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The effects of rapid and slow rates of freezing in liquid nitrogen, storage in liquid nitrogen for 12 months, and the rate of subsequent thawing on the viability and growth of M. leprae in the mouse footpad were studied. Some loss of viability of M. leprae was detected, and this was found to be associated with the freezing process, rather than with storage or thawing. Slow freezing was less deleterious than quick freezing, with a loss of viability of 90% compared with 98%. The growth pattern of M. leprae was unaffected except for a delay in the appearance of growth caused by the loss of viability, though there was some evidence of an increased lag phase of one strain, possibly due to the repair of sub-lethally damaged organisms.

The origin of autoantibodies against ubiquitous autoantigens (e.g., single-stranded (SS) DNA, cytoskeletal proteins, mitochondria) is obscure. Patients with lepromatous leprosy have many such autoantibodies in their serum. In order to study the polyspecificities of human autoantibodies expressed during infection with Mycobacterium leprae we prepared human monoclonal antibodies derived from the fusion of peripheral blood lymphocytes of a patient with lepromatous leprosy to the human lymphoblastoid line GM 4672. Hybridomas were tested for binding to a DNAse-treated sonicate of M. leprae and a panel of autoantigens. Of the primary (uncloned) cultures, 14% bound ssDNA, 35% bound M. leprae, 11% bound both M. leprae and ssDNA, and 16% bound to mitochondria. Several also bound to the acetylcholine receptor of Torpedo marmorata. Monoclonal antibodies derived from separate primary cultures revealed similar cross-reactions between several autoantigens and M. leprae. In addition, one antibody was identified which bound to mitochondria and the acetylcholine receptor, and which was recognized by an anti-idiotypic antibody which bears the "internal image" of the acetylcholine receptor. These results suggest that antigenic mimicry may play a role in eliciting autoantibody expression from the immune repertoire.

The association of Mycobacterium leprae with Schwann cells may represent an early crucial step in M. leprae pathogenesis. Using a dissociated Schwann-cell system and anti-mycobacterial monoclonal and polyclonal antibodies directed against surface and cytoplasmic components, we investigated the nature of M. leprae epitopes that mediate cytadhesion. Antibodies to polysaccharide and lipid components of M. leprae cell wall inhibited cytadhesion, whereas those directed against both surface and cytoplasmic protein epitopes did not show any such effect. No synergistic or antagonistic activity in inhibiting cytadhesion was observed when antibodies were used in combination. Thus, the association of M. leprae with Schwann cells may be mediated collectively by more than one of its lipid/polysaccharide epitopes. Also, a role for humoral immunity in intervention in the initial steps of M. leprae pathogenesis needs to be considered.

A concept has been developed in the recent years that the evenly stained 'solid' bacilli are living and the 'non-solid' forms are degenerate and dead. This communication presents the findings in experimental mice inoculated with material containing 1 to 10% solid evenly stained M. leprae and also with material containing 0% solid organisms. There was multiplication of the bacilli in both the groups. Quantitatively, the yield also was not significantly different. These fundings do not support the belief that the non-solid bacilli are necessarily dead. The non-solid bacilli were further classified on the basis of their morphology to the following forms:-- (a) short but evenly stained (b) indented (c) beaded (d) dumb-bell shaped (e) coccoid and (f) fragmented. Material without solid bacilli, but containing different proportions of the above types of bacilli also gave similar results, making it diffcult to say which types of morphological forms are non-living. It appears, therefore, that the recognition of the living status of M. leprae by its morphology is highly equivocal and subject to error.

Presence of Mycobacterium leprae in association with in vitro cultured macrophages, from bacillary negative long term treated lepromatous leprosy patients, induces reduced level of protein and lowering of hydrolytic enzymes like p-glucuronidase, Lysozyme and Lactic dehydrogenase. Alkaline phosphatase, on the other hand is increased. In the macrophages from normal healthy individuals or tuberculoid leprosy patients, presence of M.leprae increases both protein and levels of all the above enzymes. This observation shows that macrophages from lepromatous leprosy patients are unable to manifest in presence of M. leprae, the key enzymes involved in degradation of complex biological entities phagocytosed by the cells.

The influence of different frequencies of freezing-thawing cycles on the viability of in vivo grown mycobacteria was investigated. Pieces of armadillo tissues naturally or experimentally infected with Mycobacterium leprae were analyzed. The viability of M. leprae was determined by mouse foot pad titration. The viability of cultivable mycobacteria, sometimes present in armadillo tissues, was determined by culture. Electron-microscopic studies were performed on fresh or frozen-thawed armadillo tissues with natural leprosy and on livers of C57BL/6 mice experimentally infected with M. avium or M. lepraemurium. We found that the percentage of viable M. leprae bacilli is identical for naturally infected and experimentally infected tissues, frozen and thawed once. When the tissues were subjected to a second freezing-thawing cycle, a considerable loss of viability was observed (65%-97%). A third freezing-thawing cycle was lethal for most of the M. leprae cells, and after four freezing-thawing cycles no viable bacilli were found. The cultivable mycobacteria present in some armadillo tissues were found to be more resistant than M. leprae to freezing-thawing since these mycobacteria could still be cultivated after four freezing-thawing cycles. The results of the electron-microscopy study support the conclusion that M. leprae is more sensitive to freezing-thawing than the cultivable mycobacteria and show that the cytoplasmic membrane appears to be the target for the lethal action of freezing-thawing on mycobacterial cells. These results emphasize the importance of avoiding repeated thawing and refreezing of M. leprae-infected tissues when viable M. leprae cells need to be studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow-derived cultured macrophages were infected with Mycobacterium leprae. The bacteria were either used as freshly isolated organisms or incubated with M. leprae antiserum (1:5) for 30 min prior to phagocytosis. Immediately after inoculation (1 to 4 h) and at 1 to 8 days later, macrophages were stained for acid phosphatase activity to assess fusions between phagosomes and lysosomes. Inhibition of fusions was essentially apparent as an early event, which was partially reversed by antiserum treatment of the bacteria, suggesting a role for M. leprae immunogenic surface components in this early phenomenon. Later incubation times (1 to 8 days) did not show any considerable difference between antiserum-treated and nontreated bacteria. The formation of an electron-transparent zone around phagocytized bacteria and its role in phagosome-lysosome fusion was investigated, and a direct relationship could not be established.

Macrophage cultures pulsed with viable Mycobacterium leprae were assessed for erythrocyte rosetting in three groups of individuals, i.e., normal subjects, and tuberculoid and lepromatous patients. Of these, only the lepromatous group showed a reduction in rosetting ability after infection with M. leprae. The specificity of such a reduction pattern was confirmed by using various mycobacteria to infect the macrophages. A threshold effect was noted in all three groups. Although a reduction was obtained in the amount of rosetting of macrophages from lepromatous patients with 10(4) acid-fast bacilli per culture, tuberculoid and normal macrophages resisted such an effect with as large a dose as 20 X 10(6) to 30 X 10(6) and 30 X 10(6) bacilli per culture, respectively. The M. leprae-caused alterations in macrophages from lepromatous patients were reversible by treatment with trypsin and colchicine. Cytochalasin B and Tween 80 were unable to alter the pattern. Treatment of cells with neuraminidase was inconclusive since it enhanced rosetting values of both control and infected cultures. These manipulations were significant in elucidating the target point of the host (macrophage) and parasite (M. leprae) interaction and in delineation of the external and internal effects upon the macrophages. Both M. leprae and macrophages were participants in Fc reduction, as treatment of the former with rifampicin and of the latter with cyclocheximide significantly augmented the rosetting ability. In conclusion, it appears that M. leprae, upon entering a lepromatous macrophage, initiates the production of a protein which acts via the microtubules to alter membrane topography. It is possible that the altered membrane prevents effective macrophage-lymphocyte interaction. This could be one of the mechanisms by which cell-mediated immunity is suppressed in lepromatous leprosy.

Spurr's resin mixture has been found to be the embedding material most suitable for electron microscopic studies of lepromata at present. Like methacrylate, it can penetrate well into foamy structures and the leprosy bacilli within foamy structures. On the other hand, like Epon 812, it is stable against the electron beam and can preserve ultrafine structures. Additionally, we have found that the use of dimethylformamide or dimethylsulfoxide instead of propylene oxide as substituter improves the preservation of the ultrafine structures of leprosy bacilli.

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.

The liver, skeletal muscle, and adrenal gland obtained from two nine-banded armadillos infected with Mycobacterium leprae were studied using an electron microscope. M. leprae were found in varying numbers inside hepatocytes, Kupffer's cells, striated muscle cells, adrenal cortical and adrenal medullary cells, endothelial cells, and macrophages. There was evidence to suggest that M. leprae were actively phagocytosed by the liver and skeletal muscle cells. The inert nature of M. leprae and its behavior as an almost ideal parasite of parenchymal cells are emphasized. The question of whether this unique parasitism of parenchymal cells and the possible processing and presentation of M. leprae antigens by these cells could be responsible for aberrant immune responses is raised.

Mouse foot-pad experiments were carried out to study the effects of DFS and related compounds on the multiplication of M. leprae. Of the 25 cases clinically suspected Dapsone resistance, 8 were found resistant and 14 sensitive to Dapsone by mouse foot-pad experiments. Six were resistant to DFS and 16 were sensitive. Desoxy fructo 5-hydroxy tryptophane as well as Nutrition Antileprosy (NAL) diet were also found effective in suppressing the growth of M. leprae in mouse foot-pad. Of the two liposoluble derivatives of DFS tested (DFS LS-I and DFS LS-II), DFS LS-II was found more effective in suppressing the growth of M. leprae in foot-pads of mice.

Certain structures which indicate probable involvement of a filamentous phase in life cycle of M. leprae have been noted in preserved skin biopsy suspensions from lepromatous leprosy cases. These include (i) filaments with empty or pink round spaces within them (ii) conidia-like structures and (iii) membranes with acid fast bacilli. These structures were rare in the fresh material.

This study of monocyte chemotaxis in leprosy patients showed a significant inverse correlation (p less than 0.05) of chemotaxis and the bacterial index (BI) (N = 22). In addition, there was a significant inverse correlation (p less than 0.05) between chemotaxis and the serum levels of anti-phenolic glycolipid-I IgM antibodies (N = 20). Patients taking thalidomide who had a BI greater than or equal to 1 had a significantly greater (p less than 0.001) chemotaxis response than that of patients with the same BI who were not taking thalidomide. No significant decrease in chemotaxis of monocytes from healthy donors was observed when the cells were pre-incubated with serum from 18 leprosy patients. We conclude that monocytes from patients with active lepromatous leprosy not receiving thalidomide have an intrinsic abnormality when assessed by chemotaxis.

Activation of the respiratory metabolic channel (F.O.R.), responsible in part for the bactericidal effect, was measured in vitro by a chemoluminescence test on circulating phagocytes. All mycobacteria tested, except for Mycobacterium leprae, induced a significant response. Its effect on lepromatous leprosy pathology is a subject for discussion.

The recent advances in microbiology of leprosy are reviewed. Till now the leprosy bacillus had not been cultivated in laboratory media; the recent claims of success have not been confirmed. There has been a breakthrough in the experimental transmission of leprosy to experimental animals--the white mice, the immune depressed white mice, and the nine-banded armadillo. Apart from providing definite proof for the causative relationship of the bacillus discovered by Hansen and the disease leprosy, the experimental transmission to animals have considerably advances our knowledge about the disease. The mouse has provided a suitable model for screening of antileprosy drugs, detecting development of drug resistance, ascertaining viability of the bacilli and determining the genuineness of a supposed culture of the leprosy bacillus. The armadillo has provided a model for making basic studies of the disease as it occurs in man. Further, the generalised infection in armadillo has provided large amounts of leprosy bacilli for preparing standardised lepromin, for preparing a specific skin-testing antigen containing the active protein fraction of the bacillus, and a step towards the production of a vaccine against leprosy. It is interesting to note that the fact that the protein fraction of the bacillus was responsible for the positive lepromin reaction was discovered by Dharmendra far back in 1941.

The interactions of mycobacteriophage D29 and Mycobacterium leprae were examined. It was demonstrated that after adsorption D29 injected its DNA in M. leprae. While the synthesis of host proteins and lipids were inhibited in M. tuberculosis and in M. smegmatis during infection by D29, the results were inconclusive in the case of M. leprae because these bacteria did not incorporate the appropriate substrates.

Mycobacterium leprae is an intracellular pathogen that is ingested by and proliferates within cells of the monocyte/macrophage series. Mechanisms by which intracellular pathogens resist destruction may involve failure to elicit a phagocyte "respiratory burst" or resistance to toxic oxygen derivatives and lysosomal enzymes. We have studied the ability of M. leprae and Mycobacterium bovis BCG to stimulate the generation of superoxide anion (O2-) in vitro by human blood neutrophils and monocytes and murine peritoneal macrophages. M. leprae bacteria failed to stimulate significant O2- release except at high bacteria-to-cell ratios (greater than 50:1) whether or not they were pretreated with normal serum or serum from patients with lepromatous leprosy. Either viable or irradiated BCG; on the other hand, stimulated the three cell types to release significant amounts of O2- when challenged with as few as 10 organisms per cell. Serum pretreatment enhanced the release of O2- by the three cell types. Preincubation for 18 h with viable M. leprae did not inhibit the ability of monocytes to respond with an oxidative burst to phagocytic stimuli. The failure of M. leprae to stimulate phagocyte O2- generation may be an important factor in its pathogenicity.

Adherence of Mycobacterium leprae was studied in vitro in monolayer cultures of purified mouse Schwann cells. The optimum temperature and time for adherence were determined. Pretreatment of Schwann cells with lipase reduced adherence, but pretreatment with trypsin enhanced it and with four monosaccharides--L-arabinose, L-galactose, L-rhamnose and D-glucose--there was no significant effect, indicating that the receptors involved in adherence may be lipid.