . 23 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Intradermal Testing. . . . . . . . . . . . . . . . . . . . 26 BCG Sensitization. . . . . . . . . . . . . . . . . . . . . 26 Peripheral Blood Values Pre- and Postsensitization . . . . 34 DNCB Sensitization . . . . . . . . . . . . . . . . . . . . 34 Challenge of Sensitized Dogs with DNCB . . . . . . . . . . 34 Tuberculin Testing Pre- and Postsensitization. . . . . . . 34 Intradermal Testing with Tuberculin Diluted 1:50 Pre- and Postsensitization. . . . . . . . . . . . . . . . . . . 39 Direct Migration-Inhibition Pre- and Postsensitization . . 39 Migration Inhibition in Sensitized Dogs with Various Antigens . . . . . . . . . . . . . . . . . . . . . 39 iv Page Direct and Indirect Migration Inhibition . . . . . . . . . 54 Average of All Pre- and Postsensitization Direct Test Migration Inhibition . . . . . . . . . . . . . . . DISCUSSION AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . 64 SUMRY O O O O O O O O O O O O O O O O O O O BIBLIOGRAPHY. O O O O O O O O O O O O O O O O O O O O O Table LIST OF TABLES Page Gross and microscopic evaluation of the reaction to intradermal injection of various antigens (intradermal testing was done prior to DNCB or BCG sensitization) . . . 27 Size of popliteal nodes of dogs 3 weeks postinjection of BCG or DNCB in the right hind foot and presence of acid-fast organisms in right popliteal node. . . . . . . . 33 Leukocyte, lymphocyte and monocyte counts per cubic millimeter of peripheral blood of dogs prior to and after BCG or DNCB sensitization. . . . . . . . . . . . . . 35 Cross and microscopic evaluation of sites of intradermal injection of undiluted tuberculin and DNCB challenge in dogs prior to and 3 weeks after BCG or DNCB sensitization. . . . . . . . . . . . . . . . . . . . . . . 36 Compilation of gross and microscopic reactions of dogs 1-10 to undiluted tuberculin pre— and post- senSj-tization. O O O I O O O O O O O O O C O O O O O O O O 38 Cross and microscopic evaluation of intradermal tubercu- lin (1:50) injection sites prior to and 3 weeks after BCG or DNCB sensitization and direct migration inhibition of peripheral leukocytes by 1:100 tuberculin . . . . . . . 40 Compilation of gross and microscopic evaluation of intradermal injection sites of 1:50 tuberculin pre— and postsensitization. . . . . . . . . . . . . . . . . . . 42 Direct and indirect inhibition of leukocyte migration by 1:100 tuberculin prior to and after sensitization With BCG and DNCB. O O O O O O O O O O O O O O O O 0 O O O 43 Direct inhibition of leukocyte migration by 1:100 tuberculin, PPD, Band 24 and Brucellergen prior to and after sensitization with BCG and DNCB. . . . . . . . . . . 44 vi Table 10 ll 12 13 14 15 Compilation of migration inhibition by direct method with 1:100 tuberculin in dogs sensitized with BCG and DNCB 3, 6 and 8 weeks postsensitization. Comparison of the migration inhibition effects of mammalian tuberculin, PPD, Band 24 and Brucellergen 6 weeks postsensitization with BCG and DNCB. . . . Direct and indirect migration inhibition pre- and postsensitization with BCG and DNCB. Compilation of direct and indirect migration inhibi— tion presensitization and 3 weeks postsensitization in dogs sensitized with BCG or DNCB. . . . . . . . . . Average of all tests on each dog of direct migration inhibition pre— and postsensitization with BCG or DNCB Compilation of the average of all migration inhibition results by direct methods in dogs pre- and postsensi- tization with BCG and DNCB . . vii Page 53 55 56 59 60 63 Table 10 11 12 13 14 15 Compilation of migration inhibition by direct method with 1:100 tuberculin in dogs sensitized with BCG and DNCB 3, 6 and 8 weeks postsensitization. . . . . . Comparison of the migration inhibition effects of mammalian tuberculin, PPD, Band 24 and Brucellergen 6 weeks postsensitization with BCG and DNCB. . . . . . Direct and indirect migration inhibition pre— and postsensitization with BCG and DNCB. . . . . . . . . . Compilation of direct and indirect migration inhibi— tion presensitization and 3 weeks postsensitization in dogs sensitized with BCG or DNCB. . . . . . . . . . Average of all tests on each dog of direct migration inhibition pre- and postsensitization with BCG or DNCB . Compilation of the average of all migration inhibition results by direct methods in dogs pre- and postsensi— tization With BCG and DNCB O O O O O O O O O O O O O 0 vii Page 53 55 56 59 60 63 Figure LIST OF FIGURES Page Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra— tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks = weeks; PRE = presensitization. Dogs 1, 2, and 3. Inhibition Z 35% chosen empirically as positive test. . . . . . . . . 45 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks = weeks; PRE = presensitization. Dogs 4, 5 and 6. Inhibition Z 35% chosen empirically as positive test. . . . . . . . . . 46 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks = weeks; PRE = presensitization. Dogs 7, 8 and 9. Inhibition Z 35% chosen empirically as positive test. . . . . . . . . . 47 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cul- tured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks = weeks; viii Figure 10 Page PRE = presensitization. Dog 10. Inhibition Z 35% chosen empirically as positive test. . . . . . . . . . . . 48 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of M. tuberculosis (50 pgm/ml), Band 24 (50 pgm/ml) and Brucellergen 1:100. PRE = presensitization; wks = weeks. Dogs 11, 12 and 13. Inhibition 2 35% chosen empirically as positive test . . . 49 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of ML tuberculosis (50 pgm/ml), Band 24 (50 pgm/ml) and Brucellergen 1:100. PRE = presensitization; wks = weeks. Dogs 14, 15 and 16. Inhibition Z 35% chosen empirically as positive test . . . 50 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of ML tuberculosis (50 ugm/ml), Band 24 (50 ugm/ml) and Brucellergen 1:100. PRE = presensitization; wks = weeks. Dogs 17, 18 and 19. Inhibition Z 35% chosen empirically as positive test . . . 51 Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of M. tuberculosis (50 ugm/ml), Band 24 (50 ugm/ml) and Brucellergen 1:100. PRE = presensitization; wks = weeks. Dog 20. Inhibi— tion 2 35% chosen empirically as positive test . . . . . . 52 Comparison of the migration inhibition effect of 1:100 tuberculin by the direct and indirect method in dogs sensitized with BCG (Bacillus Calmette Guerin) and DNCB (Dinitrochlorobenzene) presensitization and 3 weeks postsensitization. The direct method measures the inhi— bition of migration of sensitized dog leukocytes incubated with 1:100 mammalian tuberculin. The indirect method measures the inhibition of migration of normal guinea pig peritoneal exudate cells incubated with the cell free super- nate from the dog leukocyte cultures. Wks = weeks; PRE - presensitization. Dogs 11, 12, 13, 14 and 15. Inhibi- tion 2 35% chosen empirically as positive test . . . . . . 57 Comparison of the migration inhibition effect of 1:100 tuberculin by the direct and indirect method in dogs sensitized with BCG (Bacillus Calmette Guerin) and DNCB (Dinitrochlorobenzene) presensitization and 3 weeks postsensitization. The direct method measures the inhi- bition of migration of sensitized dog leukocytes incubated with 1:100 mammalian tuberculin. The indirect method measures the inhibition of migration of normal guinea pig peritoneal exudate cells incubated with the cell free super- nate from the dog leukocyte cultures. Wks = weeks; PRE a presensitization. Dogs 16, 17, 18, 19 and 20. Inhibition Z 35% chosen empirically as positive test. . . . . . . . . 58 ix Figure 11 12 Page Averages of all direct migration inhibition studies using 1:100 mammalian tuberculin reported in Figures 1 through 8. PRE indicates an average of all migration inhibition studies pre-BCG (Bacillus Calmette Guerin) or pre-DNCB (Dinitrochlorobenzene) sensitization. POST indicates an average of all studies postsensitization. Dogs 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Inhibition 35% choseniiempirically as positive test . . . . . . . . . 61 Averages of all direct migration inhibition studies using 1:100 mammalian tuberculin reported in Figures 1 through 8. PRE indicates an average of all migration inhibition studies pre-BCG (Bacillus Calmette Guerin) or pre-DNCB (Dinitrochlorobenzene) sensitization. POST indicates an average of all studies postsensitization. Dogs 11, 12, l3, 14, 15, 16, 17, 18, 19 and 20. Inhi- bition 2 35% chosen empirically as positive test . . . . . 62 INTRODUCTION Cellular immunity or delayed type hypersensitivity plays an essential role in the immune defense system. Acquired immunity to many viral, fungal and intracellular bacterial infections, such as tuberculosis and brucellosis, is largely cell-mediated immunity, sometimes augmented by humoral or antibody-mediated immunity. Immune surveillance is a function of cellular immunity which recognizes and destroys neoplastic cells as they develop as well as foreign, trans- planted cells. The congenital lack of this system, as in DiGeorge's syndrome, generally results in death at an early age. The basic cell of the immune system is the lymphocyte, which probably originates in the bone marrow or fetal liver. The lymphocyte may then develop under the influence of the bursa of Fabricius or bursa equivalent and becomes a B-lymphocyte capable of giving rise to antibody producing plasma cells or, under the influence of the thymus, develops into a T-lymphocyte. The T-lymphocyte is the cell responsible for cell mediated or delayed type hypersensitivity responses. The responses and interactions of the B-lymphocyte (antibody system) and the T-lymphocyte (cell—mediated system) provide immunological competence and protection. The T-lymphocyte is a long-lived, small lymphocyte which circulates in the lymph, blood and other tissues, throughout most of the body. 2 The majority of the lymphocytes in peripheral blood are T-lymphocytes. After T-lymphocytes are sensitized by an antigen, they can react immunologically specifically with the same antigen. The T—lymphocyte- antigen reaction releases effector molecules or lymphokines from the lymphocyte. The lymphokines such as migration inhibition factor, macrophage activating factor, lymphocyte transformation factor and chemotactic factor appear to attract cells into the area of the reac- tion, immobilizes the cells and causes cellular proliferation. This sequence of events results in a grossly visible response which appears at 24 to 48 hours at the site of a positive skin test. The reaction is characterized by a mononuclear cell infiltrate. There are 2 methods currently used to detect and evaluate cellular immunity in vivo. The first method utilizes a ubiquitous antigen which naturally induces sensitization in a large percentage of the popula- tion. This method assumes that previous contact with the antigen has occurred. Candida is a suitable antigen in man because it produces a delayed type hypersensitivity reaction in most people tested intra- dermally. The second method involves sensitization with a chemical hapten, such as dinitrochlorobenzene (DNCB), not found in nature. The individual is then skin tested with the hapten after sufficient time for sensitization to develop. In vitro methods which became available in the last decade have expanded the understanding of cellular immunity. The in vitro methods generally measure the effects of the lymphokines produced by T-lymphocyte- antigen reaction. Currently the most accepted tests are lymphocyte transformation and inhibition of macrophage migration. The results of 3 these tests correlate well with clinical evaluation and in vivo methods. The importance of cellular immunity and methods of evaluation is reasonably well established in man. Little work has been done in the dog except in the area of transplant immunology. This study applies the methods currently used in man to evaluate cellular immunity in the dog. The response to 5 ubiquitous agents was studied, as was the response to DNCB sensitization. Dogs were infected with BCG (Bacillus Calmette Guerin) and their responses evaluated by intradermal testing and migration inhibition of leukocytes in vitro. Hopefully a system for evaluation of cellular immunity can be developed for the dog. LITERATURE REVIEW The role of cellular immunity in immune protection has been extensively investigated in the last decade. In a review of cell- mediated immunity,61 a panel of experts of the World Health Organiza- tion stated that cellular immunity plays an important role in defense against intracellular bacterial, viral, fungal and protozoal infection. David24 stated that cellular immunity is important in homograft rejection and protection against cancer cells. As the function of cellular immunity has become apparent, a variety of in vivo and in vitro methods were developed to evaluate this part of the immune system. Intradermal skin testing was among the first methods used and is still the mainstay of demonstrating delayed hypersensitivity in vivo. The tuberculin reaction is considered the classical example of delayed type hypersensitivity or cellular immune response. Tuberculin was the first antigen shown to elicit a delayed type hypersensitivity reaction. Koch,39 in 1891, first used tuberculin as a therapeutic agent because of the reaction it produced when injected into tuberculous patients. Intradermal tuberculin testing is the most widely accepted method for demonstrating sensitivity to the tuberculosis organism. The role of delayed hypersensitivity was very controversial, largely because sensitivity could not be transferred by serum. The cellular nature of the delayed hypersensitivity response was demonstrated by 5 Landsteiner and Chase41 in 1941. After inducing delayed hypersensitivity to a picryl chloride compound, peritoneal exudate cells transferred from sensitized guinea pigs to normal guinea pigs conferred the ability to respond to picryl chloride in a delayed manner. Transfer of serum or heat killed cells did not transfer the delayed type reaction. Chase15 demonstrated transfer of delayed hypersensitivity to tuberculin with leukocytes or transfer factor from leukocytes. Though the tuberculin reaction is considered the classical example of delayed hypersensitivity, many other bacterial and fungal antigens produce delayed hypersensitivity reactions. Hemolytic streptococci and their soluble extracellular products, Streptokinase-Streptodornase (SK-SD), can produce delayed type hypersensitivity reactions. Lawrence42 passively transferred cellular immunity in man with cells from sensi— tized individuals. He also found that 69% of the population had delayed type sensitivity to SK-SD without a history of prior strepto- coccal infection, which suggests previous exposure that induced a cellular immune response. The use of ubiquitous antigens to which a large percentage of the population react in a delayed manner offers a good method of in vivo evaluation of cellular immunity. If an indi- vidual failed to respond to a battery of ubiquitous antigens, his cellular immune mechanism would be suspect. A group of antigens has evolved empirically that are used in man to evaluate immune responsive- ness. Candida, Tricophyton, Streptokinase-Streptodornase and mumps antigen are commonly used.40’56’12 Intradermal tuberculin testing is presently the most frequently used method for demonstrating delayed hypersensitivity in the dog. The 6 occurrence of tuberculosis in the dog and the use of intradermal testing is well documented. Berg,S in a study of BCG infected dogs, or dogs with naturally occurring tuberculosis, reported that the use of tuberculin was not reliable. Tuberculin produced nonspecific reactions in control dogs which were equal in severity to the delayed hypersensi- tivity reactions of some infected dogs. Nonspecific reactions reached maximum intensity at 24 hours, while delayed hypersensitivity reactions were not maximum until 48 to 72 hours. The use of tuberculin had only a 50% reliability. Heat concentrated synthetic medium tuberculin pro- duced a distinctly positive reaction in all infected dogs without false positive reactions in controls. Positive skin reactions were noted as early as 9 days postinfection. Snider54 reviewed the published cases of tuberculosis in the dog and cat from 1890 to 1969 and reported that intradermal skin tests with tuberculin were unreliable. Awad2 reported experimental and Paroti46 clinical success with the use of BCG in intradermal skin testing in the dog. A study by Snider et al.55 of clinical tuberculosis in dogs and cats in Pennsylvania showed that only 50% of infected animals tested were positive to intradermal testing with tuberculin orMo bovis antigen. Delayed hypersensitivity reactions as seen in tuberculin sensi- tivity produce a characteristic type of inflammatory response. Cell and Hinde28 described the skin reactions of sensitized rabbits to tuberculin testing. Perivascular cuffing of skin vessels with mono— nuclear and polymorphonuclear leukocytes characterized the reaction at 4 hours postinfection. Mononuclear cells were more prominent in the dermis while in the subcutaneous area polymorphonuclear leukocytes 7 predominated. By 22 hours postinjection the numbers of polymorpho- nuclear cells had decreased and histiocytic hyperplasia with perivascular cuffing of mononuclear cells was the prominent feature. Focal accumu- lations of polymorphonuclear cells in the dermis were felt to be due to injection trauma and tissue necrosis. Turk and Oort58 demonstrated the histological features of delayed type tuberculin hypersensitivity in guinea pigs passively sensitized by cell transfer. Polymorpho- nuclear leukocytes were prominent at 4 hours, but decreased in numbers by 12 hours postinjection. At 12 hours mononuclear cells predominated in the dermis, but polymorphonuclear leukocytes persisted in the subcu- taneous area. Up to 12 hours postinjection there was no significant difference between the inflammatory response of sensitized and normal guinea pigs. After 12 hours the sensitized animals showed greater cellular infiltration. Cell mediated reactions in the rabbit and guinea pig are predominantly a mononuclear reaction, but polymorphonuclear leukocytes are also found in significant numbers. Skin sensitization with a chemical hapten such as DNCB is another in vivo method for evaluation of cell—mediated immunity. Skin test with hapten after adequate time for immune response produces a delayed hypersensitivity reaction. This method has been used in man in the clinical evaluation of an individual's cellular immunity.31 DNCB sensitization evaluates the ability of the immune system at that time to respond to a new antigen not found in nature. The mechanism of DNCB sensitization was clarified by experiments on guinea pigs by 25 Eisen. The ability of the hapten to form covalent bonds with skin protein was necessary to the production of a delayed hypersensitivity 8 response. Eisen and Tabachnick26 reported that DNCB combined with protein in the epidermis and not the corium. Only DNCB conjugates in the deeper parts of the epidermis produced delayed type hypersensitivity. DNCB conjugates in the cornified layers of the epidermis failed to elicit a response. A quantitative method of DNCB sensitization was developed by Catalona et al.13 The skin is sensitized with 2000 ugm in .1 ml acetone applied to a 3 cm2 area, and a challenge dose of 50 ugm is also applied at the time. After the 7 to 21 days needed for immune response, a spontaneous flare occurs at the test site where sensitized T-lymphocytes reacted with the DNCB bound in the epidermis. If no spontaneous flare occurs by 14 days, then a rechallenge dose of 50 ug DNCB is applied and the area is observed for 24 to 48 hours. Equivocal reactions are biopsied and evaluated by histopathologic examination. The reaction is graded as follows: +4 spontaneous flare at sensitization and challenge site +3 - spontaneous flare at sensitization site +2 - response at rechallenge site only +1 - equivocal reaction--positive reaction noted on histo- pathologic examination 0 - no reaction on histopathologic examination The microscopic reaction is characterized by marked lymphocytic and histiocytic infiltration at the dermal-epidermal junction and mono- nuclear perivascular cuffing in the dermis. Catalona et al.14 found that 96.5% of the individuals sensitized with DNCB responded to primary challenge by spontaneous flare. Joseph et al.38 sensitized dogs with DNCB by applying 2000 ugm in .1 ml acetone to a 3 cm2 area on the inner thigh area and challenged at 14 days 9 postsensitization with 50 and 100 ugm DNCB. All sensitized dogs reacted when challenged with 100 pgm DNCB. Others report greater difficulty in sensitizing dogs with DNCB.3 In the last decade, more in vitro methods of evaluating cell mediated immunity have been developed and are becoming available to the clinician. These methods measure the effects of the lymphokines produced when sensitized T-lymphocytes interact with specific antigen. The advantage of the in vitro system lies in the evaluation of each lymphokine individually. In contrast, the in vivo system evaluates the sum effect of all lymphokines produced. The in vitro system also eliminates many of the in vivo variables. In vitro methods have con- tributed to the understanding of the mechanism involved in cellular immune response. A recent review by Bloom et al.,9 in 1973, categorized the in vitro methods into lymphocyte-mediated cytotoxicity, lymphocyte transformation, and migration inhibition. Lymphocyte mediated cyto- toxicity measures the cytotoxic effect of the cellular immune response. It is the in vitro correlate of immune surveillance and neoplastic cell destruction. Lymphocyte transformation methods evaluate the mitotic activity of a lymphocyte culture after stimulation. Sensitized T-lymphocytes undergo blastogenesis when they encounter specific antigen. This method quantitates the magnitude of the lymphocyte response. Migration inhibition methods measure the presence of migra- tion inhibition factor (MIF). ‘Migration inhibition factor is a soluble, tunuLIalyzable substance released when sensitized T-lymphocytes encounter Specific antigen. This factor inhibits the migration of normal leukocytes . 9 postsensitization with 50 and 100 ugm DNCB. All sensitized dogs reacted when challenged with 100 ugm DNCB. Others report greater difficulty in sensitizing dogs with DNCB.3 In the last decade, more in vitro methods of evaluating cell mediated immunity have been developed and are becoming available to the clinician. These methods measure the effects of the lymphokines produced when sensitized T-lymphocytes interact with specific antigen. The advantage of the in vitro system lies in the evaluation of each lymphokine individually. In contrast, the in vivo system evaluates the sum effect of all lymphokines produced. The in vitro system also eliminates many of the in vivo variables. In vitro methods have con- tributed to the understanding of the mechanism involved in cellular immune response. A recent review by Bloom et al.,9 in 1973, categorized the in vitro methods into lymphocyte-mediated cytotoxicity, lymphocyte transformation, and migration inhibition. Lymphocyte mediated cyto- toxicity measures the cytotoxic effect of the cellular immune response. It is the in vitro correlate of immune surveillance and neoplastic cell destruction. Lymphocyte transformation methods evaluate the mitotic activity of a lymphocyte culture after stimulation. Sensitized T-lymphocytes undergo blastogenesis when they encounter specific antigen. This method quantitates the magnitude of the lymphocyte response. Migration inhibition methods measure the presence of migra- tion inhibition factor (MIF). Migration inhibition factor is a soluble, nondialyzable substance released when sensitized T-lymphocytes encounter specific antigen. This factor inhibits the migration of normal leukocytes. 10 Migration inhibition and lymphocyte transformation are the two techniques most frequently used in evaluation of cellular immunity. The two in vitro methods should be correlated with each other and with in vivo responses when cellular immunity is evaluated. The methodology of performing the in vitro techniques is described by Bloom and Glade.8 The migration inhibition method of evaluating cellular immune response has contributed greatly to the understanding of cellular immunology. Rich and Lewis,47 in 1928, were the first to report a decrease in migration of cells from tuberculous animals when the tissue culture medium contained tuberculin. An increase in cytotoxic effects of tuberculin on the cells of tuberculous animals was also observed. The use of cells in tissue culture was prompted by the inability to demonstrate an antibody involved in the tuberculin response. The observation of cytotoxicity has been confirmed in many reports, but the development of an in vitro, quantitative method of measuring migra- tion inhibition was not developed until 1957. Hall and Scherago,32 using small pieces of a buffy coat preparation of human peripheral leukocytes, showed inhibition of the leukocytes of tuberculous indi- viduals with tuberculin. Later, Hall and Scherago33 demonstrated in guinea pigs experimentally infected with M. tuberculosis that the leukocytes from infected animals were markedly inhibited in their migra- tion by tuberculin. The migration inhibition of leukocytes developed prior to the development of delayed type hypersensitivity to intradermal injection of tuberculin. Treatment of the infected guinea pigs with streptomycin and isoniazid produced an increase in the migration of leukocytes as the disease became inactive, though the skin test remained 11 positive.34 A similar response was noted with human leukocytes when the disease was in remission.30 Johnson and Scherago37 demonstrated a direct correlation between migration of leukocytes exposed to histo- plasmin and skin sensitivity to histoplasmin in guinea pigs experi- mentally infected with Histoplasma capsulatum. This experiment demon- strated the use of migration inhibition to evaluate cellular immunity in a disease other than tuberculosis. George and Vaughn,29 in 1962, developed the capillary tube method of measuring migration inhibition. Leukocytes from sensitized animals were packed in capillary tubes and incubated in culture medium with and without antigen. The leukocytes migrate out of the capillary tubes onto a coverslip in a fan-shaped pattern. Migration is measured by projection of the culture chamber on drawing paper and calculation of the area of migration with a planimeter. Percent inhibition is calculated by this formula: area of migration with antigen area of migration without antigen X 100 = % migration 100 - % migration = % migration inhibition The capillary tube method has gained wide acceptance in evaluation of migration inhibition. Moore and Scherago45 demonstrated migration inhibition of leukocytes with histoplasmin from dogs experimentally infected with Histoplasma capsulatum. David et al.,17 using guinea pigs sensitive to tuberculin, ovalbumin and diphtheria toxoid, demon— strated that migration inhibition was antigen specific. Only the cells from animals showing delayed hypersensitivity reactions to skin testing showed migration inhibition. Antigens eliciting only antibody produc- tion were incapable of producing migration inhibition. Cells from 12 unsensitized animals could not be sensitized by incubation with sera from sensitized animals. David et al.18 showed that as few as 2.5% sensitized cells in a population of normal cells would produce migra- tion inhibition when the culture was incubated with specific antigen. Sensitized cells killed by freezing failed to inhibit migration. David at al.19 demonstrated carrier specificity with migration inhibition in animals sensitized with hapten protein conjugates. This carrier spe— cificity differs from antibody-antigen reactions in that antibodies react with the hapten regardless of the protein carrier. David at al.20 demonstrated the loss of ability to produce migration inhibition in sensitized cells incubated with trypsin. The cells recovered the ability to inhibit migration suggesting the resynthesization of the material removed by trypsin. David21 showed that puromycin could prevent the migration inhi— bition of sensitized cells. Puromycin inhibits protein synthesis; therefore, it was felt that production of migration inhibition factor requires active protein synthesis. Bloom and Bennett6 and David22 independently demonstrated in guinea pigs that the lymphocyte was the cell responsible for reaction to the antigen and that the macrophage was only an indicator cell. The inhibition of macr0phage migration was due to a soluble substance(s) elaborated from sensitized lymphocytes exposed to specific antigen. This substance was appropriately called migration inhibition factor (MIF). Sdborg51 demonstrated inhibition of migration of human leuko— cytes with Brucella antigen in subjects reacting to Brucella skin test or experimentally sensitized with killed organisms. He found the l3 migration inhibition method was the best in vitro correlate of cellular hypersensitivity. Thor57 transferred the ability to produce MIF to nonsensitized lymphocytes with an RNA extract of sensitized lymphocytes. Bloom and Bennett7 detected MIF in lymphocyte cultures as early as 6 hours after addition of specific antigen. Lymphocyte transformation was not necessary for MIF production, but supernates rich in MIF increase blast transformation in normal lymphocyte cultures. MIF enriched supernates injected intradermally in guinea pigs produced inflammatory reactions with a predominantly mononuclear infiltration. The specificity of the migration inhibition test was confirmed by David and Schlossman23 using various DNP-oligolysines. This study suggests a highly specific binding site acting as a cellular receptor. Danish workers detected inhibition and enhanced migration of human leukocytes from patients with Sjogren's syndrome,52 glomerulonephritis, and Hashimoto's thyroiditis53 when their cells were incubated with respective tissue antigens. Anderson et al.1 reported inhibition of migration of human cells from patients with mammary tumors when the cells were incubated with mammary tumor extracts. Extracts of normal mammary tissue failed to produce inhibition. Hardt et al.35 demonstrated migra- tion inhibition of leukocytes from patients with biliary cirrhosis and Chronic active hepatitis with liver mitochondrial antigens. Borstaff et <2l.11 confirmed Hardt's findings. Using tolerant animals, Borel and Daxridlo demonstrated that unresponsiveness to skin testing correlated W81.1 with a lack of migration inhibition response. Rocklin et al.,49 “Sling an indirect migration inhibition system, demonstrated production 0f ldIF in cell cultures of individuals reacting intradermally to PPD, l4 SK—SD and Candida antigens. They incubated human leukocytes with the antigens for 3 days recovering a cell—free supernate. Assay for the .presence'of MIF in the supernate was done using normal guinea pig ‘peritoneal~exudate cells and evaluating the degree of inhibition. No MIF activity was demonstrated in mixed leukocyte cultures incubated for 3 days, unless the patient had received a previous transfusion. Rocklin et al.50 found that patients with thymic aplasia were unable to manifest delayed hypersensitivity reactions in vivo or migration inhibition in vitro. Mallmann et al.,44 using a tuberculoprotein found in M. bovis, were able to differentiate guinea pigs infected with.M. bovis from those infected with M. tuberculosis. The migration inhibition test used was a radial migration of a spot of cells attached to a glass surface. Winkelsteinsg found that, if 2.5% of a leukocyte population were from the lymph node of a sensitized animal, migration inhibition occurred in the presence of specific antigen. No inhibition of migration could be produced with up to a 20% population of thymic or bone marrow cells from a sensitized animal. Leu et al.43 demonstrated that MIF inhibited the migration of peritoneal macrophages but not alveolar macrophages. Killed or live peritoneal macrophages adsorbed MIF, but alveolar macrophages did not. Incubation of an MIF—rich supernate with peritoneal macrophages removed 1/2 the MIF activity in 15 minutes. MIF adsorption shows a direct dose response at low concentrations with saturation characteristics. Adsorption is time and temperature dependent and can be abolished by 15 pretreatment of macrophages with proteolytic enzymes. These character- istics suggest a receptor site on the macrophage. Remold48 reported that MIF is heterogeneous and different antigens produce MIF of slightly different molecular weights. Human MIF has a lower molecular weight than guinea pig MIF. Inactivation of MIF by chymotrypsin suggests that it is a protein. MIF was shown to be dif- ferent from lymphotoxin and chemotactic factor. Churchill et al.16 demonstrated migration inhibition with specific tumor antigens in guinea pigs with experimentally produced hepatomas. Radiation, trypsinization and freeze storage did not inhibit the ability of the tumor cells to inhibit migration. Only specific tumors produced migration inhibition, and inhibition was not noted with unre- lated tumors and normal spleen cells. Hilborg36 demonstrated tumor immunity by migration inhibition in patients with the tumor and in contact individuals. Histoincompatible tissue homogenates did not produce MIF. Waxman and Lockskin60 reported migration inhibition to tuberculin in leukocytes of patients with miliary tuberculosis and energy to intradermal tuberculin testing. This would suggest normal production of immune mediators, but deficiency of nonimmunological inflammatory response. Fauser et al.27 used the radial migration technique to detect Production of MIF by lymphocytes of chickens. Migration inhibition occurred when chickens were experimentally infected with BCG and Marek's disease virus and the leukocytes incubated with specific antigen. Migration inhibition has been shown to be a good in vitro correlate for delayed hypersensitivity reactions. It has been used in evaluating 15 pretreatment of macrophages with proteolytic enzymes. These character— istics suggest a receptor site on the macrophage. Remold48 reported that MIF is heterogeneous and different antigens produce MIF of slightly different molecular weights. Human MIF has a lower molecular weight than guinea pig MIF. Inactivation of MIF by chymotrypsin suggests that it is a protein. MIF was shown to be dif— ferent from lymphotoxin and chemotactic factor. Churchill et al.16 demonstrated migration inhibition with specific tumor antigens in guinea pigs with experimentally produced hepatomas. Radiation, trypsinization and freeze storage did not inhibit the ability of the tumor cells to inhibit migration. Only specific tumors produced migration inhibition, and inhibition was not noted with unre- lated tumors and normal spleen cells. Hilborg36 demonstrated tumor immunity by migration inhibition in patients with the tumor and in contact individuals. Histoincompatible tissue homogenates did not produce MIF. Waxman and Lockskin6O reported migration inhibition to tuberculin in leukocytes of patients with miliary tuberculosis and energy to intradermal tuberculin testing. This would suggest normal production of immune mediators, but deficiency of nonimmunological inflammatory response. Fauser et al.27 used the radial migration technique to detect production of MIF by lymphocytes of chickens. Migration inhibition occurred when chickens were experimentally infected with BCG and Marek's disease virus and the leukocytes incubated with specific antigen. Migration inhibition has been shown to be a good in vitro correlate for delayed hypersensitivity reactions. It has been used in evaluating 15 pretreatment of macrophages with proteolytic enzymes. These character- istics suggest a receptor site on the macrophage. Remold48 reported that MIF is heterogeneous and different antigens produce MIF of slightly different molecular weights. Human MIF has a lower molecular weight than guinea pig MIF. Inactivation of MIF by chymotrypsin suggests that it is a protein. MIF was shown to be dif- ferent from lymphotoxin and chemotactic factor. Churchill et al.16 demonstrated migration inhibition with specific tumor antigens in guinea pigs with experimentally produced hepatomas. Radiation, trypsinization and freeze storage did not inhibit the ability of the tumor cells to inhibit migration. Only specific tumors produced migration inhibition, and inhibition was not noted with unre- lated tumors and normal spleen cells. Hilborg36 demonstrated tumor immunity by migration inhibition in patients with the tumor and in contact individuals. Histoincompatible tissue homogenates did not produce MIF. Waxman and Lockskin60 reported migration inhibition to tuberculin in leukocytes of patients with miliary tuberculosis and energy to intradermal tuberculin testing. This would suggest normal production of immune mediators, but deficiency of nonimmunological inflammatory response. Fauser et al.27 used the radial migration technique to detect production of MIF by lymphocytes of chickens. Migration inhibition occurred when chickens were experimentally infected with BCG and Marek's disease virus and the leukocytes incubated with specific antigen. Migration inhibition has been shown to be a good in vitro correlate for delayed hypersensitivity reactions. It has been used in evaluating 16 tumor immunity, autoimmune conditions as well as identification of cellular hypersensitivity states to a variety of bacterial and viral agents. MATERIALS AND METHODS Animals Twenty dogs of both sexes, various breeds, between 5 months and 4 years of age were used in this study. The dogs were housed in concrete cages and exercised daily in accordance with Public Law 89-544 (The Animal Welfare Act). Cages were cleaned twice daily and dogs feda once daily. Fresh water was provided ad libitum. All animals were vaccinated against canine distemper and infectious canine hepatitis at least 3 months prior to the start of the study. Experimental Groups Two experimental groups of 10 dogs each were formed. All dogs were skin tested and biopsied prior to sensitization with BCG or DNCB. The first group was tested with undiluted tuberculin intra- dermally prior to sensitization, while the second group was tested with a 1:50 dilution of tuberculin. Five dogs of each group received BCG, while the other 5 dogs were sensitized with DNCB. Variations in the in vitro testing existed between the 2 groups. aKen-L—Meal, Quaker Oats Company, Chicago, Illinois. 17 18 Intradermal Testing The hair of both flanks was clipped closely with a #40 Oster blade immediately prior to injection. No cleansing of the skin was done. The skin was marked with vertical lines to identify the injec— tion sites. Intradermal injection of .1 ml of each concentration of antigen was made using a tuberculin syringe with a 25—gauge needle. The accuracy of the intradermal injection was evaluated by the presence of a small skin bleb. Injections were made in duplicate at the ends of the lines on the right and left side. The following antigens were injected: Antigen Concentration Streptokinase-Streptodornaseb (SK-SD) 50 u/.l ml Monilia MixC 1:10 and 1:100 Dermatophyton OC (Candida albicans) 1:10 and 1:100 Aspergillus MixC 1:10 and 1:100 Trichophyton MixC 1:10 and 1:100 Diluentc (control) The injection sites were evaluated at l, 4, 24 and 48 hours. The reactions were evaluated visually and by digital palpation for signs of skin thickening, induration and erythema. The following scale was used in evaluating the reactions: 0 - No evidence of inflammatory changes +1 - Slight thickening noted only on palpation +2 - Skin thickening with an area of induration less than 1.0 cm in diameter +3 - Skin thickening with induration greater than 1.0 cm in diameter usually accompanied by erythema and exudation. bVarizyme, American Cyanamid Co., Princeton, New Jersey. CHollister Stier, Downer's Grove, Illinois. 19 In addition to visual and digital evaluation, the injection sites of the second group were evaluated by measuring skin fold thickness with calipers prior to and 24 hours after intradermal injection. At 24 hours postinjection the sites on the right side were biopsied using an 8 mm Keyes cutaneous punchd after the animal had been anesthe- tized with intravenous sodium thiamylal.e The biopsies were fixed in 10% buffered formalin and tissue sections were made and stained with hematoxylin-eosin. The histological sections were examined for the degree of inflammatory changes and cellular infiltration. The reactions were graded according to the following criteria: Grade 0 - No increase in numbers of inflammatory cells in dermis or subcutaneous area. Grade I - Very slight increase in numbers of inflammatory cells. Cellular infiltrate is diffuse with no aggregates of inflammatory cells. Grade II - Slight infiltration of dermis and/or subcutaneous area with inflammatory cells. Occasional small focal accumulations of inflammatory cells (less than 20 per high power field). Grade III — Slight to moderate infiltration of dermis and subcu— taneous area with inflammatory cells. Occasional focal accumulation of moderate numbers of inflammatory cells (less than 50 cells per high power field). dAmerican Hospital Supply, Detroit, Michigan. eSurital Sodium, Parke, Davis & Company, Detroit, Michigan. 20 Grade IV - Moderate infiltration of the dermis and subcutaneous area with inflammatory cells. Moderate numbers of foci containing large numbers of inflammatory cells (100 to 200 per high power field). Grade V - Moderate to severe infiltration of large areas of the dermis and subcutaneous area with inflammatory cells. The involvement is diffuse with many areas having hundreds of cells per high power field. Edema and hemorrhage may be evident. Grade VI - Severe infiltration of the dermis and subcutaneous area with inflammatory cells. There is replacement of many of the normal skin structures with inflammatory elements. Edema, hemorrhage, and necrosis are prominent features. DTH - Indicates that the reaction shows a mononuclear infiltration especially in the perivascular and adnexal areas suggesting a delayed hypersensitivity response. In grading the reactions, injection trauma was not considered. Bacillus of Calmette—Guerinf (BCG) Sensitization Ten of the dogs were randomly selected for sensitization with BCG. Hair was clipped from the plantar surface of the right hind foot just above the pad and the area was cleansed with alcohol. Five milligrams, wet weight, of viable BCG in .5 ml of saline and .5 m1 of Freund's 8 incomplete adjuvant were injected subcutaneously. The injection site was observed every other day for 21 days. At 21 days postinjection the fViable cultures prepared by Dr. Virginia Mallmann, Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan. gDifco Company, Detroit, Michigan. 1" 3U h 30' Hi 21 right and left popliteal lymph nodes in the first group of dogs (5 BCG sensitized) were surgically removed, measured, and fixed in 10% buffered formalin. Tissue sections were made and stained with H & E and Ziehl- Neelsen acid-fast stain. The nodes were examined for histological changes and presence of acid-fast organisms. Dinitrochlorobenzene (DNCB)h Sensitization The remaining 10 dogs were sensitized with DNCB by skin applica- tion of the chemical by the method of Catalona.19 A modification was made using DNCB in DMSO1 in lieu of DNCB in acetone for sensitization. The plantar surface of the right hind leg was clipped and cleansed with acetone. A sensitization dose of 2000 ugm DNCB in .1 ml DMSO was applied to a 3 cm2 area just above the pad. A challenge dose of 100 ugm DNCB in .1 ml of acetone was applied to a 3 cm2 area approximately 4 cm proximal to the sensitization dose. The sites were observed every other day for spontaneous flare. At 21 days postsensitization, the animals were rechallenged with 100 ugm DNCB in .1 ml acetone. The site was evaluated as previously described and biopsied at 24 hours. Histo- logical evaluation was made using the criteria previously described. The popliteal lymph nodes were surgically removed, measured, and fixed in 10% buffered formalin. The tissue sections were stained with H & E and Ziehl-Neelsen stains and evaluated for histological changes. h2-4 Dinitrochlorobenzene, K and K Laboratories, Inc., Plainview, New York. iDimethylsulfoxide, J. T. Baker Chemical Company, Phillipsburg, Net» Jersey. 22 In vivo and In vitro Evaluation of BCG Sensitized Dogs Prior to sensitization blood was drawn on all dogs for complete blood counts and for in vitro migration inhibition by tuberculin. One- tenth milliliter of mammalian tuberculinj (1:50 dilution) was injected intradermally in each flank. The first group received undiluted tuberculin which produced a nonspecific inflammatory response; there- after, 1:50 dilution of tuberculin was substituted. The areas were observed at l, 4, 24 and 48 hours and were evaluated by the criteria previously described. The injection site on the right side was biopsied at 24 hours. Tissue sections were made and evaluated microscopically by the criteria previously described. Twenty—one days postsensitiza- tion, blood was drawn on all dogs for in vitro evaluation of migration inhibition with tuberculin. Blood was also drawn on the second group for a complete blood count. The same tuberculin testing procedure with biopsy was repeated on all dogs at this time using 1:50 dilution of mammalian tuberculin. The second group (10 dogs) were also given intra- dermal injections of .1 m1 of the PPD of ML bovisk (3.21 mg protein per ml) and M. aviuml (1.42 mg protein per ml) and the reactions were evaluated and biopsied at 24 hours as previously described. jMammalian Tuberculin, United States Department of Agriculture, Lansing, Michigan. kPPD-9 -.M. bovis Strain ANS, U.S.D.A., A.P.H.I.S. - U.S.D.L., Ames, Iowa. 1PPD-10 -.M. avium Strain D Ames, Iowa. 4, U.S.D.A., A.P.H.I.S. - U.S.D.L., 23 In vitro Test of Migration Inhibition of Dog Leukocytes with Tuberculin by the Radial Migration Method Nine milliliters of blood were collected from the jugular vein into a syringe containing 1 m1 of 0.1 M citrated saline. The blood was centrifuged in a capped tube for 20 minutes at 860 x g. The plasma was removed with a pipette and discarded. The buffy coat was drawn by capillary action into micro blood tubes.m The tubes were sealed with warm, sterile paraffin. The filled micro blood tubes were placed in a test tube and centrifuged for 15 minutes at 700 x g. After centrifuga- tion, the micro blood tubes were scored with a diamond pen at or slightly below the RBC-WBC interface and broken carefully. The leuko- cytes were aspirated into a syringe with a 25-gauge needle containing 0.3 ml of culture medium.n The cells were mixed gently with medium and the cell concentration adjusted using a hemacytometer to 107 leukocytes per ml. Four separate drops of a cell suspension were placed in each plastic tissue culture dish0 and allowed to attach for 5 minutes. The spots of cells were flushed gently with Hank's balanced P salt solution to remove any red blood cells and nonadherent leuko- cytes. Two milliliters of culture medium was added to the petri dishes. mMicro blood collecting tubes, Scientific Products, Evanston, Illinois. nMedium 199, Microbiological Associates, Inc., Bethesda, Maryland. 1% L-Glutamine Solution, Grand Island Biological, Grand Island, New York; 1% Amino Acid Solution, Grand Island Biological, Grand Island, New York; 1% Vitamin Mix, Grand Island Biological, Grand Island, New York; Sodium Bicarbonate to buffer to 7.0, Microbiological Associates, Bethesda, Maryland. 0Tissue Culture Dish, Falcon Plastics, Los Angeles, California. PHanks' Balanced Salt Solution, Grand Island Biological, Grand Island, New York. 24 The medium for the control cultures contained no antigen. The medium for test cultures contained 1 test antigen. In the direct test, the diameter of each spot was measured prior to incubation and after 24 hours of incubation at 37 C. The spots were measured with an eyepiece micrometer in a light microscope at 100x magnification. The following formulas were used to calculate migration inhibition: spot diameter postincubation - spot diameter preincubation = units of migration total units of migration of 4 spots in antigen medium total units of migration of 4 spots in control medium X 100 = Z migration 100 - % migration = migration inhibition In the indirect migration inhibition test, supernate fluids from direct test plates after 24-hour incubation were added to spots of guinea pig peritoneal exudate cells collected as described. Peritoneal cells were collected by injecting 3 ml of sterile mineral oil intraperitoneally into normal nonsensitized guinea pigs. Three days after mineral oil stimulation 50 cc of Hank's buffered salt solution was injected intra- peritoneally into live, nonanesthetized animals. The abdomen was massaged and as much fluid as possible aspirated with a syringe. The peritoneal fluid was centrifuged in a conical tube at 700 x g for 10 minutes. The cell pellet was resuspended and rinsed 3 times with BSS to remove the remaining mineral oil. The rinsed cells were mixed with medium and the concentration adjusted to 107 cells per ml. 25 The spots were measured, the cultures incubated for 24 hours, and the spot remeasured. Percent migration inhibition is calculated as previously described. In addition to mammalian tuberculin, PPD of.M. bovis, PPD of M. tuberculosis,q Band 24r and Brucella antigensS were also used as the antigens in some of the migration inhibition methods. qPPD, Parke, Davis & Company, Detroit, Michigan. rBand 24 - purified tuberculo-protein, Dr. Virginia Mallmann, Department of Microbiology and Public Health, Michigan State University, EastLansing, Michigan. SBrucellergen, Merck Sharp and Dohme, West Point, Pennsylvania. RESULTS Intradermal Testing The results of the gross and microscopic evaluation of the intra- dermal injection sites with various ubiquitous antigens are listed (Table 1). Most of the antigens produced an immediate type sensitivity response characterized by a polymorphonuclear infiltrate. Streptokinase- Streptodornase 50 u produced a mononuclear reaction in 10 dogs (50%), but only 4 dogs (20% had gross reactions of the delayed type. Dermato— phyton 0 1:10 produced a mononuclear response in 4 dogs (20%), but a. grossly detectable delayed type reaction in only 1 of the dogs. BCG Sensitization The subcutaneous injection site of 5 mg BCG in 0.5 ml of incomplete Freund's adjuvant was swollen in all dogs by 10 days postinjection and had ulcers of varying size 3 weeks to a month postinjection. Dog 12 had a relatively mild response to infection, and dogs 5 and 14 had severe responses. The right popliteal nodes of all BCG sensitized dogs were grossly enlarged. Microscopic examination of the right popliteal nodes of dogs 1 through 10 demonstrated the presence of acid-fast organisms in the 5 BCG sensitized dogs (Table 2). No lymphadenopathy or acid-fast organisms were found in the lymph nodes of DNCB sensi- tized dogs. 26 27 H HHH H I H AmHavHH HH AmHavHH AmaavHH .eumaoumHm I mudo: wq I I mudo: «N I I mudo: c I I Hdo: H medmm «mealdu dwm Aw won I I-Ir-iI—II-l I NI-lI-lr-l I HH >H H HH I I HHH HmsavHH AmHmvHHH .eumaoumHm I I H I I I I H I I mudo: as I I H I I I I H I I mudo: «N I I I I I I I H I I mudo: s I I H I H I I N H I udo: H memmm «onawm «quwm .m won H I HHH HH AmHHv>H .eumaoumHm I I mudo: wq I I mudo: «N I I mudo: q I I Hdo: H memmm .MHmamw .quvMIdNIwom I H MNNND’ l (VI-{HHH II II (fir—4H HH >H H AmeanH HHH H AmHHV>H .eumaoumHm I H muse; we H H m.Hdo: «N N I deo: q I I udo: H comp: vwxfia .mHmB .quvm 4H won H AmHmV>H I H H «wcvwvrI>» v-I I waoIcInI I OJOJFIFi H I N H H udodHHa OOHAH OHNH OOHuH OHHH OOHHH OHHH OHuH OOHnH d om Hmdm¢ Hodm< 0:0HHH o:oHHH o anon 0 shoe mHHHcoz m.HHHGGE QmIMm mdmeua< AGOHumNHuHmamm com no muzn ou HOHHQ meow mma wdHummu Hmaumvmuuch meowHudm meHum> wo GOHuomndH HmauwwmuudH ou dOHuommu m:u mo dOHudem>m oHdoumouofia cam mmouo .H oHan 28 HHH HH HHH HH HH H HHH HHH HH HH .:ummoumHm I H N I H I I H I I mudo: w« I H N I N I I H I I mudo: «N I H N I H I I H I I mufio: q I H m H N I I m H I Hdo: H comp: vmxHaxdewa m aquvm .w woo HH HH HHH I HH H H H HH H .:ummoumHm I H N I H I I I I I mHDOS ww I H N I I I I I I I Mw“do: «N I H H I H I I H I I mudo: « I H N I H I I H I I Hdo: H meoom .mHmamm .qucm «N mum HHH HHH >H HH HH HH I HHH HH AmanHH .:umaoumHm I H N I N I I H I H m.Hdo: m« I H N I N I I H I H undo: «N I H m H m I I N H I m.Hdo: « I H m I m H I N H I Hdo: H meoom .mHmamH .uHscm .eIwmm H AmaanHH HH H HHH HH HmHovHH HH AmHavHH HmHavHH .eumaoumHm I H N I m I I I I I mudo: w« I H m I m I I H H I MW.Hdo: «N I H m I m I I H H I mudo: « I H m H m I I N H I Hdo: H wwamm amHma .quwm .m wdn uddeHQ OOHHH OHHH COHHH OHuH OOHuH OHHH OHuH OOHHH d om Hmmm< Hmdm< o:oHHH 0:0HHH o anon o sham «HHHdoz «HHHdoz QmIMm mcuwHud< AvmddHudoov H MHAMH 29 r—INr-IN f-II-II-II-II-I NNNN MNNN> HHH NNN HHH HmnHvHH N N N m I I NNNFI> NNI-II-I> l I I NMNNI—I I NNI-II-II-I H I N I m H I .:nmdonuHm I undo: w« I undo: «N I undo: « I ndo: H vuun: vuxHa .uHuaum «u:udoa 0 .NH woo HH >H AmHmvHH H Nr-II-IN .:numonuHm I undo: w« I undo: «N I undo: « I ndo: H wuun: wuNHE .uHuaIWu:ndoa o .HHIwon HHH HH AmHaV>H H H H H NNNI-I .:nudonuHm I undo: m« I undo: «N I undo: « I ndo: H deuuBHum .uHuE .anvu .OH won HH HH AmnanHH H H NNNN .:uumonuHm I undo: w« H muse: «N I undo: « I ndo: H unuum .uHua .quwu «m won ucmaHHa OQHHH nuau< OHHH numu¢ OOHHH o:oHnH OHHH o:oHnH OOHHH o Enun OHHH o Enwm OHnH «HHHaoz OOHHH mHHHaoz d on nmIMm uduann< Avmdcfinaoov H mHan 30 I HHH I r-‘II-Ir-l NNNN I NI—INI-I'I> I AmnavHHH H H Mmmm> MNMN> I >H HmnHVHH HHH Nr-II-Ir-I l I HHH AmnmvHHH H H OIOIOIFI>’ AmnmvHHH HHH OIoIoInI>- OIOIOIOID' I I I-Ir-II-I N .:numonuHm undo: m« undo: «N undo: « ndo: H HH H «mun: vmxHa «uHmaumIdu:ndoa wInoH won HHH Nr-II-II—I HHH MNNI—I HHH NMNI—I .:numouuHm undo: m« undo: «N undo: « H I use: H memmmeuHma .anum ImH won HHH HHH .:uumonuHm undo: m« undo: «N undo: « H “so; H HH HHH H H II-II-I wuun: vmxHaxdemaIaquuujduH won .:nudonuHm undo: w« undo: «N H HH I H I I undo: « H I use: H «Human .mHmawIHHsem .mH woo ududHHQ OOHHH nuaud OHHH nudu< OOHHH o:oHnH OHuH o:UHnH OOHHH o snug OHuH o anon OHuH «HHHaoz OOHuH «HHHaoz d on QmIMm udmend< AwuddHudoov H uH:uH 31 I HH > HH >H I AmHovH >H HHHAmHQvHHH .:u~@0nuHm I I m I H I I N I I undo: w« I I m I H I I N I I undo: «N I I m H H I I N H I undo: « I I m H H I I N H I ndo: H wuun: wuxHS .mHmEuH .u:udoa OH .mHIwmn HH AmHQvHH > HH HH HH H >H HH HH .:numouuHm I I N I H I I I I I undo: m« I I N I N I I H I I undo: «N I I N I H I I H I I undo: « I I N H N I I N I I ndo: H uuun: vwaaIdema .u:ndoa w .wHdeQ H HHH > HH > >H HHH >H HHH HH .:nudonuHm I I N I I I I H I I undo: w« I H N I H I I H I I undo: «N I I N I I I I H I I undo: « I I H I H I I N I I ndo: H wumn: vuxHa .uHmsumudu:udoE w .NH won unudHHn OOHHH OHuH ooHuH OHuH OOHHH OHHH OHHH OOHHH d on nudu< nudu< o:oHnH o:oHnH o Enmn o Bnmn uHHHsoz mHHHdoz mmIMm udmend< AwuddHHGOUV H uH:uH 32 hanu>uu wdHuuunonH Ho nuwno dH H> on H I GOHuudHu>m oHdouuonon hanu>uu wdHuuunodH Ho nuwno dH m on H I dOHnude>u uuonw .dOHnomun hnH>HnHuduunud%: udhn wuhmHuw u Ho u>Hnuuwwdu dOHnunnHHHdH HHuu nuuHoddodoa u %: vuanunounu:o onus uuwdm:o I 3H: H >H H> H > H HH >H H H .:uumouuHm I I N I N I I N I I undo: w« I I m H N I I N I I undo: «N I I m I N I I N I I undo: « I I m I m I I m I I ndo: H vumn: meHE .mHuB «w:naoa w .ON won ndudHHQ OOHuH OHHH OOHHH oHuH OOHHH OHHH OHHH OOHHH d on nmmm< nmmm< onunnn oeunnn o anon o snug mHHnaoz mHHHaoz HmInm uduand< wazaHuaouV H mHsmn 33 Table 2. Size of popliteal nodes of dogs 3 weeks postinjection of BCG or DNCB in the right hind foot and presence of acid-fast organisms in right popliteal node Acid-fast Dog No. - Left popli- Right popli- organisms observed Sensitizing teal node teal node in right popliteal antigen (cm x cm x cm) (cm x cm x cm) lymph node 1 - DNCB 3.5 x .5 x .3 3.5 x 1.0 x .3 Negative 2 - BCG .5 x .5 x .5 4 x 2 x 1 Positive 3 - BCG 1.3 x .6 x .6 4.8 x 2.6 x 1.3 Positive 4 - DNCB 1 x .5 x .2 1.8 x .5 x .3 Negative 5 - BCG 1.3 x .5 x .5 3.8 x 2.0 x 1.5 Positive 6 - DNCB .75 x .5 x .38 .75 x .5 x .5 Negative 7 - BCG l x .3 x .3 1.4 x l x 1.3 Positive 8 - DNCB 1.4 x 1.3 x .3 1.8 x 1.0 x .4 Negative 9 - BCG 2 x .5 x .l 5 x 3 x 2 Positive 10 - DNCB 2 x 1 x .5 2.5 x .5 x .1 Negative 34 Peripheral Blood Values Pre— and Postsensitization The total leukocyte, lymphocyte and monocyte counts of peripheral venous blood presensitization and 3 weeks postsensitization are reported (Table 3). No significant differences were noted pre- and postsensitization. DNCB Sensitization A mild scurf of the sensitization site was found 1 to 2 days after application of DNCB-DMSO. No significant increase in size of the right popliteal nodes was noted in DNCB sensitized dogs (Table 2). Challenge of Sensitized Dogs with DNCB No gross reaction was detected at any of the sites 24 hours after challenge. Microscopically, the sites had a slight response in all 10 of the DNCB sensitized dogs (100%) and 3 of the BCG sensitized dogs (30%) (Table 4). Tuberculin Testing Pre- and Postsensitization Recording of gross and microscopic reactions to undiluted tuberculin is given in Table 4 and a compilation given in Table 5. Undiluted tuberculin caused severe microscopic inflammatory reactions characterized by edema, necrosis and polymorphonuclear leukocyte infiltration in sensitized and nonsensitized dogs. Two dogs (20%) presensitization, 3 dogs (60%) postsensitization with DNCB, and all BCG sensitized dogs had gross reactions to undiluted tuberculin. 35 Table 3. Leukocyte, lymphocyte and monocyte counts per cubic millimeter of peripheral blood of dogs prior to and after BCG or DNCB sensitization 3 3 3 Dog No. Leukocytes/mm Lymphocytes/mm Monocytes/mm 11 - Pre 10,000 2,300 400 - Post 14,700 3,675 441 12* - Pre 18,800 3,000 2,444 - Post 14,900 4,172 745 13 - Pre 6,900 1,794 414 - Post 7,700 2,464 77 14* - Pre 9,800 1,666 588 - Post 9,800 1,568 392 15* - Pre 9,460 2,914 376 - Post 10,700 3,317 107 16* - Pre 10,300 2,578 824 - Post 12,600 2,520 630 17* - Pre 9,100 3,003 728 - Post 9,800 3,332 1,274 18 Pre 15,600 3,588 312 Post 10,800 2,160 1,080 19 Pre 10,000 2,800 1,000 Post 9,200 3,404 920 20 Pre 12,700 3,048 635 Post 9,600 2,784 288 * BCG sensitized dogs. 36 Table 4. Gross and microscopic evaluation of sites of intradermal injection of undiluted tuberculin and DNCB challenge in dogs prior to and 3 weeks after BCG or DNCB sensitization Dog No. Tuberculin Sensi- Gross Microscopic DNCB 100 ugm tizing Pre- Post- Pre- Post- Postsens. Postsens. Antigen sens. sens. sens. sens. Gross Micros. l- 1* l V* V 0 I DNCB 2— 0 3 V VI 0 0 BCG 3- 0 3 V VI 0 0 BCG 4- 0 2 V IV 0 I DNCB 5- l 3 IV VI 0 I BCG 6- O 1 II II 0 I DNCB 7- 0 1 IV V O 0 BCG 8- 0 2 III III 0 I DNCB 9- 0 1 V VI 0 I BCG 10- 0 3 IV V 0 II DNCB Gross Microscopic Postsensitization Postsensitization ** 11- 0 (1 mm) I DNCB 12- 0 (0 mm) 0 BCG 13- 0 (0 mm) I DNCB 14- o (.5 mm) 0 BCG 37 Table 4 (continued) Dog No. Sensi- tizing Gross Microscopic Antigen Postsensitization Postsensitization 15— 0 (0 mm) 0 BCG 16- 0 (0 mm) I BCG 17- 0 (0 mm) 0 BCG 18- 0 (0 mm) I DNCB 19- 0 (0 mm) III DNCB 20- 0 (0 m) I DNCB * Gross evaluations are graded l to 3 and microscopic evaluation I to VI in increasing degree of inflammatory reaction. ** Increase in skin fold thickness 24 hours after DNCB challenge. 38 Table 5. Compilation of gross and microscopic reactions of dogs 1-10 to undiluted tuberculin pre- and postsensitization Presensitization Postsensitization Positive Negative Positive Negative Gross Reaction BCG Sens. 1 4 5 0 DNCB Sens. 1 4 5 0 Grade >III III or less >III III or less Microscopic Reaction BCG Sens. 5 0 5 0 DNCB Sens. 3 2 3 2 39 Intradermal Testingpwith Tuberculin Diluted 1:50 Pre- and Postsensitization Recording of gross and microscopic reactions to 1:50 tuberculin injected intradermally pre- and postsensitization is reported in Table 6 and a compilation given in Table 7. Gross reactions were 90% accurate in differentiating between BCG and DNCB sensitized dogs. Microscopically, 1:50 tuberculin produced less severe responses in unsensitized dogs and differentiation of BCG from DNCB sensitized dogs was 70% accurate. The tuberculin reaction in BCG sensitized dogs was interpreted as a delayed type hypersensitivity reaction in only half the dogs tested. These dogs had clear-cut mononuclear cell infiltration though the pre- dominant cell was the polymorphonuclear leukocyte. The other BCG sensitized dogs had minimal mononuclear cell infiltration. Direct Migration-Inhibition Pre- and Postsensitization The results of direct inhibition test of the radial migration of peripheral leukocytes by 1:100 tuberculin are reported in Tables 8 and 9 and Figures 1 through 8. After preliminary examination of the results, 35% or greater inhibition was considered positive. Only cells from 1 dog (dog 3) had migration inhibition prior to sensitization with BCG or DNCB. Compilation of migration inhibition in BCG and DNCB sensitized dogs was made in Table 10. Migration Inhibition in Sensitized Dogs with Various Antigens The results of direct inhibition test of radial migration of peripheral leukocytes by tuberculin 1:100, PPD, Band 24 and Brucellergen 6 weeks postsensitization are given in Table 9 and Figures 5 through 8. 40 M: m Anne HHH HHH I Has 8 o o HoanHH MH 0 >H I H I muanOH ow 0H HH I H I oomIm o oH H I o I moanm «m NN HHH I o I uumlm on c HH I o I muzalo o 3 is. fine HH I N I uumIm OH O >H I o I moan« w« mm >H I H I womIm «m o > I N I cumIN me 0 «*HH I «so I muzaIH N N .uduu .udmu .udmu .udmu cuwHud¢ .uduu .uduu Inuom Iunm Inuom Iunm wdHNHn Inuom Iunm onoouonon uuono IHudum 83325 .02 won GOHuuanz nounHQ dHHdonu:dn OOHuH a: uuuhu onde Hunu:dHnud Ho doHnH:H:dH dOHuuana nounHv wdu doHnuNHnHuduu mozn no mom nuuwu uxuuB m van on nOHnd umuHu dOHnoundH Homqu dHHdonm:dn HuanuwunndH Ho doHnudHu>u oHdoouonoHa can uuonw .o uH:uH 41 .hnH>HnHudmunud%: umhn wummHuw Ho u>Huuuwwdu GOHnmnnHHHdH Hku nuuHodd0doa u %: wuNHnunumnm:o «no? uuwdu:o I men «s« .dOHnouun anoumaEuHHdH Ho munwuw wdHumunodH dH H> on H dOHndeu>m 0HdoouonoHB tau m on H wmwunw mnm udOHuMdHu>u uuonu 05* .dOHnomndH dHHdonm:dn HuanuwunndH nunmm undo: «N van on nOHnd uumdx0H:n :How deu dH uuuunodH um om wH HH > Has ovo o mozanN OH Hm HH HHH Has eye 0 muanmH o o HHH HH Has ovo o muzaImH me aH Hmnav> HHH Has m.an o womInH H« mm AmnanHH H Has HvH o oumIoH me mm Hmnnv> HmnmvHHH Has m.HVm H uumImH mm HH Amnnv> HHH Has m.NVm H oomIqH o 0 HH HH Asa ovo o muzoImH mH ma > HHH Has Nvm o oomINH N N .uduu .uduu .uduu .uduu duand< .uduu .ucuu Inuom Iunm quom lunm wdHNHn Inuom Iunm unmoouonon uuonw IHudum :oHanneaH I .02 won GOHnmanz nounHQ mesaHuaoov o mHnmn 42 Table 7. Compilation of gross and microscopic evaluation of intra- dermal injection sites of 1:50 tuberculin pre- and postsensitization Presensitization* Postsensitization** Positive Negative Positive Negative Gross Reaction BCG Sens. 2 8 9 1 DNCB Sens. 0 10 1 9 Grade >III III or less >III III or less Microscopic Reaction BCG Sens. 0 5 6 4 DNCB Sens. 1 4 2 8 * Ten dogs. ** Twenty dogs. 43 Table 8. Direct and indirect inhibition of leukocyte migration by 1:100 tuberculin prior to and after sensitization with BCG and DNCB Direct Indirect Dog No. — Pre- 3 wks 8 wks 11 wks 12 wks 12 wks Sensitizing sens. Post. Post. Post. Post. Post. Antigen % % % % % % l-DNCB 0 63 42 31 0 9 2-BCG 0 34 45 34 25 69 3-BCG 59 48 40 44 12 58 4-DNCB 0 10 15 40 0 40 S—BCG 19 0 34 34 27 53 6-DNCB 0 50 50 25 0 38 7-BCG 27 34 38 80 53 48 8—DNCB 10 0 0 0 0 18 9-BCG 16 80 60 37 32 34 10-DNCB 0 13 19 0 23 24 44 Table 9. Direct inhibition of leukocyte migration by 1:100 tuberculin, PPD, Band 24 and Brucellergen prior to and after sensitiza- tion with BCG and DNCB Dog No. Sensi- Tuberculin PPD Band 24 Brucellergen tizing Pre- Pre- 3 wks 6 wks 6 wks 6 wks 6 wks Antigen sens. sens. Post. Post. Post. Post. Post. 11- 15 8 18 23 34 34 45 DNCB 12- O 23 15 0 43 29 29 BCG 13- 10 0 0 30 30 20 20 DNCB 14— 0 17 53 60 40 60 20 BCG 15- 23 28 45 58 - 43 29 BCG 16- 20 23 41 58 58 0 58 BCG 17— 0 19 45 50 38 50 50 BCG 18- 12 0 0 25 25 38 25 DNCB 19- 0 27 10 13 50 38 50 DNCB 20— 13 18 50 63 50 0 50 DNCB _Migration Inhibition % 45 100 — Direct method 90 ====Indirect method 80 70 60 50 40 30 20 10 0 u u u to In In .x ggass ggééfi 33:3. a.rI¢>FIHIFI n.6IaIFIHIFI QIFIGJFIF4F4 Dog #1 Dog #2 Dog #3 DNCB BCG BCG Figure 1. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte Cultures with l: 100 mammalian tuberculin. Wks - weeks; PRE - presensitization. Dogs 1 2 and 3 Inhibition> - 35% chosen empirically as positive test. ”Migration Inhibition Z 46 100 ~ — Direct method 90 . =Indirect method 80 70 6O 50- «o l 10 I 0 m ggiéé ggiié ggiéi n‘flidlrifiri nIvI¢>FIFIFI QIFIGJFIFIFI Dog #4 Dog #5 Dog #6 DNCB BCG DNCB Figure 2. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks . weeks; PRE - presensitization. Dogs 4, 5 and 6. Inhibition Z 35% chosen empirically as positive test. Migration Inhibition Z 47 100 —Direct method 90 ‘ =Indirect method 80 '70 60 50 40 30 20 10 ' , m m M ggéii 33:3: ggaéi Dog #7 Dog.#8 Dog #9 BCG DNCB BCG Figure 3. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra— tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte cultures with 1:100 mammalian tuberculin. Wks - weeks; PRE - presensitization. Dogs 7, 8 and 9. Inhibition Z 35% chosen empirically as positive test. Migration Inhibition Z 48 100 - — Direct method 90 I ====Indirect method 80 7O 60 50 40 30 20 10 3 wks 8 wks 11 wks 12 wks 12 wks E Dog #10 DNCB Figure 4. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin in culture media. Migra- tion inhibition before sensitization (PRE) with BCG (Bacillus Calmette Guerin) or DNCB (Dinitrochlorobenzene) and at intervals postsensitization. Indirect migration inhibition using normal guinea pig peritoneal cells cultured with cell free supernate of the dog leukocyte Cultures with 1:100 mammalian tuberculin. Wks - weeks; PRE - presensitization. Dog 10. Inhibition 2 35% chosen empirically~as~positive test. Migration. Inhibition % 49 100 —Tuberculin 1:100 90 I --PPD of M. tub. SO ugm/ml 80 “Band 24 50 Hen/ml ”Brucellergen 1:100 70 60 50 4O ' I . ----—--——' ————————— -—-———————- 30 ' I : .I . I I -f I . l 20 I j ' . I | ; ' .' ' ‘ 10 I : I 3 I I I f I : I : 0 I t I 3 l , m m (D m 0 u (D m 0 u .Q D Q aaéuuuu ggfiéfiéfi ggééaré nan-Imogene nun-ImIomxoo mmmoooo Dog #11 Dog #12 Dog #13 DNCB BCG DNCB Figure 5. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of M. tuberculosis (50 ugm/ml), Band 24 (50 ugm/ml) and Brucellergen 1:100. PRE - presensitization; wks - weeks. Dogs 11, 12 and 13. Wition 2. 35% chosen empirically as; positive test. Migration Inhibition Z 100 - 90 80 70 60 50 40 30 20 10 50 ____ " _______ __ I I I I. I I I I mm mm mm ausuéus gaugé mgmoooo mmmoo Dog #14 Dog #15 sec BCG Figure 6. 6 wks 6 wks -Tuberculin 1: 10 I-PPD of M. tub. 50 ugm/ml “Band 24 50 ugm/ml. ”Brucellergen 1: 100 -----r--- .3 3 6 wks 6 wks 6 wks 6 wks EE Dog #16 BCG Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of ML tuberculosis (50 ugm/ml), Band 24 (50 ugm/ml) and Brucellergen 1:100. PRE - presensitization; wks - weeks. Inhibition Dogs 14, 15 and 16. 2.35% chosen empirically as positive test. Migration Inhibition 2 51 100 —Tuberculin 1:10 9° --PPD of u. tub. so Ham/ml 80 - Ito-Band 24 50 ugn/ml, mBrucellergen l: 100 70 6.0 so . . I 40 f I ___ I5 . _________ . ----_-_--} _-___. | i - f .I ' 20 I : I : I I g I l ' I : I ; I 10 I : I : I , I 3 I § I o I ; I _ I n m ' on mm m a§§%%%: gggégsé aaééééé mmmoofioo 94 ”@900 294030000 Dog #17 Dog #18 Dog #19 BCG 'DNCB DNCB Figure 7. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of M. tuberculosis (50 ugm/ml), Band 24 (50 ugm/ml) and Brucellergen 1:100. PRE - presensitization; wks - weeks. Dogs 17, 18 and 19. Inhibition Z 352 chosen anpirically as positive test. Migration Inhibition Z 52 100 — Tuberculin 1:10 9° '-'PPD of M. tub. so ugm/ml 80 . Blue: Band 24 50 ugm/ml >001 Brucellergen ‘ 1:100 70 60 50 I I 40 I ---- I - -- T 30 ' I 20 I I I 10 I I o I Q m U aaggfiéé Dog #20 DNCB Figure 8. Migration inhibition of dog leukocytes (direct method) by 1:100 mammalian tuberculin, PPD of M. tuberculosis (50 ugm/ml), Band 24 .(SO pgm/ml) and Brucellergen 1:100. PRE - presensitization' wks - weeks. Dog 20. Inhibition Z 352 chosen empirically as positive test. 53 Table 10. Compilation of migration inhibition by direct method with 1:100 tuberculin in dogs sensitized with BCG and DNCB 3, 6 and 8 weeks postsensitization Dog No. Weeks Postsens. Positive* Negative 1-10 BCG (5)** 3 2 3 DNCB (5) 3 3 2 11-20 BCG (5) 3 4 l DNCB (5) 3 1 4 1-10 BCG (5) 8 4 l DNCB (5) 8 3 2 11-20 BCG (5) 6 4 1 DNCB (5) 6 l 4 1-10 BCG (5) ll 3 2 DNCB (5) ll 0 5 * Inhibition greater than 35% inhibition. ** Number in parentheses indicates number of dogs studied. 54 Compilation of their effectiveness in differentiating between BCG and DNCB sensitized dogs is given in Table 11. Tuberculin was most effective in differentiating between BCG and DNCB sensitized dogs. There was no correlation between tuberculin reactions and Brucellergen. Direct and Indirect Migration Inhibition The results of tests to compare ability of the direct and indirect migration to differentiate between BCG and DNCB sensitized dogs (dogs 11-20) are given in Table 12 and Figures 9 and 10, and a compilation is given in Table 13. The direct method was slightly better in dif— ferentiating between BCG and DNCB sensitized dogs. Fifty percent of the dogs were positive presensitization by the indirect test, while only 5% of the dogs were positive by the direct test. Averaggpof A11 Pre- and Postsensitization Direct Test Migration Inhibition The average of all pre- and postsensitization direct migration inhibition tests by 1:100 tuberculin are given in Table 14 and Figures 11 and 12, and a compilation is given in Table 15. This method was 80% accurate in differentiating BCG and DNCB sensitized dogs. 55 Table 11. Comparison of the migration inhibition effects of mammalian tuberculin, PPD, Band 24 and Brucellergen 6 weeks post- sensitization with BCG and DNCB Tub. PPD Band 24 Brucellergen Pos.* Neg. Pos. Neg. Pos. Neg. Pos. Neg. BCG Sensitized 4 l 3 l** 3 2 2 3 dogs DNCB Sensitized 1 4 2 3 2 3 3 2 dogs * Inhibition Z 35% chosen empirically as positive test. ** One dog's test contaminated. 56 Table 12. Direct and indirect migration inhibition pre— and post— sensitization with BCG and DNCB Dog No. - Presensitization Postsensitization Sensitizing Direct Indirect Direct Indirect Antigen % Z Z % ll—DNCB 8 21 18 21 12—BCG 23 37 15 24 l3-DNCB O 10 O 9 l4-BCG 17 50 53 48 lS—BCG 28 59 45 42 l6-BCG 23 47 41 35 l7—BCG 19 44 45 38 18-DNCB O 33 O 12 19-DNCB 27 17 10 0 20-DNCB 18 18 50 37 Inhibition 3 35% chosen empirically as positive testo Migration Inhibition Z 57 100 III Direct method 90 ' -= Indirect method 80 7O 60 50 40 30 10 ! + htWM—Qrfi— . _m Q Q 01’ m m 33 33 33 3 fix E35333 23:33? 2:333 aESg §§33 9.9.01a1 . 6.9.«wvw o.n.v»«1 n.o.«)«3 n.n.«wvw Dog #11 Dog #12 Dog #13 Dog #14 Dog #15 DNCB BCG DNCB BCG BCG Figure 9. Comparison of the migration inhibition effect of 1:100 tuberculin by the direct and indirect method in dogs sensitized with BCG (Bacillus Calmette Guerin) and DNCB (Dini- trochlorobenzene) presensitization and 3 weeks postsensitiza- tion. The direCt method measures the inhibition of migration of sensitized dog leukocytes incubated with 1:100 mammalian tubercu- lin. The indirect method measures the inhibition of migration .of normal guinea pig peritoneal exudate cells incubated with the cell free supernate from the dog leukocyte cultures. Wks - weeks; PRE = presensitization. Dogs 11, 12, 13, 14 and 15. Inhibition 2 352 chosen empirically as positive test. Migration Inhibition Z 58 100 — Direct method 90 == Indirect method 80 70 60 50 40 g 30 20 i ! a Q U) 0 m x 3 33 33 33 gags Easg agks 5:33 5:33 9.9.61w1 n.«w«v 9.6.616) 9.9.«16» n.n.a1aw Dog #16 Dog #17 Dog #18 Dog #19 Dog #20 BCG - BCG DNCB DNCB DNCB Figure 10. Comparison of the migration inhibition effect of 1:100 tuberculin by the direct and indirect method in dogs sensitized with BCG (Bacillus Calmette Guerin) and DNCB (Dini— trochlorobenzene) presensitization and 3 weeks postsensitization. The direct method measures the inhibition of migration of sensi- tized dog leukocytes incubated with 1:100 mammalian tuberculin. The indirect method measures the inhibition of migration of normal guinea pig peritoneal exudate cells incubated with the cell free supernate from the dog leukocyte cultures. Wks - weeks; PRE - presensitization. Dogs 16, 17, 18, 19 and 20. Inhibition Z 35% chosen empirically as positive test. ' 59 Table 13. Compilation of direct and indirect migration inhibition pre- sensitization and 3 weeks postsensitization in dogs sensi- tized with BCG or DNCB Postsensitization BCG DNCB Presensitization Posi— Nega- Posi- Nega- Positive Negative tive tive tive tive Direct (10 dogs) I 9 4 1 l 4 Indirect (10 dogs) 5 5 3 2 l 4 * Three weeks postsensitization. 60 Table 14. Average of all tests on each dog of direct migration inhi- bition pre- and postsensitization with BCG or DNCB Presensitization Postsensitization Average of Average of Direct MI Direct MI Dog No. Z Z l—DNCB 0 34 2—BCG 0 34.5 3-BCG 59 36 4-DNCB 0 16.25 5—BCG 19 23.75 6-DNCB - 31.25 7-BCG 27 51.25 8-DNCB 10 0 9-BCG 16 52.25 10-DNCB O 13.75 ll-DNCB 11.5 20.5 12-BCG 11.5 7.5 13-DNCB 5 15 14-BCG 8.5 56.5 lS-BCG 25.5 51.5 16—BCG 21.5 49.5 l7-BCG 9.5 47.5 18-DNCB 6 12.5 19-DNCB 13.5 11.5 20-DNCB 15.5 56.5 Inhibition Z 35% chosen empirically as positive test. Migration Inhibition Z 61 100 90 80 70 60 50 40 30 20 10 O 94 Pa 54 94 U: 0: VJ U) 22 E2 E2 2 Dog Dog Dog Dog #1 #2 #3 #4 DNCB BCG BCG DNCB Figure 11. 25 2 25 2‘ 2 2 .2 E2 .2 .2 .2 '° Dog Dog Dog Dog Dog Dog #5 #6 #7 #8 #9 #10 BCG DNCB BCG DNCB BCG DNCB Averages of all direct migration inhibition studies using 1:100 mammalian tuberculin reported in Figures 1 through 8. PRE indicates an average of all migration inhibition studies pre-BCG (Bacillus Calmette Guerin) or pre-DNCB (Dinitro- chlorobenzene) sensitization. studies postsensitization. POST indicates an average of all D088 1’ 2, 3, 4’. 5., 6, 7, 8, 9 and 100 Inhibition.? 352 chosen empirically as positive test. Migration Inhibition Z 62 100 90 ’ so '70 60 so 40 30 ' 20 10 ea ea an 94 a. e. 5‘ ea ea 54 U) (I) In (D a: U) U) U) l-‘I’JUI m 2 E2 .2 E2 E2 E2 E2 .2 2 22 Dog DOg' Dog Dog Dog Dog, Dog Dog Dog Dog #11 #12 #13 #14 #15, #16 #17 #18 #19 #20 DNCB BCG DNCB BCG BCG BCG BCG DNCB DNCB DNCB Figure 12. Averages of all direct migration inhibition studies using 1:100 mammalian tuberculin reported in Figures 1 through 8. PRE indicates an average of all migration inhibition studies pre—BCG (Bacillus Calmette Guerin) or pre-DNCB (Dinitro- chlorobenzene) sensitization. POST indicates an average of all studies.postsensitization. Dogs 11, 12, 13,-l4, 15, l6, 17, 18, 19 and 20. Inhibition 2 35% chosen empirically as positive test. 63 Table 15. Compilation of the average of all migration inhibition results by direct methods in dogs pre- and postsensi- tization with BCG and DNCB Presensitization Postsensitization Positive* Negative Positive Negative BCG 1 9 7 3 DNCB 0 10 l 9 * Inhibition Z 35% chosen empirically as positive test. DISCUSSION AND CONCLUSIONS This study was performed to evaluate the applicability of some of the current methods to the study of cell mediated immunity in the dog. The methods evaluated are currently employed in the clinical evaluation of the immune system in man. Ubiquitous antigens, which regularly produce delayed type sensi- tivity reactions in most individuals, are used ektensively in the evaluation of a given individual's cell-mediatbd competence. The antigens commonly used in intradermal testing in man are: Candida albicans, Trichophyton, PPD, Streptokinase-Streptodornase, and mumps.12 Five antigen preparations were selected (Monilia, Dermatophyton 0, Trichophyton, Aspergillus and Streptokinase-Streptodornase) which would be expected to produce delayed hypersensitivity reactions in the dog. Monilia, Aspergillus and Trichophyton produced immediate responses which were fully developed at 4 hours and were characterized by extensive polymorphonuclear leukocyte infiltration. These antigens were found to be unsuitable for demonstrating delayed hypersensitivity in the dog. Dermatophyton 0 (Candida albicans) 1:10 produced some mild mononuclear cell reactions of the delayed hypersensitivity type, but only 1 dog (5%) had a gross delayed hypersensitivity reaction. Streptokinase- Streptodornase 50 u produced delayed gross reactions in 4 dogs (ZOZ) and a mononuclear cell infiltrate in 10 dogs (50%). 0f the antigens 64 65 tested, only Streptokinase-Streptodornase, possibly at an increased concentration, offers a potentially useful antigen for demonstrating delayed hypersensitivity in the dog. Sensitization with DNCB is another standard method for evaluating cell mediated immune response to a new antigen. Ninety-six percent of individuals exposed experi- mentally to DNCB developed spontaneous flare reactions.14 DNCB in DMSO instead of acetone was used in the initial sensitization to achieve good penetration of the DNCB. The possibility that the anti-inflammatory effects of DMSO interfered with sensitization was considered. When challenged with a 100 ugm dose of DNCB in acetone 21 days postsensiti- zation, no gross reactions were noted, but all DNCB sensitized dogs and 3 BCG sensitized dogs (30%) had a mild microscopic response. The micro- scopic response indicates a slight sensitization, and a different method of sensitization could possibly produce gross reactions. Joseph38 reported good results by sensitizing dogs with DNCB in acetone applied near the inguinal lymph nodes. Sensitization with DNCB in DMSO is not a practical method for clinical use. Infection with BCG was accomplished by subcutaneous injection of viable BCG organisms. Lymphadenopathy and observation of acid-fast ‘r ’organisms in the right popliteal lymph node of dogs biopsied confirmed the establishment of a BCG infection. Undiluted tuberculin was unsuitable for intradermal skin testing in the dog. Undiluted tuberculin produced a strong nonspecific inflammatory reaction in unsensitized dogs. All BCG and DNCB sensi- tized dogs responded with a grossly detectable reaction. 66 Tuberculin diluted 1:50 differentiated between BCG and DNCB sensitized dogs with 90% accuracy on evaluation of gross reactions, and 70% accuracy by microscopic evaluation. Tuberculin diluted 1:50 appears to be a superior product to undiluted tuberculin for diagnostic use in the dog. The injection sites of 1:50 tuberculin were characterized by a predominantly polymorphonuclear leukocyte infiltration in BCG and DNCB sensitized dogs, but the inflammatory changes were more severe in the BCG sensitized dogs. Only half of the BCG sensitized dogs had a clear-cut mononuclear cell response, and this was overshadowed by a predominance of polymorphonuclear leukocytes. The polymorphonuclear leukocyte appears to play a greater role in the tuberculin reaction of sensitized dogs than it does in sensitized rabbits28 or guinea pigs.58 In vitro techniques have been used extensively in man in the study of cell-mediated immunity. The migration inhibition method has been shown to be a versatile method for use in a variety of immunological conditions. The direct radial migration inhibition method using 1:100 tuberculin was 80% accurate in differentiating between BCG and DNCB sensitized dogs. It offers a promising method for use in the study of cellular immunity in the dog. The direct method was superior to the indirect method of measuring migration inhibition. The indirect method had an excessive number of false positive reactions in dogs presensitization and in DNCB sensi- tized dogs. Only 1 dog (5%) had a false positive response presensi- tization by the direct method. The possibility of prior contact with 67 a Myoobacterium must be considered, but the dog was negative on intra— dermal skin testing. Mammalian tuberculin 1:100 was as effective an antigen in vitro as the more purified tuberculoproteins (PPD of M. tuberculosis or Band 24). Brucellergen was used to confirm that all reactions were not nonspecific. Brucellergen produced inhibition patterns unrelated to BCG or DNCB sensitization. Five dogs (50%) had migration inhibition to Brucellergen suggesting the possibility of previous exposure to the Brucella organism. Migration inhibition occurred in l or more tests of cells from 3 dogs of the first group (dogs 1 through 10) sensitized with DNCB. One dog in the second group (dogs 11 through 20) sensitized with DNCB had in vitro migration inhibition to 1:100 tuberculin. The possibility of infection of DNCB sensitized dogs with BCG in the first group must be considered as the dogs were exercised in the same run. The sites of inoculation ulcerated and possible transmission cannot be excluded. The second group of dogs were carefully segregated. Maximum in vitro response to BCG infection did not occur until 6 to 8 weeks postsensitization in some dogs, and began to diminish by 11 weeks. Two dogs (dogs 2 and 7) were negative at 3 weeks but positive at 8 weeks postsensitization with BCG. Two dogs (dogs 5 and 12) sensitized with BCG were never positive by migration inhibition test. Dog 5 had 2 migration inhibition values of 34% (35% or greater was selected as positive). It had a good gross response to intradermal test of 1:50 tuberculin, but microscopically poor cellular infiltration (grade II). In addition, dog 5 had the most 68 severe focal reaction to BCG infection of all the dogs sensitized. It is interesting to speculate that the dog was incapable of mounting a good immune cellular response to the organism and there was an additional compensatory nonspecific inflammatory response to contain the BCG infection. However, dog 12 had a mild reaction at the site of BCG infection and did not have even borderline positive migration inhibi- tion values. It did have a strong gross response with good cellular infiltrate to intradermal injection of 1:50 tuberculin. The possi- bility exists that dog 12 had a good cell mediated response, not detected in vitro, which prevented a larger reaction at the site of BCG infection. Much additional information is needed before the mechanism of normal cell mediated responses can be effectively analyzed and evaluated and deviations determined and explained. Averaging of the results of all the migration inhibition tests was most effective in differentiating BCG from DNCB sensitized dogs. This eliminated the variables seen on individual tests and demonstrated that the use of more than one test is preferable. Migration inhibition of leukocytes in the dog differs from other species in that the test measures mainly granulocyte migration. The macrophage is usually considered the cell influenced by migration inhibition factor. Migra- tion of normal dog leukocytes is considerably less than the migration of human and bovine leukocytes obtained in this laboratory. Poor migration makes interpretation of migration inhibition more difficult. The direct radial migration inhibition test appears to be a useful tool in the study of cell—mediated immunity in the dog. More work is 69 needed to compare findings obtained by migration inhibition with other in vitro methods such as lymphocyte transformation. SUMMARY Methods to evaluate naturally and artificially induced cell mediated immunity in the dog were tested. Twenty dogs of mixed age, sex and breed were injected intradermally with Monilia, Dermatophyton 0, Trichophyton, Aspergillus and Streptokinase-Streptodornase to determine if a delayed skin reaction occurred regularly to ubiquitous antigens. 0n the basis of visible reactions and histological examina- tion of biopsied injection sites, it was concluded that only Streptokinase-Streptodornase had promise. To test artificially induced cell mediated immunity, 10 of the dogs were injected subcutaneously with viable BCG in incomplete Freund's adjuvant, and 10 dogs were sensitized with DNCB in DMSO applied to the plantar surface of the foot. Three weeks or more after sensitization the following tests were made: intradermal tuberculin testing, skin challenge with DNCB and in vitro migration inhibition of blood leukocytes by tuberculin. Undiluted tuberculin was unsuitable for intradermal testing in the dog because of false positive reactions. Tuberculin diluted 1:50 was 90% accurate as an intradermal test in differentiating between BCG and DNCB sensitized dogs. Tuberculin diluted 1:50 produced a reaction characterized by a predominantly polymorphonuclear leukocyte infiltra- tion in BCG sensitized dogs. Half of the BCG sensitized dogs also had 70 71 a distinct mononuclear cell infiltration. The response of sensitized dogs to tuberculin appears to be different than in other species. All DNCB and 30% of the BCG sensitized dogs had microscopic but not gross responses to DNCB challenge. DNCB in DMSO is not a suitable agent for skin sensitization in dogs. The direct radial migration inhibition of peripheral dog leuko- cytes by 1:100 tuberculin was 80% accurate in differentiating between BCG and DNCB sensitized dogs. The indirect method, which measures the migration inhibition effect of the supernates of dog leukocyte cultures on normal guinea pig peritoneal cells, produced an excessive number of false positive reactions. 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