Considerable evidence indicates that events at the cell surface play a central role in the regulation of growth of normal and neoplastic cells. It is possible that modification of the surface molecules might be, in part, responsible for the processes that accompany tumorigenesis. These surface molecules are termed TSTA (tumor specific transplantation antigens) and TAA (tumor associated antigens). The purpose of this study is to raise xenogeneic antiserum which can recognize TAA. This antiserum, then, can be used as a tool to enrich or purify the antigen in question. Using the antibody feedback inhibition method which has been developed in our laboratory, an antiserum directed toward membrane components of DBA/2 mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. This antiserum was also capable of killing P815 cells in the presence of guinea pig complement but had no reactivity with another DBA/2 tumor or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected intraperitoneally into either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, apparently facilitating complete removal from the body of tumor cells when low numbers of cells were injected. The antiserum was used to monitor the purification of tumor associated antigen (TAA) of P815 cells. Membrane extracts of both P815 and normal DBA/2J spleen and peritoneal exudate cells were subjected to DEAE fractionation and gel filtration, following which fractions were tested for reactivity with the anti-P815 antiserum. Fractions from P815 extracts shown to be enriched for the TAA were used to raise a second antiserum specific for the tumor. This antiserum was also shown to have specificity for P815 and none for normal DBA/2J cells by ⁵¹Cr release assays in the presence of guinea pig complement and by surface labeling using peroxidase labeled sheep anti-rabbit immunoglobulin after treatment of either P815 or normal cells with the antiserum. This second antiserum (anti-P815-2), when allowed to react with ¹²⁵I-labeled TAA-enriched fractions of the P815 membrane extracts and passed over Sepharose-protein A columns, permitted the isolation of a single major component, detectable on autoradiographs of gradient acrylamide gels. This component was not present in equivalent normal DBA/2 tissue extracts, nor was it detectable when these tests were repeated using an antiserum raised against normal DBA/2 membrane preparations. It was thus concluded that this material constituted a TAA of the P815 mastocytoma. Then this major band was cut, eluted and used in CFA to immunize a rabbit. Three immunizations were needed to get antiserum which was specific to P815 cells and membrane extracts. P815 cells treated with this last antiserum were resistant to lysis by syngeneic cytotoxic cells but they were not when allogeneic cytotoxic cells were used. On the other hand, rabbit anti DBA/2 as well as mouse anti H-2d serum also blocked the syngeneic killing as well as having a blocking effect in the allogeneic killing. This suggests that this antiserum recognizes membrane molecules which are important for recognition by syngeneic cytotoxic cells. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The foundation of the research compilation presented here began with the derivation of an improved method for obtaining optimal in vitro mitogenic responses in salmonid lymphocyte cultures by utilizing autologous or homologous plasma as the primary serum supplement. It was observed that lymphocytes which were previously unresponsive to mitogenic challenge in vitro, when cultured in fetal bovine serum, responded well when cultured in the presence of homologous plasma. Salmonid plasma sources not only repeatedly enhanced the mitogen-specific proliferation of the lymphocyte cultures, but enhanced the antibody response as well. A prolonged kinetic response further supported the contention that former conditions of salmonid lymphocyte culture, employing only fetal bovine serum, not only fail to provide the optimal conditions for cell growth, but in many cases the essential conditions. Enhancement of the mitogenic response was observed for three distinct species, using a common plasma source, suggesting utilization of plasma as an alternate serum supplement has broad applications and may be adapted to many fish systems. With this improved culture system, examination of the regulation of lymphocytes, specifically B cells, was undertaken. Evidence for the existence of a natural regulatory cell population located in the anterior kidney is presented. Addition of anterior kidney cells to either autologous peripheral blood or spleen cell cultures resulted in significant suppression of the mitogen response. The degree of suppressor activity appears to be correlated with the anterior kidney lymphocyte's ability to respond to mitogenic stimulus. It is demonstrated that a decrease in the anterior kidney mitogen response correlates significantly with an increase in the suppressor activity observed upon coculture of these same cells with peripheral blood lymphocytes. Interestingly, while addition of anterior kidney cells to spleen plaque forming cell cultures also resulted in suppression, anterior kidney cells had either no effect or enhanced the antibody response of the peripheral blood lymphocyte cultures. It is postulated that the mediator of this anterior kidney activity is a suppressor cell population which may possess an important immunoregulatory function. / Graduation date: 1992

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Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2008. / Includes vita. Includes bibliographical references (leaves 225-240).

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1997. / Titre de l'écran-titre (visionné le 28 août 2006). Publié aussi en version papier.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2002. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (M. Phil.)--University of Hong Kong, 1991.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 177-205). Also available online.