Effect of immunization or cyclophosphamide treatment on growth of EL-4 leukemia cells in syngenic C57BL/6 mice.

Prolonged survival of C57BL/6 (B6) mice bearing syngenic EL-4 leukemia cells resulted from immunization with irradiated EL-4 cells on the day of inoculation of live leukemia cells. No prolongation of survival was observed if the irradiated cells were injected 6 or 13 days after live cell inoculation. Protection also was observed in EL-4-bearing mice that were treated with cyclophosphamide (CY) rather than by immunization, however, the protective effects were observed only when CY treatment was instituted 6 days after inoculation of live leukemia cells. No protection was seen when CY was administered on the day of, or 13 days after inoculation of live leukemia cells. In fact, administration of CY on the day live EL-4 cells were inoculated appeared to enhance the lethal effects of the tumor. In mice that underwent combined treatment, i.e., immunization and CY, prolonged survival was seen only in the group that received combined treatment 6 days after inoculation of live leukemia cells. No protection was seen in mice receiving combined treatment on the day of or 13 days after inoculation of live leukemia cells. The role that suppressor T cells might play in the observed results is discussed.

Effect of a single injection of cyclophosphamide on immunization therapy of metastases remaining after excision of dermal primary tumors in guinea pigs.

Guniea pigs with established (7 or 14 days old) syngeneic dermal tumors and metastases in the draining lymph nodes were unsuccessfully treated by excision of the dermal tumors and specific immunization. The vaccines consisted of killed BCG in oil in an emulsified form admixed with mitomycin C treated or irradiated tumor cells. The therapeutic failure to eradicate the metastases was overcome by an additional treatment with a single injection of cyclophosphamide prior to excision of the primary tumor and immunization. It is assumed that cyclophosphamide destroys suppressor elements in the tumor-bearing guinea pigs and, in this way, augments the therapeutic effects of specific immunization.

Immunotherapy of guinea pig line 10 hepatoma with nonliving BCG cells in aqueous medium.

Killed BCG cells suspended in 1.5% carboxymethylcellulose cured guinea pigs with established line 10 tumors in a high percentage of cases. The bacterial preparation of BCG in carboxymethylcellulose displayed a stronger tumor regressive activity and the process of healing was accelerated when endotoxin from a rough (Re) strain of Salmonella typhimurium was added to the BCG bacilli.

Treatment of mycosis fungoides with heat-killed BCG and cord factor.

6 patients with mycosis fungoides in different stages of the disease were successfully treated with intralesional injections of 1% mineral oil emulsions of killed BCG and cord factor, and/or topical application of an ointment containing killed BCG and cord factor.

Stimulation of macrophages by cord factor and by heat-killed and living BCG.

Trehalose-6,6'-dimycolate (cord factor; CF) injected into the peritoneal cavity of mice induced stimulation of the peritoneal macrophages, evidenced both by increased activity of the lysosomal enzyme, acid phosphatase, and by increased phagocytosis of Listeria monocytogenes. The increase in enzyme activity and phagocytosis was similar to that induced by killed or living BCG. When administered intravenously, CF or BCG did not induce stimulation of peritoneal macrophages. CF when added to tissue cultures of normal peritoneal cells did not induce increased acid phosphatase activity or increased phagocytosis of L. monocytogenes.

Chemotactic response of leukocytes to cord factor (trehalose-6,6'-dimycolate).

Cord factor (trehalose-6,6'-dimycolate), a glycolipid extractable from myocobacteria, was chemotactic for peritoneal cells of mice at concentrations from 5 to 25 mug/ml medium, as well as for peripheral white blood cells of mice and humans. At higher concentrations, presumably only macrophages were attracted.

Nonspecific resistance against infection with Salmonella typhi and Salmonella typhimurium induced in mice by cord factor (trehalose-6,6'-dimycolate) and its analogues.

Mice pretreated intraperitoneally with trehalose-6,6'-dimycolate (cord factor) were protected against an intraperitoneal challenge with Salmonella typhi strain Ty2 or Salmonella typhimurium strain SR 11. The nonspecific resistance to S. typhi and S. typhimurium was still detectable 7 and 14 days, respectively, after administration of cord factor. The effect of cord factor was local. Synthetic analogues of cord factor--trehalose-6,6'-dipalmitate and trehalose monopalmitate--also induced nonspecific resistance to the above virulent bacteria. The results are discussed.

Immunotherapy of cancer with nonliving mycobacteria and cord factor (trehalose-6,6'-dimycolate) in aqueous medium.

Heat-killed BCG, cord factor (trehalose-6,6'-dimycolate), or killed BCG plus cord factor in aqueous medium, admixed with tumor cells and injected into the skin of guinea pigs, inhibited the growth of a hepatocellular carcinoma. Intralesional administration of killed BCG or Mycobacterium kansasii coated with cord factor, in the same medium, caused regression of established tumors in 48 and 45% of the treated animals, respectively.

Stimulation of lymphocyte proliferation by killed mycobacteria and other bacterial species.

Killed mycobacteria and some mycobacterial fractions induced spleen cells from normal C3H mice to incorporate tritiated thymidine to a relatively high degree. Thymocytes under the same conditions were not activated. However, incorporation of thymidine was significantly increased when mixtures of thymus and spleen cells were cultured in the presence of the inducers. A similar activation of spleen cells was displayed in the presence of gram-positive and gram-negative non-mycobacterial species.

Immunotherapy and vaccination against cancer with non-living BCG and cord factor (trehalose-6,6'-dimycolate).

A vaccine containing non-living BCG and living tumour cells prevented the growth of 106 tumour cells introduced distally into the skin of guinea-pigs at the time of vaccination. A similar effect was achieved when living tumour cells of the vaccine were replaced by tumour cells pretreated with mitomycin C. The efficacy of the vaccine was significantly increased when cord factor was added to the vaccination mixture. The percentage of cures was about 88% (17 cases) in experiments in which the vaccine contained living tumour cells, 100% (10 cases) when mitomycin C-treated cells were used. The significance and implications of the above findings are discussed.

Immunotherapy of Cancer with Nonliving BCG and Fractions Derived from Mycobacteria: Role of Cord Factor (Trehalose-6, 6'-Dimycolate) in Tumor Regression.

Delipidated and deproteinized cell walls from Mycobacterium tuberculosis H37Ra suspended in 1.25% mineral oil emulsion cured established tumors in the skin and metastases in draining lymph nodes of guinea pigs (strain 2) after intratumoral administration in 33% of the cases examined. This was increased to 83% when a mixture of cord factor and the delipidated cell walls was used. A similarly high percentage of cures was obtained after administration of lyophilized, killed BCG alone or with addition of cord factor in 1% mineral oil emulsion. Lyophilized, killed BCG dispersed in a solution of tocopherol acetate in peanut oil or in peanut oil alone showed a limited tumor-regressive activity (36%). However, a mixture of BCG and cord factor suspended in the above media cured 80 and 69% of the treated animals, respectively.

Suppression of growth of Ehrlich ascites tumor cells in mice by trehalose-6,6'-dimycolate (cord factor) and BCG.

Growth of Ehrlich ascites tumor cells in mice pretreated with cord factor was compared to growth of the tumor cells after pretreatment with Calmette-Guérin bacilli. Growth of Ehrlich ascites cells was strongly inhibited in the peritoneal cavities of mice pretreated with 80 mug of cord factor. The median survival time of the animals was prolonged (70 versus 17 days), and 40% of the mice survived more than 90 days. Tumor suppression was still detectable 36 days after administration of cord factor. The effect of cord factor was local. Comparable results were obtained with living or killed Calmette-Guérin bacilli. The results are discussed.

Granulomatous hypersensitivity to trehalose-6,6'-dimycolate (cord factor) in mice infected with BCG.

Cord factor in the form of emulsion is unable to sensitize mice to react with a more extensive granulomatous response to a subsequent challenge with the same substance. Mice infected with BCG bacilli are sensitized to cord factor. Such animals react to administration of cord factor with a very extensive granulomatous response, much stronger than normal ones. This hypersensitivity seems to be specific and is distinct from delayed hypersensitivity to PPD. PPD, administered to mice sensitized with BCG bacilli, has no effect on the granulomatous response. Our findings and their importance are discussed.

Suppression of urethan-induced lung adenomas in mice treated with trehalose-6,6-dimycolate (cord factor) and living bacillus Calmette Guérin.

Trehalose-6,6-dimycolate (cord factor), a glycolipid from mycobacteria, suppressed the development of urethan-induced tumors in the lungs of mice to a similar degree as living Mycobacterium bovis (strain BCG) bacilli. The inhibition was apparently due to the host cellular reaction caused locally by cord factor.

Cellular reaction in the footpad and draining lymph nodes of mice induced by mycobacterial fractions and BCG bacilli.

Ten micrograms of trehalose-6, 6'-dimycolate (cord factor), injected into the footpad of mice, induced histological changes similar to those following injection of living BCG bacilli. Both materials induced in the draining lymph nodes the formation of granulomas composed of epitheloid cells, macrophages, and small numbers of lymphocytes. Apart from the granulomatous inflammatory process, marked hyperplasia of the lymphoid tissue in the paracortical zone of the nodes and accumulations of macrophages were evident. In some cases, the macrophages were very numerous and replaced part of the lymphoid tissue. Compared to cord factor, wax D showed weak granulomagenic activity. Only slight and transient inflammation was found in the footpads as well as transient slight lymphoid hyperplasia. Wax D also induced small accumulations of macrophages. Complete Freund's adjuvant induced, under the same experimental conditions, large accumulations of macrophages in the draining lymphnode and lymphoid hyperplasia in the paracortical zone. No cellular reaction was seen in the liver, spleen, and lungs after injection of cord factor and BCG into the footpads of the animals. The results and implications are discussed.

Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions.

Ten micrograms of trehalose-6,6' -dimycolate (cord factor) injected into the footpads of mice increased the antibody response to sheep red blood cells (SRBC) subsequently injected into the same sites. There is a relationship between the antibody response and the cellular reaction induced locally and in the draining lymph nodes by cord factor, as judged by a much weaker response when antigen is injected into the contralateral footpads. The time intervals between injection of cord factor and antigen were from 5 to 20 days. A similar increased antibody response to SRBC was evident after preliminary administration of Freund's complete adjuvant or living BCG bacilli into the footpads. There was no increased antibody response in mice pretreated with living BCG or Freund's adjuvant to SRBC injected into the contralateral footpads. Administration of wax D from human strains Peurois and Test was without any effect. Administration of SRBC emulsified in incomplete Freund's adjuvant containing 5 mug of cord factor induced a very strong antibody response in the mice as compared to that after injection of the same amount of antigen in incomplete Freund's adjuvant containing wax D or mycobacteria. The results are discussed.