lHllHHlel |: 1 I §E§ [Hill THS STUDEEE$ ON THE ANTEGENKIW OF LYMF‘HOMATQUS" TESSUE FROM THE DOG The“: for fine Domes me M. 5. MECEEGAN STATE UNWERSET? James A. Osburn 1967 fl ' LIBRARY L' Michigan 3 rate A FEW 5‘ univemi ty ' 'V- ABSTRACT STUDIES ON THE ANTIGENICITY OF LYMPHOMATOUS TISSUE FROM THE DOG by James A. Osburn The object of this study was to determine if lymph node cells from dogs with malignant lymphoma had an antigen or antigens that were not present on normal lymph node cells. The results indicated a change in the anti- genic structure of canine lymphomatous lymph node cells. This change was evidenced by the absence of ”normal” antigens or by the presence of a unique antigen or antigens on the lymphomatous lymph node cells. The antigenic changes were detected using normal canine serum antiserum (rabbit origin), lymphomatous canine serum antiserum (rabbit origin), lymphomatous canine lymph node antiserum (rabbit origin) and normal canine lymph node antiserum (rabbit origin) to develop immunoprecipitates with normal canine lymph nodes and lymphomatous canine lymph nodes using immunoelectrophoretic techniques. STUDIES ON THE ANTIGENICITY OF LYMFHOMATOUS TISSUE FROM THE DOG By James A. Osburn A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1967 ACKNOWLEDGEMENTS My sincere appreciation and gratitude are expressed to my major professor, Dr. D. A. Schmidt of the Department of Pathology, for his counseling, advice, encouragement and enduring patience throughout the course of this research and manuscript preparation. The counseling and advice of Drs. S. D. Sleight and V. A. Mallmann has been greatly appreciated. To my personal friend and colleague, Mr. Robert A. Brooks, I wish to express my thanks for his help with the many technical procedures involved with this study. I wish to thank Dr. L. S. Goyings for procuring the lymph nodes from dogs with lymphoma used in this study. Also, I would like to thank Drs. E. S. Feenstra and R. G. Carlson of the Department of Pathology at the Upjohn Company, Kalamazoo, Michigan, for supplying the normal lymph nodes. I would like to express my thanks to Dr. C. C. Morrill, Chairman of the Department of Pathology, Michigan State University, for allowing me to purchase the necessary animals and equipment used in this study. ii TABLE OF CONTENTS Page INTRODUCTION ............................................ 1 LITERATURE REVIEW ....................................... 2 MATERIALS AND METHODS ................................... 7 Source of specimens .............................. 7 Preparation of anti-normal canine serum antiserum (rabbit origin) (NCSAS) ............... 7 Preparation of anti-lymphomatous canine serum - antiserum (rabbit origin) (LCSAS) ................ 5 Preparation of anti-lymphomatous lymph node antisera LLNAS and anti—normal lymph node antisera NLNAS (rabbit origin) ................ 9 Preparation of extract of lymph nodes for electrophoresis .................................. lO Immunoelectrophoresis ............................ 10 RESULTS ................................................. 1A Lymphomatous canine serum antiserum (LCSAS) ...... IA Normal canine serum antiserum (NCSAS) ............ l4 Lymphomatous lymph node antiserum (LLNAS) ........ 18 Normal lymph node antiserum (NLNAS) .............. 18 DISCUSSION .............................................. 28 SUMMARY ................................................. 33 REFERENCES CITED ........................................ 3A VITA .................................................... 37 iii Table [0 LIST OF TABLES Results of immunoelectrophoretic studies lymphomatous canine serum antiserum ............. Results of immunoelectrophoretic studies normal canine serum antiserum ................... Results of immunoelectrophoretic studies lymphomatous lymph node serum antiserum ......... Results of immunoelectrophoretic studies normal lymph node serum antiserum ............... iv using using using using Page 15 PO \. LIST OF FIGURES Figure Page 1 Modification of electrophoresis chamber ......... ll 2 Agar cutter for cutting reproducible antigen wells and antisera troughs ...................... 12 DO Photographs of immunoprecipitates developed with lymphomatous canine serum antiserum ........ l? A Photographs of immunoprecipitates developed with normal canine serum antiserum .............. 5 Photographs of immunoprecipitates developed . with lymphomatous lymph node antiserum .......... 24 6 Photographs of immunoprecipitates developed with normal lymph node antiserum ................ 27 INTRODUCTION The antigenicity of malignant tissues has been studied with gel diffusion techniques (BjBrkland, 1956; Cryan _£.§l-> 1966; DeCarvalho, 1960; and Korngold, 1957). It has been generally conceded that malignant tissue cells do possess a unique antigenic structure. McKenna and Prier (1966) were unable to detect a unique antigen in lymphomas from five dogs. However, DeCarvalho (1960) did detect unique antigens on malignant cells from human patients with chronic lymphatic leukemia. Lampkin-Hibbard and McCain (1965) were able to produce antibodies in guinea pigs against lymphomas by using a nucleoprotein extract from malignant lymphoma cells. The objective of this study was to determine if lymph node cells from dogs with malignant lymphoma had antigens that were not present on normal lymph node cells that could be detected by immunoelectrophoretic techniques. LITERATURE REVIEW Malignant lymphoma is one of many neoplasms that invade man and lower animals. Extensive research has been done on the antigenic properties of numerous malignant neoplasms, spontaneously and chemically induced, including the lymphomas. In this review of the literature, only information relating to the antigenic properties of lymphomas and other neoplastic diseases will be included. Specific tumor antigens have been detected by various investigators. These include Bjfirkland and Paulsson (1962), Cryan et_a1, (1966), DeCarvalho (1960), Greenspan et_a1, (1963), Korngold (1957), Korngold and VanLeeuwen (1957), McKenna and Prier (1966), McKenna t al. (1962), McKenna §£.il- (196A), Strausser and Goldman (1965), Taylor _t_al. (1959) and Yagi and Pressman (1961). Foley (1953) induced fibrosarcomas in mice by im- planting methylcholanthrene crystals subcutaneously in C3H (Hestar subline) mice, or by subcutaneous injection of 1 mg of methylcholanthrene in 0.05 ml of lard. After the tumors had grown to a sufficient size, they were transplanted to other mice. The same transplantation procedure was used for spontaneously developed mammary carcinomas. After several transplantations, the tumors which developed were ligated and 2 eventually died. The animals were then challenged with another tumor transplant. The animals that had received the chemically induced fibrosarcoma were immune to an additional transplant. The animals receiving the mammary carcinoma transplants did not develop immunity against additional transplants. Thus, some malignant tissues may contain an antigen or antigens that are not present in normal tissue. Prehn (1960) reported similar results by inducing fibrosarcomas with dibenz (a,h)-anthracene. Old §t_al. (1962) noted that 3-methyichoianthrene-induced fibrosarcomas were more antigenic than fibrosarcomas induced with 3,4-9,1o-dibenzpyrene. Using hemagglutination techniques, Blakemore and McKenna (1962) reported a serologically diStinct G antigen from the HeLa and Jlll cell lines. McKenna £3 21. (1966) tested numerous cell lines from malignant tissues for the presence of the HeLa G antigen. The antigen was present in approximately 50% of the malignant tissue cell lines. The antigen was absent from all normal tissue cell lines except for the Minnesota embryonal esophagus cell line. Taylor 23 31. (1959) prepared rabbit antisera against HEp No. 2 tissue culture cells, human sarcoma, germfree (G. F.) chicken tumor, Rous sarComa and normal G. F. chicken muscle cells. Half of each of the antigen preparations were treated with fluorocarbon to remove any nonviral or host tissue antigens. The use of fluorocarbon to remove normal tissue antigen was first reported by Gessler gt 31. (1956, 1956a). Antisera against the non-fluorocarbon—treated antigens cross-reacted with each type of tumor cell line antigen and with the normal tissue. Antisera against the fluorocarbon-treated antigens reacted only with their respective antigens. MacKenzie and Kidd (19A5) used complement fixation tests to demonstrate an antigen unique to the Brown-Pearce carcinoma. The antigen was not present in normal rabbit tissues or other neoplastic tissues. ijrkland (1956) pooled tissues from several different carcinomas and several normal tissues. The pools were extracted twice with diethyl ether and the extract injected into a horse. The resulting antisera were adsorbed with human serum to remove any normal tissue antigens. Four different cellular antigens were demonstrated using a modified Ouchterlony gel diffusion technique. The antigens were not tumor specific, however, as they cross—reacted with other tumors and normal tissue even after adsorption. t 31. (1966), Using gel diffusion techniques, Cryan demonstrated specific antigens in spontaneously occurring mammary carcinomas of Swiss Webster mice. These antigens were not present in normal mammary tissue. DeCarvalho (1960) studied the antigenic characteristics of fluorocarbon- purified extracts of carcinomas, sarcomas, acute stem cell leukemia cells and chronic lymphocytic leukemia cells. Antisera against the tissue extracts were prepared in rabbits. The anti—carcinoma and the anti—sarcoma antisera reacted with both the carcinoma and sarcoma extracts, but not the acute stem cell leukemia or chronic lymphocytic leukemia cell extracts. The anti—acute stem cell leukemia anti- serum and the anti-chronic lymphocytic leukemia antiserum reacted only with their respective antigens. This sug- gested the presence of a distinct antigen for the acute stem cell leukemia cell and for the chronic lymphatic leukemia cell. Carcinoma and sarcoma appeared to have the same or a similar antigen or antigens. Greenspan et 21. (1963) prepared anti-leukemic brain, anti—normal brain and anti-Hodgkin's disease lymph node antisera in human volunteers. The antisera were adsorbed with normal brain tissue. Each antiserum reacted with its own antigen. The anti-leukemic brain antiserum also reacted with Hodgkin's disease lymph nodes, human leukemic reticulo-endothelial cells and leukemic mouse brain. Lampkin-Hibbard and McCain (1965) extracted the nucleo— proteins from various mouse and human lymphomas and prepared antisera against each nucleoprotein. By injecting the anti- sera into mice, the authors were able to protect the mice from lymphoma transplants. McKenna and Prier (1966) used canine neoplasms to study neoplastic antigens. Normal tissues were pooled for use in adsorption procedures and in antisera preparation. The authors used several types of malignant neoplasms. Specific antigens were demonstrated in adenocarcinomas, osteosarcomas, basal cell carcinomas, bile duct carcinomas and squamous cell carcinomas. No specific tumor antigens were demonstrated in the lymphomas used. Paradise and Nungester (1966) reported a soluble antigen from a particulate fraction of mouse lymphosarcoma GC3HED. The antigenic substance appeared to be a protein- lipid-carbohydrate complex. All three components were necessary for maximum adsorption of rabbit antilympho- sarcoma antiserum. Strausser and Goldman (1965) evaluated eleven different tumors as to antigen specificity. Normal tissues were used to adsorb the anti—tumor antisera. Antigens specific for the tumors were demonstrated in all tissues. Tumor specific antigens have been demonstrated by many investigators using various methods. In this study an attempt was made, using immunoelectrophoretic techniques, to demonstrate antigens unique to lymphomatous lymph node tissue. *I'IZI‘I: hid. . nu MATERIALS AND METHODS Source of specimens. Normal lymph nodes (NLN) were obtained from 11 normal dogs owned by the Upjohn Co., Kalamazoo, Michigan. After removal, the lymph nodes were placed in a container with dry ice and stored at —70 0. Normal canine serum was obtained from 10 normal dogs prior to surgery at the Michigan State University Veterinary Clinic. Lymph nodes were obtained from 24 dogs with histolo— gically diagnosed malignant lymphoma. These lymph nodes will be abbreviated (LLN). The lymph nodes were placed in containers with dry ice and stored at -70 0. Serum was obtained from 7 dogs with histologically diagnosed malignant lymphoma. The lymph nodes and serum from dogs with lymphoma were obtained from the NIH project (PH 43—65-100) for leukemia transmission studies in the dog at Michigan State University. Preparation of anti—normal canine serum antiserum (rabbit origin) (NCSAS). Blood was obtained from the dogs by venipuncture, placed in tubes and allowed to clot. The tubes were centrifuged, the serum removed, pooled, dispensed in 13 ml amounts and stored at —70 C until used. Inocula were prepared as follows (Hirschfeld, 1960): 12.5 ml canine serum 40.0 ml distilled water 45.0 ml 10% KAl(SOh)2'l2H20 The mixture was adjusted to pH 6.5 with 5N NaOH, centrifuged and washed twice with a 0.85% saline solution. The sediment was made up to a volume of 50 ml with the saline solution. The mixture was injected into 6 Dutch rabbits according to the following schedule (Hirshfeld, 1960): Day 1: 4 ml intramuscularly into each buttock Day 1A: 4 ml intramuscularly into each buttock Day 28: 4 ml intramuscularly into each buttock Day 38: 1 ml whole serum intraperitoneally Five days after the last injection all rabbits were bled by cardiac puncture. After the blood clotted, it was centri— fuged and the serum removed. Each serum was tested by the ring precipitin test for cross-reactions with other sera. Two sera that cross—reacted were discarded. The other four sera were pooled, dispensed in 1 ml amounts and stored at —70 0. Preparation of anti—lymphomatous canine serum antiserum (rabbit origin) (LCSAS). Blood was obtained from 7 dogs with histologically diagnosed malignant lymphoma by catheterization of the carotid artery following terminal surgery. After the blood clotted and was centrifuged, the serum was removed, pooled, dispensed in 13 ml amounts and stored at -70 C. Sera were prepared, tested and stored as described in the previous section. Preparation of anti-lymphomatous lymph node antisera (LLNAS) and anti—normal lymph node antisera (NLNAS). The lymph nodes were trimmed free of fat while frozen, minced and ground in a tissue grinder. To each milliliter of lymph node tissue, 0.02 ml of 10% AlC13 was added and the pH adjusted to 7.0 with 10% NaOH. A volume of the material containing 0.2 gm of lymph node tissue was injected sub- cutaneously into four areas on the back of each of 6 rabbits according to the schedule given for the preparation of NCSAS. The fourth inoculum did not contain A1013 and was injected intraperitoneally. Five days after the last injection the rabbits were bled by cardiac puncture. After the blood clotted and was centrifuged, the serum was removed and tested for cross- reactions as described previously. Two of the sera from rabbits inoculated with the lymphomatous lymph nodes cross— reacted and were discarded. The sera from the other A rabbits were pooled, dispensed in 1 ml amounts and stored at -70 C. No cross-reactions occurred with the sera from rabbits injected with normal lymph nodes. They were pooled, dispensed in 1 ml amounts and stored at -70 C. 10 Preparation of extract of lymph nodes for electrophoresis. Lymph nodes were trimmed free of fat, minced and ground with a small amount of 0.85% saline in a tissue grinder. The ground lymph node material was centrifuged and the supernatant fluid was used for electrophoresis. Immunoelectrophoresis. The modified Spinco Durham cella electrophoresis chamber was modified by reducing inside air space to lessen evaporation (Figure 1). Standard lx3-inch glass microscope slides were coated with 2.5 ml of a 0.7% Agaroseb solution in 0.0375 ionic strength veronal buffer of pH 8.5. The buffer was prepared by diluting 1 part of B-2 buffera with 1 part of distilled water. After the Agarose solidified on the glass slides at A C, antigen wells and an antiserum slot were cut into the hardened Agarose with a device made for cutting reproducible patterns of the wells and anti- serum troughs (Figure 2). An extract of ground lymphomatous lymph node (LLN) was placed in one antigen well and an extract of normal lymph node (NLN) was placed in the other antigen well. The slides were placed across the end baffles of the chamber. Contact between the buffer and Agarose was accomplished by the use of filter paper wicks. The slides were electrophoresed for 60 minutes at 40 ma of constant current. After separation of the protein components of aBeckman Instruments, Inc., Fullerton, Calif. bBausch and Lomb, Inc., Rochester, New York ll ‘$ Figure l. Electrophoresis chamber modified to accommodate lx3-inch agar-coated glass slides. Top, cell cover with a plastic sheet taped inside to reduce evaporation by reducing the air space. Bottom, cell showing paper wicks and slides in place. 12 Figure 2. Agar cutter. An agar-coated slide (front, center) was placed in the chamber (arrow) and the plunger was pressed down to cut wells and trough for immunoelectrophoresis. 13 each of the lymph node extracts, the agar which up to now had been in the pre-cut antisera trough was removed and 0.1 ml of the antiserum being studied was added to the trough of each of the slides. The slides were placed in a moist, airtight container and incubated at 37 C for 18 hours for development of immunoprecipitates. After devel- opment of the arcs, the slides were photographed. Following photography, the slides were washed with several changes of an 0.85% saline solution. Washing was completed in approx- imately 24 hours. The slides were then washed in distilled water for 4 to 6 hours. After drying at 37 C, the slides were stained with a trichrome stain described by Crowle (1961) in order to permanently preserve the results previously photographed. RESULTS Lymphomatous canine serum antiserum (LCSAS). The results using LCSAS were quite variable especially when individual normal lymph nodes were used instead of a pool of normal lymph nodes (Table 1). Immunoprecipitates occurred with four of five normal lymph node pools which did not occur with the lymphomatous lymph nodes. Using individual normal lymph node extracts, only two of seven lymph nodes developed arcs with the antiserum which were not present with the lymphomatous lymph node extracts. Three of the 12 lymphomatous lymph node extracts formed immunoprecipitates with this antiserum that did not form with normal lymph nodes. These arcs were found in the intermediate (between gamma and albumin) region. Representative immunoelectrophoretic patterns developed with LCSAS are depicted (Figure 3). Normal canine serum antiserum (NCSAS). The results using this antiserum were similar to those using LCSAS. 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