Nicotinamide-adenine dinucleotide in tubercle bacilli exposed to isoniazid.

There is a decrease in the content of nicotinamide-adenine dinucleotide of tubercle bacilli grown in the presence of isoniazid. In extracts of tubercle bacilli, the activity of nicotinamide-adenine dinucleotidase is nil or very small; after incubation with the drug the enzyme becomes active. Isoniazid also increases the activity of the enzyme after it is partially activated by heating. There may be a correlation between the capacity of isoniazid to activate the enzyme and the decrease in the dinucleotide content of the tubercle bacilli.

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.

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.

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.

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.

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.

Acute granulomatous response produced in mice by trehalose-6,6-dimycolate.

Trehalose-6,6-dimycolate, a toxic lipid derived from tubercle bacilli, injected intravenously into mice in 10% emulsions of Bayol F caused a three- to fourfold increase in the weights of lungs compared with those of mice injected with emulsions of Bayol F alone or emulsions of Wax D. This change is due to a heavy infiltration of the lungs with granulocytes and large numbers of macrophages, with formation in some of the infiltrates of round foci, resembling granulomas.

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.

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.

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.

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.

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.

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.

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.