The effect of exogenous peroxidase on the evolution of murine leprosy.

Mycobacterium lepraemurium (MLM) is a successful parasite of murine macrophages; in vitro, this microorganism infects macrophages without triggering these cells' ability to produce either the reactive oxygen intermediaries (ROI) or the reactive nitrogen intermediaries (RNI), and ends up lodging within these cells, that, in addition, do not contain myeloperoxidase (MPO). In this study, we analyzed the effect of exogenous peroxidase on the evolution of murine leprosy. Bacilli were intraperitoneally injected, either alone (MLM) or precoated with horseradish peroxidase (MLM-PO), into two different groups of mice. At two-week intervals, the groups were blood-sampled to measure the levels of antibodies to protein- or lipid-MLM antigens. The extent of the disease was also assessed by looking at the histopathologic changes that occurred both in the liver and the spleen of the infected animals. We found that the animals injected with MLM-PO developed a disease that evolved at a slower pace than the disease that occurred in the animals injected with intact MLM. The difference between groups, both in terms of antibody levels and histological changes, was clearly evident at the intermediate stages of the disease (2 to 2.5 months), but was not so obvious at the more advanced stage of 3 months. Several possibilities to explain how the PO-coated bacilli might have regained their infectiousness are discussed. Lowering the infective dose of MLM and MLM-PO from 5 x 10(7) bacilli to 5 x 10(6) bacilli would, probably, have resulted in a different outcome of the disease: more extended in the MLM-group than in the MLM-PO group.

[For the growth of Mycobacterium lepraemurium in cell-free liquid medium, the key essential factor may be the pH (optimal 6.0-6.2) of the culture medium, rather than the presence of alpha-ketoglutaric acid].

Since the success of the multiplication of Mycobacterium lepraemurium in cell-free liquid medium by the author in 1972, various factors affecting its growth have been reported. In particular, it was emphasized that the key essential factor for the growth was alpha-ketoglutaric acid(alpha-kg). However, recent data now indicate that the critical factor for the growth of M. lepraemurium is not alpha-kg, but the optimal pH of the liquid culture medium, quite similar to the condition of egg-yolk solid medium. The recent experimental results clearly indicated that an abundant multiplication of M. lepraemurium took place in an acid liquid medium containing egg-yolk extract, without alpha-kg. This finding means that the cells of M. lepraemurium can multiply in a liquid medium, if the pH of the medium is adjusted to 6.0-6.2. The reason why the cells of M. lepraemurium multiplied in NC medium, and NC-5 medium, containing alpha-kg was that the pH of neutral liquid base medium of the NC or NC-5 medium, was adjusted to 6.0-6.2, optimal for the growth of M. lepraemurium by addition of the 10% alpha-kg (where 10% alpha-kg is strongly acidic; pH:1.22). Other supplements, such as cytochrome c, l-cysteine, and hemin, which are routinely added to the base medium of NC and NC-5 medium, have shown stimulatory effects on multiplication of M. lepraemurium, rather than a key essential effect. In addition, recent experimental data have suggested a second key essential component for growth present in egg-yolk extract.

Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.

Propagation of Mycobacterium lepraemurium on supplemented minimal medium and its experimental pathogenesis.

The splenic tissue of a mouse experimentally infected with M. lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M. lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc. This could be maintained, by serial passages in vitro with good growth. Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible. The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.

Alpha-ketoglutarate dehydrogenase in the in vitro-grown Mycobacterium lepraemurium.

The Hawaiian and Kumato strains of Mycobacterium lepraemurium were cultivated on Ogawa egg-yolk medium, and the alpha-ketoglutarate dehydrogenase activity was investigated in cell-free preparations of this mycobacterium. The enzymatic activity was mainly localized in the particulate fraction (150,000 x g pellet), and extremely low activity was found in the soluble fraction (150,000 x g supernatant). alpha-Ketoglutarate dehydrogenase was not stable; the activity was lost completely when the enzyme was kept at 45 degrees C for 1 hr or stored at -70 degrees C. The enzyme reduced only NAD+ but not NADP+ by alpha-ketoglutarate, indicating the presence of NAD(+)-dependent alpha-ketoglutarate dehydrogenase in cultivated M. lepraemurium.

Modulation of Mycobacterium lepraemurium growth in murine macrophages: beneficial effect of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor.

Mycobacterium lepraemurium grew progressively in monolayers of Proteose Peptone-elicited macrophages from C57BL/6 mice. Treatment of macrophage monolayers with gamma interferon led to an enhancement of growth of M. lepraemurium in macrophages. Treatment with tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor led to restriction of mycobacterial growth in macrophages.

Activated murine natural killer cells control growth of Mycobacterium lepraemurium in mouse macrophages; in vitro and in vivo evidence.

The role of natural killer cells (NK) in murine leprosy was investigated in vivo and in vitro. In a first set of experiments, it was found that IL-2 (interleukin-2) activated NK cells reduced Mycobacterium lepraemurium (MLM) growth in mouse C57BL/J peritoneal macrophages which had phagocytosed low numbers (MOI of 10 : 1) of MLM (P less than 0.0001 at day 20). There was no cytotoxicity exerted by the NK cells against the infected cells in these conditions. Conversely, macrophages heavily infected with MLM (multiplicity of infection of 1000 : 1) were found to be susceptible to lysis by activated NK cells in vitro. In vivo, progressing murine leprosy was associated with a sharp increase in splenic NK cell activity, which was abrogated by treatment with a monoclonal antibody against NK cells. Administration of this monoclonal antibody against NK cells enhanced C57BL6/J mouse susceptibility to mouse leprosy, as seen by a decrease in survival time of mice infected with 10(7) MLM i.v. (81 days vs 110 days, P less than 0.0005). Overall, these findings suggest that NK cells may play an important role in resistance to leprosy, either by reducing MLM growth in macrophages or by lysing heavily infected macrophages.

Cytokine modulation of Mycobacterium lepraemurium infection in mice; important involvement of tumor necrosis factor, interleukin-2 and dissociation from macrophage activation.

Susceptible BALB/c and resistant A/J mice were infected by the intravenous route with 10(7) Mycobacterium lepraemurium (MLM), and mortality was followed in controls and in experimental animals infused i.p. with interleukin-2 (IL-2), tumour necrosis factor alpha (TNF alpha) and interferon-gamma (IFN gamma). BALB/c mice injected with buffer and 10(7) M. lepraemurium i.v. died significantly earlier than A/J mice (111 days vs 158 days, respectively P less than 0.001), confirming earlier reports. Infusion of IFN gamma (1 or 2 micrograms every day) led to no significant increase in survival of both strains of mice infected with M. lepraemurium. Injection of IL-2 (1 microgram/day) led to a moderate increase in survival time in both strains of mice (P less than 0.01) although not changing the differences between resistant and susceptible strains of mice. Injection of tumour necrosis factor (1 microgram/day) significantly enhanced survival time in both strains (P less than 0.001), although all infected mice eventually died. The beneficial effect of IL-2 and TNF alpha on progression of the disease was seen as a modest reduction in bacterial growth in the lymph nodes and livers of BALB/c mice injected subcutaneously (approximately a one-log reduction in bacterial numbers). There was no evidence that the beneficial effect seen in vivo in mice was related to superior macrophage activation, inasmuch as peritoneal macrophages from untreated infected mice released similar amounts of superoxide anion, upon PMA triggering, as macrophages from infected and cytokine-treated mice. Moreover, macrophages from infected mice responded better to IFN gamma than cells from uninfected mice, in terms of developing tumour cytotoxicity in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant interleukin-1 infusion increases resistance of BALB/c mice to murine leprosy.

BALB/c mice were infected by the subcutaneous route with 10(7) Mycobacterium lepraemurium (MLM), and the progression of the infection followed in mice injected i.p. with diluent or recombinant human recombinant interleukin-1 alpha (IL-1 alpha). It was observed that infusion of 1 microgram of IL-1 alpha per day led to a reduction of bacterial growth in the livers and popliteal lymph nodes of MLM injected mice (2-3-log reduction at 6 months, P less than 0.0001). There was no indication that IL-1 alpha infusion was acting by enhancing macrophage activation. Indeed, resident peritoneal macrophages from control infected mice were as competent as macrophages from infected mice treated with IL-1 alpha in generating superoxide anion (O2-) (approximately 400 nM O2-/h/mg at 2 months post-infection). Moreover, they were no more permissive than those of IL-1 alpha infused mice for MLM in vitro as both groups of cells allowed progressive MLM growth, i.e. a 20-fold enhancement of intramacrophage MLM growth. Infusion of IL-1 alpha during MLM infection was not associated with any abrogation of the suppression of the T-cell response to T-cell mitogens or specific stimulation with antigens which is complete at 1 month post-infection. It is concluded that IL-1 alpha has immunotherapeutic potential in leprosy with the mechanism(s) of action still unclear.

The disease of CBA and BALB/c mice that follows inoculation of a small number of Mycobacterium lepraemurium into the hind foot pad.

To learn if the lack of an immune response in mice infected with Mycobacterium lepraemurium (MLM) was a consequence of the organisms, we studied the disease that followed inoculation of less than or equal to 5000 organisms into the hind foot pads of CBA and BALB/c mice. The mice of both strains demonstrated a rapid increase of bacterial numbers soon after inoculation, with a slowing of the rate of multiplication once the number of organisms per foot pad passed 3 x 10(7). By 1 year after inoculation, the numbers of organisms had reached levels greater than or equal to 10(11) in the spleen and liver, and greater than or equal to 10(8) in the femoral bone marrow. In mice that had been inoculated with as few as 5 MLM or 50 MLM, the organisms had multiplied to numbers greater than 10(8) in the foot pads and to greater than or equal to 10(9) in the spleens, suggesting that the ID50 of viable MLM may be less than or equal to 5 organisms per foot pad. No protection against superinfection could be demonstrated. On the other hand, initial multiplication of MLM in the foot pads was followed virtually immediately by the death of at least 97% of the organisms.

Biological properties of factors secreted by antigen-reactive suppressor cells in mice infected with Mycobacterium lepraemurium.

Antigen-reactive cells were isolated from the spleens of Mycobacterium lepraemurium-infected C57BL/6 mice on petri dishes coated with mycobacterial antigens. When adoptively transferred to syngeneic mice, the mycobacterial antigen-reactive cells were found to depress the induction and expression of the delayed-type hypersensitivity (DTH) reaction to M. lepraemurium antigens. The adoptive transfer of soluble suppressor factors (SF) secreted by these cells inhibited only the expression of DTH. The cells depressing the induction of DTH mainly belonged to the L3T4+ (CD4+) T-lymphocyte subset, whereas those depressing its expression differed from the L3T4+ and Lyt-2+ (CD8+) subsets. Treatment of M. lepraemurium-infected mice with SF reduced their mean survival time and enhanced the multiplication of bacilli at the site of infection and their dissemination to the spleen and liver. In vitro at least, SF appeared to interfere at the level of mycobacterial antigen recognition by T lymphocytes rather than at the levels of antigen processing and presentation by macrophages.

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.

Enhancement of growth of Mycobacterium lepraemurium in macrophages by gamma interferon.

Gamma interferon, an immune lymphokine that protects mouse macrophages against infection by several parasites, was ineffective against Mycobacterium lepraemurium. On the contrary, it significantly stimulated multiplication of M. lepraemurium in the macrophages. Simultaneous treatment of macrophages with gamma interferon and interleukin-4 or interleukin-2 or a combination of all three did not enhance the macrophage resistance to infection with M. lepraemurium, but instead stimulated growth of M. lepraemurium.

Doubling time of Mycobacterium lepraemurium in mouse footpads.

The doubling time of Mycobacterium lepraemurium (MLM) was measured in CBA/Ca mice. In eight experiments, 5 X 10(3) MLM were inoculated into the hind footpads of groups of mice, and the organisms were harvested from 1-45 days later. The harvested organisms were enumerated, and the doubling time was calculated assuming that MLM had multiplied without a lag phase and that multiplication continued at a constant rate from inoculation to harvest. Simultaneously, the proportions of viable organisms in the inocula were determined by inoculation of serially diluted suspensions into the footpads of other mice, harvesting 4 months later and calculating the most probable number. MLM were observed to multiply rapidly during the first several days, and more slowly thereafter; the mean initial doubling time was determined to be 0.5 days, a value much smaller than those previously reported by other workers.

A histopathological study of pulmonary infection of mice with Mycobacterium lepraemurium.

Intranasal instillation of Mycobacterium lepraemurium (MLM) into mice produced pulmonary infection. MLM multiplied rapidly in the lung tissue during the first few weeks without involvement of other organs. The increase in number, size and confluence of lung granulomas paralleled the multiplication of MLM which could be found both intracellularly and extracellularly. It is postulated that extracellular bacteria may find their way to the bloodstream and thus spread to other visceral organs. Extensive destruction of alveoli and occupation of airspaces by lepra-like cells invariably occurred as the disease progressed.

Transitory macrophage activation in the granulomatous lesions of Mycobacterium lepraemurium-induced lepromatoid leprosy in the mouse.

A kinetic study on the evolution of granulomas that appear in the liver of NIH mice inoculated with 10(8) Mycobacterium lepraemurium by the intraperitoneal route has been performed. The liver was chosen because of its nonlymphoid histology which allowed us to visualize the appearance and maturation of the cell infiltrates generated as a consequence of the mycobacterial infection. The study analyzed both the macrophage activation within the granulomas and the fate of bacilli within the macrophage. The results showed that this mycobacteriosis induces a relatively early macrophage activation (a very likely result of a cell-mediated immune response triggered by the bacilli) that peaks between 45 and 60 days postinoculation, fades thereafter, and practically disappears several days later. Bacilli are susceptible to the microbicidal effects of activated macrophages, but when the macrophages are turned off (probably due to active suppressive mechanisms), the surviving bacilli reinitiate the infection with no further macrophage opposition. As a result, more phagocytes are attracted to the infection sites and the cell infiltrates grow steadily to become confluent, increasing the granuloma fraction and eventually replacing the liver parenchyma. The findings suggest that in murine "leprosy" infection, early immunological changes occur that enable the macrophages present in the granulomas to kill the infecting M. lepraemurium regardless of the eventual lepromatoid evolution of the granulomas. Lepromatoid granulomas in the mouse and lepromatous granulomas in man are equivalent structures in regard to their histology and bacteriology.

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.