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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.

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.

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.

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.

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.

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.

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.

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

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.

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 (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.

The guinea pig migration inhibition technique, an accepted in vitro correlate of delayed hypersensitivity, has been adapted to a murine system. Peritoneal exudate cells from CF-1 mice vaccinated with viable cells of the H37Ra strain of Mycobacterium tuberculosis were inhibited in vitro by purified protein derivative (PPD) or whole H37Ra microorganisms. Peritoneal exudate cells from the inbred C57Bl/6 mice immunized with H37Ra cells also were inhibited in vitro by PPD or whole H37Ra microorganisms. Migration inhibitory factor (MIF) was produced by splenic lymphocytes from the H37Ra-immunized C57Bl/6 mice when incubated with either antigen. Intravenous injection of PPD or viable H37Ra organisms into H37Ra mice resulted in MIF production in vitro by splenic lymphocytes without further antigenic stimulation. Peritoneal exudate cells from nonimmunized C57Bl/6 mice and supernatant fluids from cultures of lymphocytes from nonimmunized C57Bl/6 mice were not inhibited in the presence of antigen. The production of MIF by splenic lymphocytes from immunized C57Bl/6 mice depended upon the conditions under which the lymphocytes were cultured, the time of exposure to antigen (3 days), the use of a higher concentration of PPD for stimulation of lymphocytes than that required for guinea pig cells, and also the use of cells from a highly inbred mouse strain.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate. J. Bacteriol. 91:2139-2145. 1966.-Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 x g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 mug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10(-4)m MgCl(2) to the final diluent increased immunogenic activity, whereas larger concentrations (10(-3)m) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Two-week-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.

Layton, Herbert W. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of dietary factors upon the resistance of albino mice to experimental infection with Mycobacterium tuberculosis. J. Bacteriol. 90:958-964. 1965.-Each of the major nutritional components of a synthetic diet was quantitatively altered to determine its effect upon the resistance of albino mice to infection with Mycobacterium tuberculosis. The animals were challenged after the first 2 weeks of feeding, and the percentage that survived acute death was determined statistically. The level of protein which provided the greatest percentage of survival was 30%; increases or decreases from this level were detrimental. The optimal fat level was found to be 5% for either corn oil or coconut oil. Survival decreased when greater amounts of oil were added, and this effect was especially marked for 40% coconut oil. Vitamin A enhanced survival when increased from the normal level of 20,000 units per kg of diet to 160,000 units, but further increases were harmful. An amount of 75 g/kg of a vitamin B complex mixture afforded optimal resistance, but 25-g shifts in either direction lowered this resistance. Vitamin K-free diets resulted in high levels of survival, and addition of the vitamin resulted in proportional decreases in resistance.

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.

Data are presented which show that the syngeneic C57B1/6 mouse strain is far more susceptible to infection with Mycobacterium tuberculosis strain H37Rv than are the other allogeneic mouse strains used in this laboratory, particularly the Strong A and the CF-1 strains. Not only are the C57B1/6 mice more susceptible to tuberculous infection, but also they respond to infection more uniformly than do the allogeneic strains. C57B1/6 mice develop immunity to challenge with small infecting doses of the virulent H37Rv strain when they are vaccinated with viable cells of the attenuated H37Ra strain and with ribonucleic acid (RNA) preparations isolated from the H37Ra strain. Mice vaccinated with viable cells of the H37Ra strain, however, may die more rapidly than nonvaccinated mice when given a large infecting dose (1.0 mg). This accelerated type of disease is not seen in mice vaccinated with mycobacterial RNA. Since C57B1/6 mice are known to develop tuberculin hypersensitivity more readily than many other mouse strains, the possibility is discussed that the increased susceptibility to tuberculous infection of mice vaccinated with viable cells of the H37Ra strain may be due to a superimposition of a pronounced acute inflammatory response due to tuberculin hypersensitivity upon the infectious process. The several advantages that may be gained in the study of certain host-parasite interactions in tuberculosis by the use of a highly susceptible syngeneic mouse strain such as the C57B1/6 are discussed.

The five polybasic amines, methylated bovine serum albumin (MBSA), protamine sulfate, neomycin sulfate, streptomycin sulfate, and diethylaminoethyl-dextran (DEAE-dextran), were examined to determine their effects on the immunogenic activity of mycobacterial ribonucleic acid (RNA) preparations, and whether they could substitute for Freund incomplete adjuvant (FIA) by protecting the mycobacterial RNA in vivo. These compounds were either emulsified in FIA or not emulsified in FIA. Different results were obtained when these compounds, complexed with mycobacterial RNA, were emulsified in FIA. MBSA, in ratios of mycobacterial RNA-MBSA of 1:0.2 to 1:0.4, had no effect on immunogenic activity. However, when the ratio was increased to 1:1, 1:2, or to 1:4, marked inhibition of the immunogenic activity occurred. Protamine sulfate and neomycin sulfate also inhibited the immunogenic activity of mycobacterial RNA; however, neither streptomycin sulfate nor DEAE-dextran had any effect on immunogenic activity. Without being emulsified in FIA, these five polybasic amines, with the exception of DEAE-dextran, acted only as weak adjuvants for mycobacterial RNA and, therefore, could not be used as substitutes for FIA for the protection of mycobacterial RNA from host nucleases. DEAE-dextran, although not as effective as FIA, afforded some protection for the mycobacterial RNA. DEAE-dextran alone also produced a low degree of nonspecific immunity against tuberculosis. Since MBSA, protamine sulfate, and neomycin sulfate reduce the biological activity of mycobacterial RNA after complexing with it, it is probable that these compounds "mask" the immunogenic sites on the mycobacterial RNA structure.

Five to 20% linear sucrose gradients were used to obtain sedimentation patterns of mycobacterial ribosomes, ribosomal subfractions, and ribonucleic acid (RNA) preparations. Classical 70S ribosomes were obtained when 10(-1)m magnesium chloride was used. These, when dialyzed against 10(-4)m MgCl(2), yielded typical 50S, 30S, and smaller ribosomal subunits. The 30S subunits were the most immunogenic under these conditions. A ribosomal preparation containing subunits which varied from 2.5 to 40S was fractionated by collecting five fractions from a sucrose gradient; based upon the amount of nucleic acid present, the fraction containing the 40S particles was most immunogenic. Physical and chemical evidence suggested that mycobacterial RNA preparations extracted with 65% ethyl alcohol from the ribosomes and diluted in distilled water, were either double-stranded, or mostly double-helical, or had a highly organized secondary structure. This was based on the following observations. (i) Native RNA was resistant to trace amounts of ribonuclease. (ii) The approximate T(m) value in SSC buffer (0.15 m NaCl plus 0.015 m sodium citrate) was greater than 85 C and in 0.1 SSC buffer was 55 C; the RNA diluted in SSC buffer produced a hypochromic effect on cooling at room temperature. (iii) Formaldehyde, in the presence of SSC buffer, decreased the T(m) of the RNA to approximately 55 C, and there was no hypochromic effect on cooling. (iv) Formaldehyde did not increase the wavelength of maximal adsorption of the RNA. (v) The purine/pyrimidine ratio was close to one. (vi) The major peak of the RNA sedimented in the more dense zones of the sucrose gradients. There was a relationship between the sedimentation pattern obtained with the RNA-protein subunits on sucrose gradients and immunogenicity; several examples are given. RNA-protein complexes of approximately 14 to 20S, and occasionally 23S in the major peak, appeared to produce the highest immune response. Smaller RNA-protein complexes such as 6S, which were obtained when the RNA preparation was diluted in certain buffers, were much less immunogenic. This was confirmed by collecting five fractions from sucrose gradients and finding the third fraction (containing RNA-protein complexes approximately 15 to 16S) the most immunogenic. Immunogenic activity was apparently related to the structure of the RNA since it was maximal when the RNA appeared to be either double stranded, double helical, or had a highly organized structure.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.) and Guy P. Youmans. Effect of mycosuppressin on the respiration and growth of Mycobacterium tuberculosis. J. Bacteriol. 84:708-715. 1962.-A substance, called mycosuppressin, was found in the lungs of guinea pigs and rabbits vaccinated with BCG or with a particulate immunizing fraction isolated from mycobacterial cells, and was not found in lungs of unvaccinated animals. Mycosuppressin inhibited the endogenous respiration and the growth of the virulent H37Rv strain of Mycobacterium tuberculosis. It also inhibited the endogenous respiration of the avirulent H37Ra strain and the saprophyte, M. smegmatis, but it increased the respiration of M. phlei. The oxidation by the H37Rv strain of lactate, pyruvate, glycerol, and glucose was also inhibited. Cytochrome oxidase activity was suppressed. Mycosuppressin was most stable at pH 6 to 7. It was nondialyzable, stable at 98 C, and not affected by lyophilization or freezing. It was soluble, in alcohol and acetone, insoluble in ether and water. Under appropriate conditions, mycosuppressin combined with, or was adsorbed to, mycobacterial cells, and was inactivated by serum and bovine serum albumin. It did not inhibit but, instead increased markedly, the respiration of Staphylococcus aureus and Escherichia coli.