René Verloes

Tumor growth inhibition mediated by trypsin inhibitor or urokinase inhibitors

Abstract When testing in vitro, we often observed that erythrocytes taken from Ehrlich ascites tumor bearing mice displayed enhanced agglutinability by the lectin Concanavalin A, suggesting that protease activity operates in vivo. In several studies, we were able to inhibit Ehrlich ascites tumor growth by the repeated administration of soya bean trypsin inhibitor. Studies of dosages and schedules of treatment showed that for 20,000 initially grafted cells, treatment resulted in 0–70% of long-term survivors and induced a significant increase in mean survival time of treated mice over control mice. For 200,000 grafted tumor cells, 0–40% of long-term survivors were recorded. In a comparative study, we found that different inhibitors of urokinase displayed a similar chemotherapeutic effect against 200,000 inoculated cells. Our results corroborate the idea that a plasminogen activator monitored chain of fibrinolytic and proteolytic activity controls tumor growth and metastasis enzymatically.

Antitumour immunoprotection by an immunobacterial lectin approach

Abstract By in vivo screening against lymphoid leukemia L1210 in BDF1 mice and Ehrlich ascites carcinoma in BALB/c mice, we found that a vaccination with a synthetic antigen, built up by the cell wall of Micrococcus lysodeikticus (ATCC 4698) on which we coupled oligosaccharides of poly-N-acetyl-D-glucosamine, was able to protect to a fairly good extent against both tumours in the case of L1210 leukemia, hyperimmunization induced an increase in life span of 75% over controls and long term survivals of 25%; in the case of Ehrlich carcinoma, it induced an increase in life span of 104% and long term survivals of 25%. Under the present conditions, we observed that antibodies to the less immunogenic N-acetyl-D-glucosamine polymer were ineffective. Different antigens, antigen doses and immunization schemes were investigated. This system can be considered as an immunobacterial equivalent of both the lectins wheat germ agglutinin and concanavalin A. Although bacterial vaccines are widely used now as non-specific immunoadjuvants that stimulate the T helper cell function and non-specific macrophage immunity, we present evidence that subpopulations of antibacterial antibodies may be tumour specific.

Successful immunotherapy with micrococcus, BCG or related polysaccharides on L1210 leukaemia after BCNU chemotherapy.

The experiments aimed at evaluating the optimal parameters in the chemo-immunotherapeutic treatment of the L1210 lymphoid leukaemia grafted to [female BALB/c (H2d) X male DBA/2 (H2d)]F1 hybrid mice, hereafter referred to as CDF1 mice. in vitro irradiation of leukaemic ascites cells by X- or gamma-rays and subsequent inoculation in mice showed that optimum immunogenicity is radiation dose-dependent. Grafting mice with 10(7) leukaemic ascites cells irradiated at optimum dose (80 GyX- or gamma-rays) delays mortality of the animals when challenged later with untreated L1210 cells, but is unable to cure mice. By contrast, specific immunoprophylaxis induced by Micrococcus, complement-triggering polysaccharides or BCG and irradiated leukaemic cells was able to protect mice against grafts of 10(4) L1210 cells. The i.p. route was notably superior to the i.v. route. When mice bearing advanced L1210 tumour were treated by chemotherapy (12 mg/kg of BCNU) on Day 6.5 after grafting 10(4) L1210 cells and subsequently treated by immunotherapy, a very high percentage (up to 90%) of mice with 10(8) leukaemic cells could be cured by repeated 1mg injections of bacterium or polysaccharide, and challenge with irradiated leukaemic cells was unnecessary. Because of the high cure rate obtained, the very regular response pattern and the non-pathogenicity, the bacterium Micrococcus lysodeikticus would seem a promising new candidate for chemo-immunotherapeutic antitumour strategies.

Influence of anti-Micrococcus immunoglobulins on the proliferation of normal immunocompetent cells in inbred mice☆

We have developed an immunization schedule that leads to the production of anti-Micrococcus antibodies of clonal dominance in CDF1 and Balb/c mice. Micrococcus vaccinated mice displayed a significantly impaired immunoresponsiveness to sheep erythrocytes (p < 0.001) and this was not related to the presence of homogeneous antibodies in the serum. Anti-Micrococcus sera transferred to preimmune mice simultaneously with sheep erythrocytes decreased the number of anti-sheep-erythrocyte plaque-forming cells, suggesting that,in vivo also, the anu-Micrococcus serum plays an immunoregulatory role. Immune serum was fractionated on an immunoadsorbent, and sheep erythrocytes suspended in the different fractions were used for sensitization of mice. By analyzing the number of plaque forming cells four days after priming, we found that immune (IgM) responses were only suppressed in mice treated with the anti-Micrococcus antibody containing fraction, indicating that the immunosuppression was specifically caused by the anti-Micrococcus antibody. A hypothetical mechanism invoked to explain the production by bacterial vaccines of antibodies with limited electrophoretic heterogeneity is described in detail.

The obtention of high-titer antibodies of restricted heterogeneity against lysozyme methyl ester in rabbits vaccinated with Micrococcus lysodeikticus

Abstract Vaccination of rabbits with the Gram-positive bacterium Micrococcus lysodeikticus results in the production of antimicrococcus antibodies of restricted heterogeneity (Van Hoegaerden et al. , 1975). Similarly, we observed that intravenous vaccination of rabbits with Micrococcus cells, partially covered with lysozyme methyl ester, induces the production of large amounts of antilysozyme methyl ester antibodies (up to 40 mg per millilitre of serum) of limited heterogeneity. Our results clearly demonstrate that rabbits, hyperimmunized for months with Micrococcus and yielding homogeneous antimicrococcus antibodies, were incapable of mounting any antilysozyme methyl ester antibody response, when vaccinated with free Micrococcus cells and lysozyme methyl ester bound to Micrococcus. In rabbits, immunized from the beginning with free Micrococcus cells and lysozyme methyl ester bound to Micrococcus, the elicited immune response showed a restricted heterogeneity and was predominantly directed at Micrococcus antigenic epitopes. However, those rabbits were equally capable of producing later a high titer of antilysozyme methyl ester antibodies of limited heterogeneity and clonal dominance, under conditions of mixed vaccination with a low free Micrococcus dose and progressively increasing lysozyme methyl ester doses.

Influence of micrococcus, BCG and related polysaccharides on the proliferation of the L1210 Leukaemia

A comparative study of the effects of BCG, Micrococcus lysodeikticus, and a series of structurally related polysaccharides (complement triggers) on the non-specific and specific immune resistance against L1210 lymphoid leukaemia was carried out and commented on. In contrast with authors of earlier reports, we were unable to generate any effective non-specific or specific immunotherapy after the graft of 10(4) leukaemic cells to 8--10-week-old CDF1 mice. However, when mice were prevaccinated with irradiated (8 krad X-rays) cultured cells combined with 1 mg of bacterium or polysaccharide one month before grafting 10(4) cells, they were given an immunoprotection that was more pronounced with the i.p. than with the i.v. route. Prevaccinated mice were afforded a stronger immunoprotection when boosted repeatedly with 1mg injections of bacterium or polysaccharide after tumour challenge.

Comparison between the in vitro interaction of lectins (PHA and con A) and antimicrococcus antibodies on normal and malignant cells.

Abstract Rabbit sera and purified antibodies directed against Micrococcus lysodeikticus (a Gram-positive, non-pathogenic and easy-to-eliminate bacterium) do not bind at 37°C to autologous, isologous or heterologous (mouse and human) erythrocytes, as measured by agglutination assays, heterologous (guinea pig) complement cytotoxicity or Coombs test reactivity. However, rabbit and mice antimicrococcus sera agglutinate Ehrlich carcinoma cells and human leucocytes. Both lectins, Con A and PHA, agglutinate Ehlrich carcinoma cells, human leucocytes and erythrocytes of different species but only ConA is able to agglutinate Micrococcus cells. Biological effects of cell surface binding are discussed.

Tumour immunoprophylaxis exerted by the antimicrococcus immunity II. Influence on the proliferation of murine plasmacytoma (MOPC 173) and Ehrlich carcinoma cells

Abstract The influence of the micrococcal vaccine on the proliferation of normal immunocompetent and neoplastic plasma (MOPC 173 ) and of carcinoma (Ehrlich ascites) cells is studied. BALBc mice and CDF 1 mice, hypervaccinated with Micrococcus Lysodeikticus , developed significantly ( 0.001 0.005 ) lower primary immune responses against sheep erythrocytes and also displayed impaired secondary immune responses. Administration of Bacillus Calmette-Guerin could partially restore the immunologic potential of vaccinated mice as measured by the capability of mice to answer with primary or secondary immune responses to sheep erythrocytes but did not increase immunoresponsiveness of normal mice sensitized at nearly optimal antigenic dose. Switching on a secondary antimicrococcus immune response after grafting 200,000 Ehrlich carcinoma cells in the peritoneum of BALBc mice resulted in a 30% increase in mean life span over control mice. However, mice hyperimmunized for months with Micrococcus lysodeikticus were offered the strongest immunoprotection and we found 67% of mice surviving to day 90 whereas the other vaccinated but tumour bearing animals showed a 29% increase in life span over control mice. Similarly, we demonstrated that 44% of BALBc mice, hyperimmunized for months, were able to reject a tumour challenge of 200,000 grafted MOPC 173 plasmacytoma cells, whereas the other micrococcus vaccinated but tumour bearing animals died with a 26% increase in mean survival time over control mice.

Tumour immunoprophylaxis exerted by the antimicrococcus immunity. I. Influence on the proliferation of murine leukaemic L1210 cells.

Abstract An immunization schedule that leads to the production of antimicrococcus antibodies of restricted electrophoretic heterogeneity in (female BALB/c × male DBA/2) F1 mice (CDF1) is described and commented on. Although mouse antimicrococcus sera agglutinate cultured L1210 cells in vitro, micrococcus vaccinated mice never showed any anti-leukaemia immunoprotection. However, vaccinated mice, given booster doses of 1 mg heat-killed Micrococcus lysodeikticus on days 4, 8 and 12 after a tumour challenge of 104 cells, were considerably protected (P Our results underline the idea that, after establishment of tumour growth, the humoral antitumour immunity requires an adequate trigger of immunocompetent effector cells to be operationally effective.

Comparison between the effects of Micrococcus Lysodeikticus, bacterial cell wall and related polysaccharides in the non-specific tumour immunotherapy of Ehrlich ascites tumour growth

Abstract By analyzing, at different times after grafting 300,000 Ehrlich ascites cells to BALB/c mice, the primary immune response to 2.10 8 sheep erythrocytes, the carcinoma monitored immunosuppression was outlined. The fact that murine ascites fluid contains less immunoglobulins and less pre-albumin migrating components than serum of tumour bearing mice suggests that the tumour site may be the consuming focus. When different treatment schedules were assayed, we found that the intraperitoneal route (intratumoral) was better than other treatment routes: for 30,000 initially grafted tumour cells, i.p. treatment with 0.08 and 0.4 mg heat-killed Micrococcus on day 1, 3, 5, 7 and 9 resulted respectively in a 120 and 148% increase in mean life span over control mice and 50 and 20% of long-term survivors were recorded. However, the administration of 1 mg Micrococcus on days 1, 5, 9 and 12 after grafting 300,000 Ehrlich carcinoma cells considerably enhanced tumour growth. The administration of 1 mg of Micrococcus, cell wall, cell wall conjugated chitin, chitin and zymosan A on days 1, 2, 3, 4 and 5 resulted in a 71, 45, 29.5, 68 and 82% increase in mean life span over control mice and 30, 20 and 30% of long-term survivors were recorded for chitin, cell wall conjugated chitin and zymosan A respectively.