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.

Protection against tuberculosis by bone marrow cells expressing mycobacterial hsp65.

Although mice acquire only a slight degree of protection against tuberculosis by immunization with Mycobacterium leprae (M. leprae) hsp65 in incomplete Freund's adjuvant, protection is substantial following immunization by injection with J774 macrophage-like tumour cells that express the protein from the mycobacterial gene via a retroviral vector. We here took the same vector, used it to transfect the gene into normal murine bone marrow cells in vitro, and then used the transfected cells to reconstitute haematopoiesis in lethally irradiated mice. Bone marrow-cell clonal expansion and production of the protein in vivo resulted in specific delayed-type hypersensitivity and protection against challenge with Mycobacterium tuberculosis (M. tuberculosis) in about half of recipients. Counts of live bacteria in liver at 3 weeks were fivefold lower in delayed-type hypersensitivity (DTH)-positive than in DTH-negative mice. Other mice acquired neither DTH nor protection despite the presence of the protein in peripheral blood.

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.