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Six-month-old Fischer rats (F344) were given the carcinogen methylnitrosourea in their drinking water. Of the induced brain tumors, four were established in culture and propagated as 78FR-G-219 (pleomorphic glioma), 78FR-G-299 (astrocytoma), 78FR-G-284 and 78FR-G-344 (mixed glioma) permanent lines. All cell lines produced S-100 protein and grew as tumors when inoculated s.c. or i.c. in syngeneic hosts. A comparative study of the antigenicity of these lines at different passage levels was carried out using native and chemically modified cells. Syngeneic rats were immunized with cells conjugated with dimethylsulfate and trinitrobenzene sulfonic acid. The immune response was characterized and quantified by an indirect immunofluorescence method and by a complement-dependent microcytotoxicity test. Chemical modification of the tumor cells enhanced antigenicity of the treated cells. The best results were attained with trinitrobenzene sulfonic-acid-treated cells and constituted a two-fold increase in the cytotoxicity index. Cytotoxicity values varied in the different cell lines. Antisera raised with trinitrobenzene sulfonic-acid-modified cells of all lines cross-reacted with cells of all lines. Cytotoxicity values were insignificantly reduced by absorbing the antisera with a variety of syngeneic tissues. Antisera raised against native syngeneic brain cells showed virtually no cytotoxicity for glioma cells. Antisera raised against syngeneic brain cells treated with trinitrobenzene sulfonic acid, however, were slightly cytotoxic for normal brain cells and glioma cells as well. The results of the present studies show that antigenicity of glioma cells can be definitely raised with trinitrobenzene sulfonic acid treatment. Furthermore, it would seem that haptenization of glioma-associated antigens may be a promising approach ot the study of glioma-host interactions.

Radiolabelled monoclonal antibodies (McAbs) raised against membrane components of an experimental rat glioma (79FR-G-41) were administered parenterally to immunodeficient mice bearing glioma grafts for tumor radioimmunodetection by external imaging. Purified McAbs (14AC1) of IgG2a isotype were labelled with Na131I (2mCi/50ml) using the Chloramin-T method. As control, for non-specific uptake of proteins in the tumor, normal mouse IgG were also iodinated. For radioimaging, nude mice bearing gliomas in the thigh muscle were injected intravenously with 15 micrograms of the 131I-McAb with an activity of approximately 150 mu Ci. Control tumor-bearing animals received the same amount of mouse 131I-IgG. Scans obtained immediately after injecting the intact 131I-14AC1 antibody and at 24, 48, 72, and 96 hours demonstrated accumulation in the tumor. The tumor was clearly visible 48 hours following injection of 131I-labelled antibody. At 96 hours after injection, the McAb showed a clearly higher uptake into the tumor as the control IgG. The biodistribution of the injected antibody was studied at 96 hours after injection following the last gamma-imaging. At this time the blood activity was still high, but the maximum activity was found in the tumor for the specific McAb. Using the 131I-14AC1 to image glioma transplants, it could be shown that grafts are permeable for the McAb. The time-course experiments administering 131I-14AC1 antibody and normal mouse 131I-IgG, demonstrated that the localization of 131I-I4AC1 antibody in glioma grafts is the result of specific antigen binding. The scintigrams using intact antibody without background subtraction provided adequate tumor visualization, but the activity in the blood was high even 96 hours after injection. More rapid clearance of blood - pool radioactivity would possibly be achieved with F(ab')2 fragments. These in vivo glioma imaging studies, together with related in vitro binding tests, indicate the potential value of monoclonal antiglioma antibodies not only for clinical tumor radioimmunodetection, but also for the evaluation of immunotherapeutic approaches to the glioma disease of man.

The production of hybridomas between X63-Ag8.653 myeloma cells and spleen cells from female BALB/c mice immunized with 79FR-G-41 glioma cells is reported. One hundred and six hybridoma clones were obtained secreting monoclonal antibodies (McAbs) against different target cells. Specificity tests (RIA, micro-ELISA) showed that McAbs produced by three hybridoma clones (13GC1, 14BC1, 14FC3) bound 79FR-G-41 glioma cells but did not react with fibroblasts, kidney and brain cells of newborn F344 rats. From the specificity analysis of two McAbs (13GC1, 14BC1) it is evident that these did not only react with 79FR-G-41 cells but also with other glioma cell lines (78FR-G-219, 78FR-G-284, 78FR-G-299 and 78FR-G-344) established from chemically induced rat brain gliomas. These results suggest, in accordance with the findings in human neuroectodermal tumors, the expression of common reactivity antigens in different brain tumors of glial origin. However, the McAbs obtained against experimental rat glioma cells did not recognize glioma cells derived from spontaneous brain tumors of dog or man. Immunofluorescence and immunoperoxidase tests indicate that there is a remarkable heterogeneity among cells of experimental glioma lines with respect to the expression of glioma associated determinants recognized by McAbs. The fact that only a variable number instead of the totality of tumor cells in any asynchronous and uncloned tumor cell population expresses, at a certain time, recognizable antigenic determinants has to be taken into account, particularly if they are considered to be employing McAbs as carrier molecules for diagnostic and therapeutic purposes.

Brain tumors were induced in adult inbred Fischer rats (F-344) by systemic administration of N-methyl-N-nitrosourea mixed in the drinking water (pH 6,2). Four of these tumors, one pleomorphic glioma (78FR-G-219), two pleomorphic mixed gliomas (78FR-G-284, 78FR-G-344) and one grade I to II astrocytoma (78FR-G-299) were established in vitro and maintained as permanent cultures. The glial nature of all cell lines was ascertained by demonstrating the presence of the S-100 protein in the cultured cells. All cell lines grow as tumors when isografted in syngeneic animals. Glioma cells were conjugated with trinitrobenzene sulfonic acid (TNBS) under standard conditions. Syngeneic adult rats were immunized with TNBS-modified or unmodified irradiated glioma cells by s.c. inoculation of 1 X 10(6) cells on days 1,8 and 15. Two weeks later the animals received a s.c. booster of 5 X 10(6) native cells. Using the complement-dependent microcytotoxicity test glioma cytotoxic titers were measured 5, 6 and 7 weeks after the first immunization. The results indicated that trinitrophenylated glioma cells induced a cytotoxic antibody response against native glioma cells which was higher than that induced by untreated cells.

Brain tumors were induced in inbred Fischer rats (F344) by administration of methylnitrosourea in the drinking water. One of the induced tumors, a pleomorphic glioma (78-219), was established in vitro and propagated as 78FR-G-219 permanent cell line. The tumorigenicity of the established line was investigated by intracerebral or subcutaneous inoculation of 1X10 (6) - 10X10(6) cells. Lymphocytes infiltrating secondary tumors (TIL) were enriched by a single step centrifugation on different discontinuous Percoll density gradients, while blood lymphocytes (PBL) and lymphocytes from spleen and lymph node (SL and LNL) were enriched by Ficoll - Isopaque flotation. The reactivity spectrum of the isolated lymphocytes raised against cultured 78FR-G-219 cells was monitored by means of two different assays: Lymphocyte microcytotoxicity test (LMC) and colony inhibition assay (CIA). Reactivity of PBL, SL and LNL of glioma-bearing animals was clearly reduced, while TIL showed no natural killer (NK) activity, no cytotoxicity against syngeneic 78FR-G-219 pleomorphic glioma cells and no colony inhibition in mixed lymphocyte/target cell cocultivation. NK activity of TIL was only slightly reduced against target cells of a non-cross-reacting syngeneic astrocytoma line (78FR-G-299).

Brain tumours were induced in inbred Fischer rats (F344) by methylnitrosourea. A pleomorphic glioma (78-219) was established in vitro and propagated as 78FR-G-219 permanent cell line. These cells were modified either by dimethylsulfate or by trinitrobenzene sulfonic acid. Antisera were raised against both native an chemically latered cells in syngeneic rats. The cytotoxicity of these sera was tested against the native glioma cell line as target, by means of the 14C-nicotinamide release. The methylated cells induced a complement--dependent humoral cytotoxicity in the range of that produced by native cells (20%); trinitrophenylation, however, resulted in a two-fold increased cytotoxic humoral immune response. Effects of chaotropic salts on methylated cell surface structure suggested a mode of action different from that of trinitrophenylation, which could be further substantiated by scanning electron microscopy. Furthermore, a new tool for the evaluation of cell surface structure, secondary ion mass spectrometry, was applied on our cell system. Significantly different ionized fragments were obtained from normal brain cells and glioma cells, respectively.

In context with the first light and electron microscopic description of a spontaneous esthesioneuroepithelioma in a dog (10 year old female Labrador) general morpho- and etiopathogenetic problems concerning olfactory neoplasias were discussed. Because of its specific location and growth pattern, especially the development of true rosettes and pseudo-rosettes, the tumor was evaluated light microscopically as an esthesioneurogeneic one. The final classification was based upon ultrastructural peculiarities, as occurrence of ciliar structures, characteristic intercellular junctions, and the - although rare - appearance of dense cored vesicles.

Iodinated F (ab')2-fragments of monoclonal IgG2a-antibodies (McAbs) 14 AC1 raised against membrane components of an experimental rat glioma (79FR-G-41) were used for the radioimmunodetection of glioma xenografts in athymic mice. For gamma imaging, nude mice with intramuscular glioma grafts received 10-20 micrograms of 131I-labelled whole immunoglobulins and F (ab')2-fragments of the 14AC1 McAb, respectively, with an activity of 50-150 mu Ci (1.85-5.55 MBq). Scans obtained at 24, 48, 72 and 96 hours after injection demonstrated tumor accumulation of F (ab')2-fragments at 24 hours, and adequate tumor visualization without background subtraction at 48 hours. The rapid blood clearance of F (ab')2-fragments resulted in a whole body half life of 6 hours. Scintigrams using intact antibody provided comparable tumor localization at 96 hours, but also high bloodpool radioactivity and a half life period of more than 4 days. 131I-labelled normal mouse-IgG as control for non-specific uptake of immunoglobulins in the tumor revealed no tumor visualization at any time. Rapid tumor permeation, short blood clearance and a markedly reduced background radioactivity let F (ab')2-fragments appear superior to whole immunoglobulins in immunoscintigraphy, thus indicating their potential value for the improvement of radioimmunodiagnosis of glioma disease.