Lysosomal enzymes and microbicidal capacity of activated macrophage.

A relation was sought between acid phosphatase contents and the presence of tubercle bacilli inside the peritoneal exudate cells (PEC) of normal guinea pigs and those immunized with BCG. This was done to investigate the role lysosomal enzymes play in the microbicidal capacity of the cell. In both normal and immune animals tubercle bacilli were present only in those PEC that contained acid phosphatase. Cells without acid phosphatase did not contain bacilli. Thus, only activated cells ingested bacilli. Under the conditions of these experiments, macrophage activation, as indicated by the presence of acid phosphatase, was not related to the immune status of the animal. Similarly, stimulation by ingestion of tubercle bacilli was not significant. Also, the number of acid phosphatase grains/cell did not influence the number of bacilli/cell. Thus, the acid phosphatase content of the cell did not correlate with the number of bacilli inside the cell. It was concluded that acid phosphatase may not be one of the factors that contribute to the microbicidal capacity of the cell.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Granuloma Formation Induced in Mice by Chemically Defined Mycobacterial Fractions

Amounts of trehalose-6,6-dimycolate as small as 1 to 5 mug can, after intravenous injection, induce in the lungs of mice formation of tubercles in which the cellular composition is indistinguishable from that in tubercles formed after an infection with living BCG bacilli. The strongest cellular response in mice was induced by cord factor from Mycobacterium kansasii; the weakest was induced by cord factor from the BCG strain of M. bovis. It was found that three intravenous injections of cord factor induced a more extensive cellular response than did one injection of the same total amount of cord factor. Mice treated intravenously with cord factor were protected against an intravenous challenge with the virulent H37Rv strain of M. tuberculosis. The cellular response in the lungs of mice to intraperitoneal injections of living BCG and cord factor was very weak compared with that after intravenous injections. Intraperitoneal vaccination of mice with cord factor did not protect the mice against a challenge with virulent tubercle bacilli. Mice vaccinated intraperitoneally with BCG were immunized although no granulomas, or very few, were present in the lungs at the time of the challenge. The significance of the cellular response induced by cord factor is discussed.     

Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).     

Probability Distribution of Drug-Resistant Mutants in Unselected Populations of Mycobacterium tuberculosis

The fluctuation test shows that Mycobacterium tuberculosis mutates to resistance to isoniazid, streptomycin, ethambutol and rifampin spontaneously and at random. The average mutation rates for the drugs, in the same order, were calculated to be 2.56 × 10⁻⁸, 2.95 × 10⁻⁸, 10⁻⁷, and 2.25 × 10⁻¹⁰ mutation per bacterium per generation. The relatively high mutation rate to ethambutol resistance and the low mutation rate to rifampin resistance were confirmed by analyzing the increase in the proportion of mutants with time in a growing population of the tubercle bacilli. The highest proportions of mutants to be expected in unselected populations of the tubercle bacilli were calculated from the results of fluctuation tests.     

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.     

Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4⁺ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.     

Experimental study on the relapse of tuberculosis after the termination of antituberculous chemotherapy using immunodeficient nude mice.

The effect of cellular immunity induced by tuberculous infection on bacteriological relapse after the termination of antituberculous chemotherapy was studied experimentally using immunodeficient nu/nu mice and immunocompetent dd mice. The efficacy of intensive chemotherapy was excellent even in nu/nu mice; tubercle bacilli in the organs decreased below the detectable limit, but formidable regrowth of bacilli was seen after the termination of chemotherapy. On the other hand, in the case of dd mice that established antituberculous cellular immunity through tuberculous infection, bacteriological relapse was generally very slight. It was concluded that bacteriological relapse was related closely with the established cellular immunity induced by the infected tubercle bacilli.     

Pulmonary Tuberculosis Apparently Caused by the Avian Tubercle Bacillus

Avian tubercle bacilli were repeatedly isolated from the sputum of a 65-year-old man who had cavitary disease in the upper lobe of the right lung. The clinical picture resembled that of pulmonary tuberculosis caused by the human type of tubercle bacilli, but the response to antituberculosis chemotherapy was unsatisfactory and the patient''s sputum remained positive. The bacilli were markedly pathogenic to chickens and rabbits but failed to produce progressive disease in guinea pigs. This is in accord with the properties of tubercle bacilli of the avian type.     

Inflammatory responses in experimental tuberculosis pleurisy

A model of tuberculous pleurisy in New Zealand white rabbits was developed to describe the sequential cellular and biochemical changes in pleural fluid. Bacille Calmette-Guérin (BCG) in 4 X 10(7) colony-forming units was introduced into the right pleural space of rabbits previously sensitized by intradermal BCG. Pleural fluid was obtained via serial thoracenteses. A normal-pH, normal-glucose, exudative effusion was seen through 144 hours. Polymorphonuclear leukocytes were the first cells to respond to the introduction of tubercle bacilli in the pleural space; they remained the predominant cell for the first 24 hours and were followed by macrophages, which peaked at 96 hours, and then by lymphocytes. Numerous granulomata were observed on both the visceral and parietal pleura ten days following intrapleural instillation of BCG. We propose that the polymorphonuclear leukocyte influx is not a nonspecific response to pleural injury and that such a leukocyte response, either itself or through its interaction with the macrophage, plays a role in host defense mechanisms against the tubercle bacilli.     

Immunogenicity of viable tumor cells

Summary The immunogenicity of KMT-17 fibrosarcoma cells which had been xenogenized by infection with FV was compared to that of KMT-17 cells which had been admixed with BCG. We report here that 10⁵ and 10⁶ KMT-17 cells also grew progressively to kill rats, but when 10⁵ KMT-17 cells were administered with BCG the tumor cells did not grow in the majority of rats. The strength of immunogenicity (ETD₅₀), as measured by the number of immunizing cells required for a suppression of growth of 10⁷ KMT-17 cells in 50% of the rats, was 2.1×10³ for FV-KMT-17 and 36.3×10³ for BCG+KMT-17. The tumor cell dose (LTD₅₀) which was required to kill 50% of the rats immunized with 10⁵ FV-KMT-17 was more than 10,000 times higher than that found in normal rats, whereas the number of tumor cells required to kill 50% of the rats immunized with the same number of BCG+KMT-17 was only 3,680 times higher than the amount found in normal rats. Thus the immunogenicity of FV-KMT-17 is much stronger than that of BCG+KMT-17.The difference in immunogenicity between the two vaccines was also observed in the tumor-neutralizing activities of spleen cells obtained from rats which had been immunized with both vaccines, as measured by a Winn assay. Moreover, the antitumor activity of spleen cells from rats immunized with FV-KMT-17 was concentrated in the carrageenan-resistant and plastic nonadherent cells, while that of spleen cells from rats immunized with BCG+KMT-17 was observed in carrageenan-sensitive and plastic adherent cells as well as in nonadherent cells. The involvement of different effector cells indicates that different mechanisms operate in the antitumor resistance in rats immunized with either FV-KMT-17 or BCG+KMT-17. Abbreviations used: FV, Friend leukemia virus; FV-KMT-17, Friend leukemia virus infected KMT-17 cells; EDT₅₀, a 50% effective tumor dose; LTD₅₀, a 50% lethal tumor dose     

Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.     

Efficacy of a Vaccine Formula against Tuberculosis in Cattle

“Test-and-slaughter” has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10⁶ CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10⁵ CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.     

Potentiation and modulation of the immune response of guinea pigs to poorly immunogenic protein-hapten conjugates by pretreatment with the MER fraction of attenuated tubercle bacilli

The MER fraction of attenuated tubercle bacilli of the BCG strain was shown capable of stimulating and modulating the immunological responsiveness of guinea pigs to immunization with DNP conjugates of allogeneic globulin (DNP-GPG) and xenogeneic albumin (DNP-HSA). These antigens are very poorly immunogenic and fail to evoke detectable immune responses following single administration alone.When incorporated in incomplete Freund's adjuvant (IFA) together with the conjugates, MER could substitute for whole tubercle bacilli in the adjuvant mixture, and cause the conjugates to evoke both cellular and humoral reactivity, the former indicated by the development of skin reactions of delayed type (DH) to test injections of the antigens, the latter by the formation of humoral antibodies detected by an indirect hemagglutination (HA) test. When administered in saline together with antigen, MER was ineffective.Pretreatment with MER by any of several different routes 7 or 14 days prior to sensitization enabled a large number of the animals to respond with either DH, circulating antibody formation, or both. Under similar circumstances, pretreatment with Freund's complete adjuvant (FCA) elicited no such preparatory effect. In order to be efficacious in pretreatment, MER had to be given in a saline suspension; activity was lost when it was applied in IFA.MER pretreatment modulated the immune response to subsequent sensitization with the conjugates preferentially towards DH or antibody production, depending on the parameters of treatment and specific immunization. It appeared that when the specific immunogenic stimulus was weak, pretreatment with MER strongly favored DH.     

In vitro studies on the mechanism of acquired resistance to tuberculous infection. II. The effects of the culture supernatants of specifically stimulated-sensitized lymphocytes on the growth of tubercle bacilli within macrophages.

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

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.