Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Primary and secondary anti-sheep red blood cell antibody formation by BALB/c mice was reduced by administration of cyclophosphamide shortly following primary and secondary immunization. Treatment with the MER tubercle bacillus fraction by several schedules potentiated markedly and significantly the recovery of ability to respond to the antigen, as indicated by the production of specific plaque-forming cells and free hemagglutinating antibody.     

Effect of treatment with the MER tubercle bacillus fraction on syngeneic plasma cell tumors of BALB/c mice

Summary MER administered prophylactically prolonged the survival of BALB/c mice challenged with transplants of syngeneic plasmacytomas to a moderate but significant extent. In contrast, MER exerted little therapeutic action when given alone at or after tumor implantation.Combined treatment, with MER introduced prophylactically and cyclophopshamide (CY) after tumor implantation, decreased the incidence of recurrence of one of the tumors tested (MPC-11 NP, a non-myeloma protein producer) and prolonged host survival significantly as compared with animals subjected to CY therapy alone. When, instead, MER was introduced at the time of tumor challenge or thereafter to animals also treated with CY, the therapeutic response was not appreciably different from that of mice under therapy with CY only. With regard to the second plasmacytoma (MPC-11 P, a myeloma protein producer), mice treated with CY and given MER prior to or after challenge showed similar responses to animals given chemotherapy only; when MER was injected at the time of challenge and CY thereafter, the chemoimmunotherapy was somewhat inferior to chemotherapy alone.This work was supported by US Public Health Service Contract NO1-CM-12127, and by grants from Mrs. J. H. Hazen, Ruth Estrin Goldberg Memorial for Cancer Research, Leukemia Research Foundation, Inc., Concern Foundation, Inc., and Mr. and Mrs. M. Gordon     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Development of cellular anti-tumor immunity in chickens bearing tumors induced by Rous sarcoma virus.

The technique of peripheral lymphocyte stimulation in response to antigen was used to demonstrate the existence of cell-mediated anti-tumor immunity in chickens bearing tumors induced by avian sarcoma viruses. The expression of this anti-tumor response against virus-containing transformed cell culture supernatant fluids and cell extracts varied among animals in terms of time after inoculation with the oncogenic agent. Animals whose tumors had completely regressed rapidly lost the ability to mount such continuing immunity. In addition, we found evidence that some normal animals express endogenous levels of natural immunity against neoplasms induced by these viruses and/or against the viruses themselves.     

Effects of the methanol extraction residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. III. Consequence of prior sensitization to MER

Mice repeatedly immunized with the methanol extraction residue fraction of tubercle bacilli (MER) in incomplete Freund's adjuvant produced high titers of circulating antibodies against MER, as assessed by the enzyme-linked immunosorbent assay (ELISA) method. Spleen cells derived from these animals failed to respond to the usual nonspecific immunopotentiating influence of MER on the primary production of antibodies (generation of specific plaque-forming cells) in vitro to sheep red blood cells. The defect was expressed by B lymphocytes and splenic macrophages, but not by splenic T lymphocytes or peritoneal exudate macrophagic cells. Impaired responsiveness by spleen cells from MER-immunized animals to nonspecific immunostimulation was also expressed with regard to another, unrelated biological response modifier, lipopolysaccharide. There was no impairment of responsiveness to polyclonal mitogenic stimulation. Possible mechanisms of the effects described are discussed.     

Reversal by the MER tubercle bacillus fraction of the suppressive effects of heterologous antilymphocytic serum (ALS) on the allograft reactivity of mice

Treatment of immunized C57B1 mice with the MER fraction of BCG prevented wholly or in large part the strong immunosuppressive effects elicited by heterologous ALS against allograft reactivity, as measured by the in vitro cytotoxic activity of spleen cells against the immunizing allogeneic cells (a BALB/c plasmacytoma). Moreover, the heightened cytotoxic capacity of spleen cells bestowed by treatment of the donor with MER was manifested in some instances despite exposure to ALS. Spleen cells from MER-treated hosts were also more refractory to the direct cytotoxic action of ALS and complement in vitro.     

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Effect of treatment with the MER fraction of tubercle bacilli on hydrolytic lysozomal enzyme activity of mouse peritoneal macrophages

Treatment of $\text{BALB}\text{c}$ mice with MER by both the intraperitoneal and intravenous routes brought about a pronounced and significant elevation of hydrolytic lysozomal enzyme activity of their peritoneal macrophages for up to 30 days after administration of the agent.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.