Light and ultrastructural study of sciatic nerve lesions induced using intraneural injection of viable Mycobacterium leprae in normal and immunosuppressed Swiss white mice.

Freshly harvested M. leprae were microinjected into the sciatic nerves of nonimmunosuppressed (non-TR) and immunosuppressed (TR) mice using the technique described by Wisniewski and Bloom. The lesions thus induced, on bypassing the blood-nerve barrier, were biopsied at regular intervals beginning 24 hr and followed up to one year. The fate of M. leprae and the ensuing inflammation and nerve damage were studied using light and electron microscopy. The lesions in both non-TR and TR mice at 24 hr showed an influx of polymorphonuclear leukocytes and an increase in mast cells. The influx and peaking of lymphocytes were delayed by two weeks and 6 weeks, respectively, in TR mice, but the density of lymphocytes at the peak intervals was comparable in both. The plasma cells denoting the humoral response were seen in both, but there was a delay of 3 weeks in non-TR mice. The lesions in non-TR mice showed differentiation of macrophages into epithelioid cells and the formation of giant cells depicting borderline tuberculoid leprosy (BT), Whereas in TR mice, the macrophages showed foamy cytoplasmic changes depicting borderline lepromatous leprosy (BL). Other significant observations common to both non-TR and TR mice were: a) The lesions remained highly localized and showed signs of regression at the 6th and the 12th month intervals. b) The characteristic segmental demyelination and some attempt at remyelination were seen at the site. c) The influx of lymphocytes concorded well with demyelination. d) Bacteria were only seen in the macrophages and never in the Schwann cells or endothelial cells. e) Bacteria persisted in the macrophages, but appeared progressively degenerate at the 6th and 12th post-inoculation months, suggesting loss of viability. The study shows that there was a very effective containment of the infection and that the Schwann cells were resistant to M. leprae infection in the neural milieu. Nerve damage and Schwann cell bacillation do not go hand-in-hand.

Dosage and site of entry influence growth and dissemination of Mycobacterium leprae in T900r mice.

The role of dosage of Mycobacterium leprae and the environment of the inoculated site, in producing leprosy lesions in immunologically-suppressed, highly-susceptible T900r mice, was investigated. Various doses of M. leprae, i.e., 10(7), 10(6), 10(5), 10(4), were inoculated into both flanks and footpads of two different groups of mice. The sites of inoculation were biopsied for histopathological examination and for M. leprae counts at the end of 6, 8 and 12 months. M. leprae multiplied at the infected site and disseminated [figure: see text] to other parts of the body at all concentrations in the mice that were infected in the footpad with a temperature of 31 degrees C. In animals inoculated at the flanks with a temperature of 37 degrees C, multiplication was recorded only when the dosage of M. leprae was high and there was no dissemination of the organism in any of them. The temperature at the site of entry and the dose of infecting M. leprae may play an important role in the development of leprosy in susceptible individuals exposed to M. leprae.

Sciatic nerve of normal and T200x5R Swiss white mice fails to support multiplication of intraneurally injected M. leprae.

In a preliminary study we have shown that freshly harvested Mycobacterium leprae, when injected into the sciatic nerve in normal and immunosuppressed (TR) mice, induce massive but localized epithelioid and macrophage granuloma, respectively, in 3-4 weeks. In order to determine the fate of M. leprae injected intraneurally into normal and TR mice, in the present study we measured sequentially the viability, fold increase and clearance, if any, using semi-quantitative methods. The average M. leprae yield per nerve assessed at regular intervals, beginning at 24 hr and including 72 hr, 1 week, 2, 3, 4, 12, 24 and 48 weeks, showed neither a significant increase nor a decrease in either the normal or the TR mice. The viability of M. leprae, assessed using the standard mouse foot pad method, showed a significant decrease as compared to baseline growth effective at 24 hr and remained static until approximately 4 weeks. A further decline and total loss of viability was noted by 12 months. The results show that injection of M. leprae via the intraneural route in both normal and TR mice failed to sustain the viability and failed to support the multiplication of the organisms.

Effect of in vitro formed immune complexes on macrophage functions of Mycobacterium leprae infected mice.

Nonspecific macrophage functions were studied in Mycobacterium leprae infected and preformed immune complex (IC) administered normal (NI) and thymectomized/irradiated (TRI) mice at different time periods. Uninfected controls given IC were also included. Significant decrease in the chemotaxis, phagocytosis and bactericidal activities of macrophages obtained from infected groups compared to their controls were observed. Phagocytic and chemotactic activities of macrophages were normal but intracellular killing was seen to be depressed in studies conducted in normal and thymectomized immunosuppressed groups (Vaishnavi et al., 1985, Kumar et al, 1987) which were not administered with preformed IC.

Circulating immune complexes in normal and immunosuppressed mice infected with Mycobacterium leprae.

Mycobacterium leprae infection was produced through the footpads in normal and immunosuppressed mice. Circulating immune complexes were detected by specific binding test and by conglutinin binding assay for specific and total immune complexes respectively in the sera of these mice during different periods of infection. Out of the total 30 samples tested from the infected groups, 3 were positive by specific binding test and 5 by conglutinin binding ELISA. The implications of the findings in relation to human leprosy are discussed.

Superiority of the neonatally thymectomized Lewis rat (NTLR) to monitor a clinical trial in lepromatous leprosy of the two regimens of rifampin and dapsone.

The ability of the neonatally thymectomized Lewis rat (NTLR) and the congenitally athymic (nude) rat systems to detect low numbers of viable Mycobacterium leprae in tissues from lepromatous leprosy patients undergoing short-course chemotherapy was compared with that of the commonly employed mouse foot pad assay. Fifteen previously untreated lepromatous patients were randomly assigned to treatment regimens of either a single initial 1500 mg dose of rifampin plus daily doses of 100 mg of dapsone, or weekly doses of 900 mg of rifampin plus daily doses of 100 mg of dapsone. Four skin biopsies from each patient taken sequentially up to one month after initiation of therapy were used as the source of the M. leprae inocula. Only 2 of 57 skin biopsies (2%) proved positive for viable M. leprae following direct inoculation into mouse foot pads. However, 30 of 58 patient biopsies (52%) provided positive for viable M. leprae following direct passage into NTLR foot pads or in subsequent mouse subpassage. In contrast, the nude rat was observed to be a poor monitor of such trials. Although not statistically significant, the regimen consisting of a single dose of rifampin plus daily dapsone resulted in a lower percentage of biopsies found to contain viable M. leprae at each of the four sampling intervals.

Levels of the third component of complement in Mycobacterium leprae infected intact and immune compromised mice.

Normal and immunosuppressed mice were infected with Mycobacterium leprae and the bacillary counts were made from the footpads at 3, 6 and 9 months post inoculation. A decrease in the serum C3 level was observed in the infected groups of animals compared to controls.

Intracellular location of Mycobacterium leprae in macrophages of normal and immune-deficient mice and effect of rifampin.

Soon after more than 10(6) Mycobacterium leprae, freshly harvested from armadillo liver or harvested and 60CO irradiated, were inoculated into the hind footpads of either normal or thymectomized and irradiated (T900R) mice, the organisms were found to reside within phagosomes of polymorphonuclear and mononuclear cells. On the other hand, 7 and 8 months after 10(4) freshly harvested M. leprae were inoculated into the footpads of normal or T900R mice and the organisms had multiplied to their maximum in the normal mice, many organisms, largely intact by electron-microscopic criteria, were found to reside free in the cytoplasm of the footpad macrophages, whereas damaged organisms were contained within phagosomes. After 11 months, many intact organisms were found to lie free in the cytoplasm of the macrophages of T900R mice, whereas only damaged intraphagosomal M. leprae cells were observed in the macrophages of normal mice. Finally, a remarkably large proportion of damaged extraphagosomal M. leprae was found in T900R mice administered rifampin for 2 days in a bactericidal dosage. It appears that M. leprae multiplies free in the cytoplasm of the footpad macrophages of infected mice, whereas the M. leprae cells resident within the phagosomes of the macrophages are dead. As the result of treatment with rifampin, the organisms appeared to have been killed in their extraphagosomal location, only afterwards being incorporated into phagosomes. However, the intracellular site in which M. leprae is killed in the course of an effective immune response remains unclear.

Relationship between T-cell population in neonatally thymectomized Lewis rats and susceptibility to infection with mycobacterium leprae.

The neonatally thymectomized Lewis rat (NTLR) is highly susceptible to infection with M. leprae. However, a significant percentage of NTLR respond to infection with M. leprae in much the same way as do intact rats, yet show no evidence of residual thymus. To determine whether there was a correlation between the number of remaining T-cells and susceptibility to infection with M. leprae, a direct fluorescent antibody test was performed using a highly specific, absorbed antithymocyte globulin labeled with fluorescein isothiocyanate. Both total circulating white blood cells and T-cells were significantly depressed in all NTLR examined. Although the greatest numbers of M. leprae were found in NTLR from the groups having the lowest percentage of circulating T-cells, these groups also contained NTLR infected with small numbers of M. leprae. The groups containing NTLR with the highest percentages of circulating T-cells also contained animals with both moderate and severe M. leprae infection. The response of cultured splenic lymphocytes from NTLR and normal rats to the T-cell mitogen concanavalin A was investigated to determine whether there was any correlation between T-cell activity and susceptibility to M. leprae infection. The mean stimulation index for normal rats was five to ten times greater than indices for NTLR, but there were no significant differences between NTLR with a well developed, generalized infection and those with a poorly developed infection. it was concluded that since there was no apparent relationship between T-cell depletion and susceptibility to infection with M. leprae, an additional, unknown mechanism was also involved.

Myco. leprae infection in normal, thymectomised irradiated and thymus transplanted mice.

Normal mice and thymectomised, X-irradiated and thymic transplanted groups of mice were challenged with 10(3) Myco. leprae in the foot pad. Course of infection was studied for a period of seven months by sacrificing animals every month and counting the bacilli from the pooled tissues. In the thymectomised irradiated group the counts showed a plateau from 5th month onwards, whereas in control and thymus implant group the counts rose in 6th and 7th months.