Liselotte Graf

Identification of glial fibrillary acidic protein by the immunoperoxidase method in human brain tumors.

The immunocytochemical localization of glial fibrillary acidic protein within glioma cell bodies and their processes by the immunoperoxidase method is demonstrated to be of diagnostic value. This method has advantages over “special” stains because it is not so dependent upon color alone, and because it identifies a specific protein in the cells. The immunoperoxidase method using antiserum to glial fibrillary acidic protein is shown to be useful for the differentiation of mixed glial and mesenchymal tumors, and for the diagnosis of tumors in which a glial or mesenchymal cell origin is in doubt.

Glial fibrillary acidic protein in giant cell tumors of brain and other gliomas

SummaryThe giant cell tumor of brain (glioblastoma/sarcoma) has been considered a glioma by some and a sarcoma by others. This study shows that glial fibrillary acidic protein (GFAP), a specific “marker” for astrocytes, is present in the tumor cells, thus indicating that the cell of origin is the astrocyte and that the tumor should be called a giant cell glioblastoma. GFAP is present in the smaller cells of this tumor and in larger mononucleated cells, but little if any is detectable in multinucleated giant cells.In a different kind of tumor, the giant cell astrocytoma assonciated with tuberosclerosis, GFAP is restricted in most cells to a narrow peripheral zone.Immunocytochemical localization of GFAP is superior to “special stains” to differentiate giant cell glioblastomas from true sarcomas and giant cell bone tumors, since the latter are both negative for GFAP.Comparison of GFAP in all tumors of astrocyte origin shows that the cells that appear to contain the most GFAP include low grade well differentiated stellate cells, elongated “piloid” cells, and gemistocytic astrocytoma cells. Highly malignant undifferentiated cells, with less well developed processes, are less densely positive.Although there is in general an inverse relationship between GFAP content and degree of tumor malignancy, a more complex relationship exists with respect to individual cells; more GFAP is present in well differentiated cells with well-developed processes and filaments than in undifferentiated cells and large multinucleated cells. It is suggested that the pleomorphism of more malignant cells may relate to their relatively low GFAP content and perhaps to the disassembly of their glial filaments.

Antibodies to total brain gangliosides: titer and specificity of antisera.

Our sustained studies of the immunological activity of lipidsl,2 led to the important generalization that such activity was intimately related to glycosphingolipid structures. We therefore had an early interest in the immunological activity of gangliosides and an early appreciation that they exhibited only weak immunogenic properties, much weaker than those of neutral glycosphingolipids.

Immunogenicity of cytolipin H aggregated with Acholeplasma laidlawii membrane proteins.

The list of immunologically active lipids found in membranes of normal and malignant cells is growing steadily since the isolation of cardiolipin in 1942 and of cytolipin H in 1958 (1, 2). The purified lipids usually function as haptens, failing to elicit an antibody response when injected into animals (2). Antisera to the lipid haptens have, therefore, been prepared either by injection of tissue fractions or of the purified lipids mixed with protein of a different species. Both methods are not completely satisfactory as tissue fractions may contain several serologically active lipids resulting in sera of low specificity, and the injection of the purified lipid hapten mixed with swine serum or albumin and adjuant frequently fails to elicit a good antibody response to the lipid (2). A new approach to the preparation of antibodies to lipid haptens has recently been proposed by us (3). Purified glycolipid haptens of Mycoplasma pneumoniae were bound to membrane proteins of Acholeplasma laidlawii by a reaggregation process (4) which consisted of solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glycolipids in 20 mM sodium dodecyl sulfate (SDS) followed by dialysis of the mixed solutions against 20 mM Mg2+ . The reaggregated material elicited the production in rabbits of antibodies which fixed complement and precipitated the purified glycolipids (3, 5). Free glycolipids or their mixture with A. laidlawii membrane proteins were far less effective in evoking these antibodies (3). The main advantage in utilizing hybrid reaggregates consisting of protein and lipid derived from membranes of two different organisms lies in the ability to select the lipid and the protein components of the reaggregate and thus control the type of antibodies produced. The reaggregation process seems to involve an intimate binding of lipid to protein, presumably by the same kind of bonds responsible for their association in biological membranes.

Immunochemical studies of organ and tumour lipids. XVII. The existence of two complement-fixing systems involving cerebroside.

ORGAN specificity of brain antigens has been a well-recognized phenomenon among immunologists. Antisera prepared against various brain fractions of one species are found to react with brain fractions of most other species, but not with similarly prepared fractions from other tissues. These observations are particularly decisive when attention is focussed on the tissue lipids with the method of complement-fixation. Several years ago it was shown that galactocerebroside, a major lipid constituent of brain and other nervous tissue, is the substance that accounts for these ‘brain specific’ reactions (JOFFE, RAPPORT and GRAF, 1963). Four findings were offered to support this conclusion by using an antiserum against bovine brain which gave good reactions with 1-2 pg of brain lipid from ten mammalian species but did not react with 50 pg of lipid from other bovine tissues. First, the antiserum gave equally good reactions with 3-5 mpg of cerebroside, and this reactivity was more than sufficient to account for that of total brain lipid. Second, the antiserum did not react with other established lipid haptens such as cytolipin H, cardiolipin and Forssman substance, nor with substances of closely related structure, such as glucocerebroside. Third, an equal degree of reactivity was obtained with pure phrenosine, cerasine, and mixed galactocerebrosides of bovine cord. And finally, similar reactivity was found with galactocerebroside prepared by organic synthesis. Brain specificity was entirely consistent with the known distribution of galactocerebroside in this organ. As was the case with other lipid haptens, pure galactocerebroside did not give complement-fixation reactions with the antiserum in the absence of auxiliary lipids, and optimal reactions (high sensitivity as well as marked intensity) were obtained in the presence of a mixture of lecithin and cholesterol (1 : 1, w/w) added to cerebroside in a ratio of 100: 1. In examining the reactivity of a large number of anti-brain sera we discovered that, whereas most of these reacted well with total white matter lipids of the brain, only a small number reacted with galactocerebroside in the test formulation described above. The immediate implication of these results was that some other reactive lipid was present in white matter. A systematic study of this problem has ’

Immunochemical Studies of Organ and Tumor Lipids Xxi. Sensitivity and Specificity of the Cytolipin F – Sheep Erythrocyte System

The sensitivity and specificity of the sheep erythrocyte - anti-sheep erythrocyte system to inhibition by pure cytolipin F has been studied with 5 antisera, in order to compare it with the rat erythrocyte-anti-rat lymphosarcoma system and its inhibition by pure cytolipin R. The cytolipin F - sheep erythrocyte system is much more sensitive than the cytolipin R - rat erythrocyte system, inhibition of hemolysis of 6 x 10(6) sheep cells being produced by 10 ng of cytolipin F (combined with a four-fold quantity of lecithin) compared with inhibition of hemolysis of 10(6) rat cells by 50 to 100 ng of cytolipin R (also combined with lecithin). Differences in sensitivity are attributed to the larger number of available cytolipin F determinants on sheep erythrocytes compared with cytolipin R determinants on rat erythrocytes. Studies of the auxiliary lipid enhancement of cytolipin F activity by galactocerebroside, lactosyl ceramide (cytolipin H), and lecithin are also reported.