Effect of metabolic inhibitors on the formation of antibody to sheep erythrocytes, on development of delayed hypersensitivity, and on the immune response to infection with Mycobacterium tuberculosis in mice.

The effect of a number of metabolic inhibitors was determined on: (i) the production of cellular immunity to infection with Mycobacterium tuberculosis in mice by vaccination with mycobacterial ribonucleic acid (RNA), (Ii) the production of cellular immunity to infection with M. tuberculosis in mice with viable H37Ra cells, (iii) the induction of antibody formation to sheep erythrocytes, and (iv) the induction of delayed hypersensitivity in mice to purified protein derivative. The pattern of inhibition produced by metabolic inhibitors on cellular immunity to infection with M. tuberculosis produced by mycobacterial RNA was entirely different from the pattern of inhibition produced by the same metabolic inhibitors on antibody formation to sheep erythrocytes. The effect of the metabolic inhibitors on the induction of delayed hypersensitivity to purified protein derivative did not correlate with the pattern of inhibition produced by the same compounds on antibody formation or on the development of immunity produced by mycobacterial RNA. Cellular immunity to infection produced in mice by viable H37Ra cells was not reduced by any of the metabolic inhibitors except actinomycin D. The possible reasons for the lack of activity of the metabolic inhibitors on the immune response to viable H37Ra cells and the lack of correlation with the pattern of inhibition found in mice vaccinated with mycobacterial RNA is discussed.

Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge.

Mycobacterial ribonucleic acid: comparison with mycobacterial cell wall fractions for regression of murine tumor growth.

Mycobacterial ribonucleic acid (RNA) and cell wall skeleton fraction isolated from H37Ra caused P-815 mastocytoma regression in DBA/2 mice provided the animals were presensitized with freshly harvested living H37Ra cells. In the absence of presensitization, only the RNA fraction inhibited. Cell wall skeleton fraction, under these conditions, stimulated tumor growth. Cell wall lipids (from H37Ra) added to H37Ra cell wall skeleton fraction did not increase the inhibitory activity of cell wall skeleton fraction alone. Mycobacterial RNA appeared to be an effective inhibitor of P-815 mastocytoma metastases as shown by (i) the inhibition of a second footpad lesion distant from the one treated and (ii) increase in survival time.

Effect of rifampin on immunity to tuberculosis and on delayed hypersensitivity to purified protein derivative.

Mice vaccinated with mycobacterial ribonucleic acid (RNA) produced a high immune response and did not develop delayed hypersensitivity to purified protein derivative (PPD), and rifampin had no effect on the immune response. Mice vaccinated with viable H37Ra cells produced a high immune response and did develop delayed hypersensitivity to PPD. Rifampin had no effect on this immune response, but reduced the footpad reactions to PPD. Both mycobacterial RNA and poly(A:U) served as adjuvants for induction of hypersensitivity to PPD. This hypersensitivity was reduced by the administration of rifampin. Rifampin had no effect on the production of mycobacterial growth inhibitory factor, which is produced following vaccination of mice with mycobacterial RNA or viable H37Ra cells. Rifampin had no effect on the nonspecific phase of the granulomatous response, but did inhibit the secondary allergic phase of this response. The action, therefore, of rifampin that inhibits the induction of delayed hypersensitivity but had no effect on the immune responses against tuberculosis leads to a separation of tuberculin hypersensitivity from cellular immunity to tuberculosis.

Mycobacterial RNA. A comparison with intact mycobacteria for suppression of murine tumor growth.

Our experimental data imply the following: (1) An RNA-rich fraction isolated from H37Ra, and intact H37Ra, are effective agents for suppression of tumor growth. (2) For suppression of two chemically-induced tumors with both RNA and H37Ra, direct contact with tumor is necessary. This may not be the case with other tumors in different strains of syngeneic mice. (3) The host's immunological system is a necessary component for inhibition of tumors by our test materials since there is no tumor suppression in immunologically impaired mice.

Conditions for production, and some characteristics, of mycobacterial growth inhibitory factor produced by spleen cells from mice immunized with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis.

Mycobacterial growth inhibitory factor (MycoIF), found in supernatant fluids of mouse spleen cell cultures that have been stimulated in vitro with homologous antigen, inhibited the intracellular multiplication of virulent tubercle bacilli within normal mouse peritoneal macrophages in vitro. Antigenically stimulated H37Ra-immunized mouse spleen cells required 72 h of incubation to produce supernatant fluids that would cause intracellular inhibition. Supernatant fluids from 48-h mouse spleen cell cultures were not able to produce intracellular inhibition. Investigation of the culture conditions showed that at lease 1.0% human serum was required in the tissue culture medium for the production of MycoIF by spleen cells from immunized mice. MycoIF activity was noted only in supernatant fluids from spleen cell cultures incubated with antigen for 72 h. MycoIF was nondialyzable and unaffected by freezing, lyophilization, or incubation at 60 C for 30 min. However, MycoIF was inactivated after incubation at 80 C for 30 min. MycoIF was unaffected by low hydrogen ion concentrations (pH 7 to 12), but exposure to higher hydrogen ion concentrations (pH 6, pH 5) significantly decrease MycoIF activity, and exposure to pH 4 to 2 abolished all activity. Supernatant fluids diluted 1:32 were still able to produce significant intracellular inhibition of growth of virulent tubercle bacilli.

Molecular weight and other characteristics of mycobacterial growth inhibitory factor produced by spleen cells obtained from mice immunized with viable attenuated mycobacterial cells.

Exposure of mycobacterial growth inhibitory factor (MycoIF) to trypsin, chymotrypsin, or neuraminidase decrease its ability to produce intracellular inhibition of mycobacterial growth within macrophages, suggesting that MycoIF was a glycoprotein. MycoIF was unaffected by deoxyribonuclease or ribonuclease. Supernatant fluids from antigenically stimulated H37Ra-immunized mouse spleen cells exposed to puromycin were unable to produce significant intracellular inhibition. This indicated that the presence of MycoIF activity in supernatant fluids required protein synthesis. The filtration of MycoIF-containing supernatant fluids on Sephadex G-150 demonstrated that significant MycoIF activity appeared only in those fractions which eluted on the downward side of the serum albumin peak. Based on protein standards filtered through the Sephadex gel, the molecular weight of MycoIF was calculated to be between 20,000 and 35,000. These calculations assumed that MycoIF is a globular protein. Attempts to purify MycoIF by anion exchange chromatography (diethylaminoethylcellulose) was not successful.

Macrophage migration inhibitory activity of mycobacterial growth inhibitory factor and the effect of a number of factors on mycobacterial growth inhibitory factor activity.

Mycobacterial growth inhibitory factor (MycoIF), which inhibits the intracellular multiplication of virulent tubercle bacilli within normal peritoneal macrophages in vitro, was tested for its ability to inhibit the migration of normal peritoneal exudate cells. The migration of peritoneal exudate cells was not inhibited by MycoIF. It was also shown that normal peritoneal macrophages infected with virulent Mycobacterium tuberculosis, strain H37Rv, required 72 h of incubation with spleen cell culture supernatant fluids containing MycoIF in order to inhibit intracellular bacillary multiplication. Treatment of infected macrophages with trypsin before their exposure to MycoIF abolished the ability of MycoIF to inhibit intracellular mutiplication of tubercle bacilli. Incubation of infected macrophages with goat anti-mouse globulin before their exposure to MycoIF also blocked the action of MycoIF.

The induction of delayed hypersensitivity in guinea pigs to poly U and poly A:U.

Guinea pigs were sensitized to poly U and poly A:U so that subsequent stimulation of spleen cells from these immunized animals with poly U and poly A:U resulted in the production of migration inhibitory factor (MIF). MIF was also produced when spleen cells from animals immunized with poly A:U were cultured in the presence of mycobacterial RNA or whole viable H37 Ra cells. Negative dermal reactions were observed when guinea pigs immunized with poly A, poly A:U were skin tested wtih these same synthetic nucleotides.

The adjuvant activity of mycobacterial RNA preparations and synthetic polynucleotides for induction of delayed hypersensitivity to purified protein derivative in guinea pigs.

The adjuvant activity of mycobacterial RNA and synthetic polynucleotides for the induction of delayed hypersensitivity to PPD was determined. It was shown that when mycobacterial RNA or synthetic polynucleotides are injected together with purified protein derivative (PPD), delayed hypersensitivity to PPD developed as compared to no detectable delayed response when PPD was administered alone without adjuvant into guinea pigs. Four different criteria were employed to detect delayed hypersensitivity responses. These were the time, appearance, and magnitude of dermal reactions, histologic examination of dermal sections, passive transfer of sensitivity with sensitized spleen cells and the elaboration of migration inhibitory factor (MIF) by sensitized spleen cells. When synthetic polynucleotides were used as adjuvants and were injected into guinea pigs in combination with PPD, dermal reactions as well ad MIF assays gave evidence that these animals exhibited delayed-type hypersensitivity. Poly U alone exhibited adjuvant activity for induction of delayed hypersensitivity to PPD. Trypsin and pronase treatment did not affect the adjuvant activity of mycobacterial RNA whereas KOH treatment completely abolished any adjuvant effect, suggesting that ribosomal protein did not contribute to the adjuvant characteristics of mycobacterial RNA. Titration experiments indicated that the adjuvant activity of mycobacterial RNA was greater than that of poly A:U.

Nonspecific inhibition of growth of intracellular Listeria monocytogenes by lymphocyte culture products.

Supernatant fluids from mycobacteria-immune and mitogen-stimulated lymphocyte cultures were able to inhibit the intracellular growth of listeria in listeria-infected macrophages.

Relationship between tuberculin hypersensitivity and cellular immunity to infection in mice vaccinated with viable attenuated Mycobacterial cells or with Mycobacterial ribonucleic acid preparations.

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.