G. Bandlow

Cytostatic effect of gangliosides present in the membrane of macrophages

Stimulated macrophages are known to inhibit the growth of certain tumor cells. Using mouse peritoneal exudates as a source of macrophages and the mastocytoma cell line P815 as the target, the inhibition was found to depend on direct contact between the macrophages and the growing cells. Cytostatic activities were detected in extracts of macrophages as well as in membranes of macrophages bound to substances of low molecular weight. Physical and biochemical characteristics of the cytostatic activity hint toward N-acetylneuraminic acid containing glycosphingolipids (gangliosides). The different macrophage gangliosides were separated by thin-layer chromatography. All types showed cytostatic activity, but the most effective gangliosides were identified as monosialoganglioside GM1 and disialoganglioside GD3.

Cytostatic effect of macrophages from non-immunised mice on mastocytoma P-815 cells in vitro.

SummaryPeritoneal macrophages from non-immunised C57/bl, C3H, and DBA/2 mice were examined for their capability of inhibiting the growth of mastocytoma P-815 cells in vitro. The rate of tumour cell growth was measured by the uptake of 125IUDR into the DNA of the tumor cells. Sodium thioglycollateinduced macrophages as well as resident macrophages inhibited the growth of allogeneic tumor cells distinctly, when incubated for 48 h. The effect depended on the effector to target cell ratio. Tumor cell growth was still reduced at a ratio of one macrophage to one tumor cell. A decrease in the proliferative activity of mastocytoma cells was also observed in the presence of non-stimulated an non-immune macrophages from DBA/2 mice. This decrease in target cell proliferation, however, occurred more slowly.

Increased cellular immunity against host cell antigens induced by vacciniavirus

Guinea pigs show an increased cell-mediated immunity against host cell antigens after active immunization with vaccinia virus infected tissue culture cells. This immunologic adjuvant effect was observed in a heterologous system comprising primary rabbit kidney cells, primary fibroblasts from the mouse, permanent monkey kidney cells andin vivo infected mouse brain cells. The quantitative determination of the cellular immune response was carried out both in macrophage migration inhibition and in lymphocyte transformation tests. Cell-mediated immunity appears on the third day after immunization. A prerequisite for the adjuvant effect is the reproduction of the virus in the cell against which the increased cell-mediated immune response is directed.

Gesteigerte Immunogenität von Plasmamembranen vaccinia virusinfizierter BHK-Zellen

Vacciniavirusinduzierte Zelloberflächenveränderungen können durch den zytoziden Effekt virusspezifischer Antikörper auf infizierte Zellen sowie durch die Agglutination virusinfizierter Zellen durch Concanavalin A demonstriert werden. Diese Veränderungen treten zeitlich korreliert mit einer gesteigerten Immunogenität der Wirtszelle 2–3 Stunden nach Infektionsbeginn bereits vor der Produktion reifer infektiöser Viruspartikeln auf. Eine gesteigerte Antikörperproduktion gegen Wirtszellantigene wird auch nach Immunisierung mit isolierten Plasmamembranen vacciniavirusinfizierter Zellen erreicht, während Zellkernfraktionen diesen Effekt nicht auslösen. Bei den zytotoxischen Antikörpern, die zu den 7 S-Immunglobulinen gehören, handelt es sich — wie durch Absorption mit BHK-Zellmembranen gezeigt werden kann — um spezifisch gegen BHK-Zellen gerichtete Antikörper. Cell surface alterations induced by vaccinia virus can be demonstrated either by the cytocidal effect of specific antibodies against vaccinia virus on the infected cells or agglutination of the infected cells by Concanavalin A. This change occurs 2–3 hours after infection, that is before any mature particles of vaccinia virus were produced. It coincides with an increased immunogenicity of the host cell. The production of cytotoxic antibodies against host cell antigens was also increased after active immunization with plasma membranes isolated from vaccinia virus infected BHK-cells, whereas the fraction of cell nuclei did not show up this effect. The cytotoxic antibodies which proved to be 7 S immunoglobulines, are specifically directed against BHK-cells as could be shown by absorption tests with membranes of BHK-cells.

Abnormal lysozyme production of peritoneal macrophages from mastocytoma P-815 bearing C3D2F1-mice

Sodium thioglycollate-induced peritoneal macrophages from C3D2F1 mice were examined for their ability to produce and secrete lysozyme in vitro. Lysozyme was quantitated by use of a ‘lysoplate assay’, in which lyophilizedMicrococcus lysodeicticus suspended in agar were lysed by the enzyme. The ability to secrete lysozyme decreased in mice bearing a subcutaneous or intraperitoneal mastocytoma P-815 with progressive tumour growth.

Untersuchungen zum Mechanismus der immunologischen Adjuvanswirkung des Vacciniavirus

Vacciniavirus wirkt immunologisch adjuvierend bei der Produktion heterologer, gegen Wirtszellantigene gerichteter zytotoxischer Antikörper. Dieser Adjuvanseffekt konnte bei aktiver Immunisierung von Meerschweinchen mit verschiedenen Wirtszellen, i. e. BHK-Zellen, Affennierenzellen, Kaninchennierenzellen, humanen embryonalen Fibroblasten, primären menschlichen Zellen aus Hirntumoren, embryonalen Mäusefibroblasten und Mäusetumorzellen demonstriert werden. Nach schonender Inaktivierung der Infektiosität des Virus mit Formalin bleibt der adjuvierende Effekt voll erhalten. Die gesteigerte Immunogenität der Wirtszelle ist schon vor der Produktion reifer infektiöser Viruspartikeln nachweisbar. Sie ist zeitlich mit dem Auftreten spezifischer Virusantigene in der Zellmembran korreliert. Durch Vorbehandlung des virushaltigen Antigenmaterials mit virusspezifischen Antikörpern in vitro kann die adjuvierende Wirkung unterdrückt werden. Vacciniavirus acts as an immunological adjuvant in the production of heterologous cytotoxic antibodies against host cell antigens. This adjuvant effect was demonstrated by active immunisation of guinea pigs with different host cells, such as BHK-cells, monkey kidney cells, rabbit kidney cells, human embryonic fibroblasts, cells of human brain tumours, fibroblasts of embryonic mice and tumourcells of mice. After careful treatment of the virus-cell antigen with formalin the adjuvant effect is still present, although the infectivity of the virus was abolished. Increased immunogenicity of host cells could already be observed before any mature vaccinia particles were produced. It coincides with the appearence of virusspecific antigens on the surface of the cell membrane. The adjuvant effect could be suppressed by treating in vitro the material that containes the virus and the host cell antigens, with specific antibodies against the virus.

Virusinfizierte Lymphoblasten als Antigen für die Herstellung von heterologen Antilymphocytenseren

SummaryAfter active immunisation with virus infected lymphoblasts the production of guinea pig antibodies against antigens of not infected lymphocytes is increased. This immunological adjuvant effect was observed when human and mice lymphocytes were infected with herpes simplex virus, VSV and vacciniavirus. The increased production of antibodies was demonstrated by an in vitro micro-lymphocytotoxicitytest and in vivo by its immunsuppressive effect when allogeneic tumor cells (Mastocytoma P-815-x2 DBA/2→NMRI) were transplanted.ZusammenfassungNach aktiver Immunisierung mit virusinfizierten Lymphoblasten bilden Meerschweinchen vermehrt Antikörper gegen Antigene nicht infizierter Lymphocyten. Dieser immunologische Adjuvanseffekt wurde nach Infektion von menschlichen und Mäuselymphoblasten mit Herpes simplex-Virus, VSV und Vacciniavirus beobachtet. Die vermehrt gebildeten Antikörper wurden im Mikrolymphocytotoxicitätstest in vitro und in vivo durch ihre immunsuppressive Wirkung bei Tumorverpflanzung (Mastocytoma P-815-x2 DBA/2 → NMRI) nachgewiesen.

Selection and isolation of a new variant of DBA/2 mastocytoma P 815 X 2

SummaryA tumor model was developed in DBA/2 mice for studying the progression of the mastocytoma P 815 X 2 tumor. Tumor cells obtained from ascites were grown in vitro. The number of cells derived from a single clone was increased by in vitro culture. Cells were then injected either i. v. or i. p. into DBA/2 mice. Large volumes of tumor ascites were observed after i. p. but not i. v. injection. The latter led to tumor growth at multiple sites, especially in the liver. Mastocytoma cells were released from liver tissue and then injected i. p. into other recipients. For cloning of liverinvading tumor cells, this procedure was repeated for >20 generations of mice. Tumor infiltration of the liver increased strongly during this period, but ascites volume clearly decreased.

Selection of a metastatic mastocytoma P 815 variant and characterization of its ganglioside pattern

In vitro cultured Mastocytoma P 815 cells were injected into DBA/2 mice either i.p. or into a tail vain. After intravenieus injection tumour cells predominantly proliferated in the liver. The liver was broken up and the released Mastocytoma cells were now transplanted and further passaged i.p. After several passages the volume of tumour ascites gradually decreased whereas the rate of tumour proliferation increased. This selected tumour cell variant could also be differentiated from the original primary tumour line using histological, immune histological and electronmicroscopical methods. Both tumour lines differed in rive in their enzyme pattern in the peripheral blood. The cell surfaces of the two cell lines showed a high content of GSL in the lipid fraction, mainly NANA containing GSL, respectively gangliosides. The gangliosides from Mastocytema P 815 cells, from liver tissues and from liver infiltrated by tumour cells were analysed by HPTLC. A characteristic pattern was found for liver tissue and Mastocytoma P 815. The infiltrated liver showed a combined pattern of both. Furthermore the ganglioside content was determined in the peripheral blood. Tumour bearing mice showed a higher amount of gangliosides than normal animals. A steady increase of the ganglioside level was observed during tumeur growth.

Cytotoxische Reaktionen an Sendai-Virus infizierten Zellen

Monolayerkulturen von. primären Mäusefibroblasten werden mit Myxovirus parainfluenza 1 (Sendai) infiziert. Während der Virusvermehrung wird homologes Sendai-Immunserum und Komplement zugefügt. An den infizierten Zellen können dann parallel zum Auftreten hämadsorbierender Oberflächenantigene immuncytotoxische Reaktionen beobachtet werden. Cytotoxicitätsparameter sind die Freisetzung von Laktatdehydrogenase und von3H nach Markierung der Zellen mit3H-Uridin. Die immuncytotoxische Aktivität, die in den IgM-und IgG-Fraktionen lokalisiert ist, läßt sich durch Sendai-Virus absorbieren. Monolayer cultures of primary mouse fibroblasts were infected with parainfluenza-1 (Sendai) virus. During virus replication homologous Sendai-immuneserum and complement were added. On infected cells immunocytotoxic reactions could be observed as soon as hemadsorbing surface antigens were appearing. Parameters of cytotoxicity were the liberation of lactic dehydrogenase and the3H-release after labeling the cells with3H-uridine. The immunocytotoxic activity localized in the IgM and IgG fractions could be absorbed by Sendai virus.