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The association (attachment and/or uptake) of Mycobacterium leprae with cultured Schwann cells was studied at 8 and 72 h in the presence of a new antileprosy compound, deoxyfructoserotonin (DFS), as well as conventional antileprosy drugs such as rifampin (RFP) and 4,4'-diaminodiphenyl sulfone (DDS). DFS significantly inhibited bacterial association with Schwann cells at 8 h. RFP also affected the association of M. leprae but not to the same extent as DFS. A similar inhibition at 8 h was noted when M. leprae but not Schwann cells were pretreated with DFS or RFP for 5 days before infection of cultures, implying that modulation was achieved through some form of drug action on bacteria. DDS had no effect on M. leprae association; however, the combination of DFS and DDS was neither antagonistic nor additive. At 72 h postinfection, when attached but noninternalized bacteria were removed with trypsin-EDTA from Schwann cell cultures containing DFS or RFP, a 50% reduction in the number of bacteria in the drug-treated group was obtained as compared with the numbers in drug-free cultures. This indicated a slow entry of M. leprae into Schwann cells in the presence of these drugs. Collectively, these observations point to differing requirements for late and early association of M. leprae with Schwann cells, besides suggesting a role for DFS and RFP in the prevention and minimization of M. leprae-induced nerve damage in vivo.

The presence and distribution of Mycobacterium leprae-specific and cross-reacting antigens within the peripheral nerves of multidrug-treated patients with leprosy was investigated to gain a better understanding of the mechanism of nerve damage and the effect of multidrug therapy (MDT) on it. There was no specific qualitative difference in the type of antigens in the leprosy spectrum. However, our results indicate that there may be differential handling of antigens by the macrophages as compared to Schwann cells. This could play a key role in the pathogenesis of the disease. Multidrug treatment is effective in arresting the progression of the disease process as well as in reducing the viable bacterial load, both in borderline lepromatous and lepromatous (MB) and borderline tuberculoid and tuberculoid (PB) cases. However, the presence of M. leprae antigens in all the multidrug-treated MB nerve lesions and 87% of PB nerve lesions suggest that the antigens persist for a prolonged period. Hence, the risk of immunological reaction and antigen-associated silent nerve damage may continue even after majority of M. leprae were killed. The findings give further support to the view that most of the nerve damage is due to bacterial antigens.

Myelin changes were observed in fibres of nerves from cases of leprosy. The myelin had a ‘loosened’ appearance caused by increased and irregular separation of the intraperiod line. ‘Loosening’ might affect all, or only some, of the lamellae forming a myelin sheath. There was a pathcy distribution of fibres with abnormal myelin, and they were seen only in nerves showing other marked pathological changes including the presence of oedema. The appearances are suggestive of intramyelinic oedema which may be related to the presence of endoneurial oedema.

Twenty nerve biopsies of tuberculoid leprosy patients who showed no acid-fast bacilli in their skin smears or in tissue biopsies, were stained for mycobacterial antigens using anti-bacille Calmette-Gúerin (BCG) by the peroxidase-antiperoxidase method. Adjacent parts of some of these nerves were examined for the presence of osmiophilic bacilli under the transmission electron microscope. Eight of the 20 nerves were both clinically and histologically uninvolved. All the 20 involved nerves showed presence of antigen located, mainly intracellularly, in the cytoplasm of epithelioid cells and to a lesser degree in Schwann, endothelial and plasma cells. A few nerves with caseated nerve abscesses showed clusters of antigen deposits in both the caseous mass as well as the wall of the abscess. In six of the nine nerves processed for electron microscopy, electron-dense bacilli were noted within the cytoplasm of Schwann cells but not within the infiltrating cells. The uninvolved nerves showed neither antigen deposits nor osmiophilic bacilli despite fine ultrastructural changes. Our observations indicate that (a) the specificity of the immune response in paucibacillary nerve lesions is probably against bacterial components. (b) There is a differing antigen handling by Schwann cell and the inflammatory epithelioid cell. (c) Plasma cells may play a role in presenting antigen. (d)Mycobacterium leprae may be acting as an adjuvant in causing damage to uninvolved nerves at distal sites.

This study describes a novel method which could be developed into a test system of evaluating the efficacy of antileprosy drugs. The method estimates incorporation of [14C]acetate into lipids of Mycobacterium leprae maintained within the 33B Schwannoma cell line. Schwannoma cell-resident M. leprae cells incorporated significant levels of radiolabel within their lipids during 12 days of incubation in vitro. This incorporation was markedly reduced by 5 micrograms of rifampin per ml (decrease, 81.62%); this decrease was observed within 24 h of addition of the drug. Dapsone also reduced the radiolabel incorporation into the lipids, but to a lesser extent (decrease, 27.58%). This system was also able to differentiate between rifampin-sensitive and -resistant strains of mycobacteria. It is suggested that since the effect of bacteriostatic (dapsone) and bactericidal (rifampin) drugs could be detected by using this technique, it may prove useful in screening novel drugs acting against M. leprae.

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.

The interaction betweenM. leprae-infected cultured Schwann cells and sensitized splenic cells was noted both under light and electron microscopy. No evidence of cytomorphological changes in infected Schwann cells was obtained. However, sensitized splenic cells were noted to undergo degenerative changes suggestive of the phenomenon of apoptosis. Subsequently a large number of these degenerated cells were observed within the Schwann cell. Such a process has not been hitherto reported in the histopathology of leprous nerves. Nevertheless, these findings indicate an aberrant metabolic function inM. leprae-infected Schwann cells.

Electron micrographs have been presented, demonstrating myelination around multiple axons. This is an exception from the normal 1:1 relationship of axon to Schwann cell in a myelinated peripheral nerve fibre.

The killing of Mycobacterium leprae by resting and gamma interferon (IFN-gamma)-activated macrophages in normal subjects and leprosy patients was assessed. Resting macrophages from normal individuals demonstrated the ability to kill M. leprae. For macrophages from tuberculoid patients, killing of M. leprae was only achieved in the presence of IFN-gamma, suggesting that initial T-cell activation occurs prior to the killing of M. leprae. In contrast, though activation with IFN-gamma rendered the lepromatous macrophages microbicidal, it failed to induce lymphocyte proliferation, suggesting a defect at either the antigen-presenting cell or the lymphocyte level or both. The concept that T-cell anergy is primarily due to lack of lymphokine generation was ruled out by our results, since responsiveness was restored in only a small proportion of lepromatous patients after exogenous lymphokine addition. In conclusion, this study demonstrated that killing and antigen presentation are two independent events. It appears that the ability of the macrophages per se to kill M. leprae may be of greater importance than lymphocyte-mediated activation for protection against M. leprae infection.

Human peripheral blood mononuclear cell proliferation induced by Mycobacterium leprae could be inhibited by the suppressor factor in the lysate of the macrophages of lepromatous leprosy patients. Macrophages from normal subjects and tuberculoid patients did not show production of a suppressor factor. Inhibition occurred only when the factor was present in the initial stages of lymphocyte culture. The factor is heat stable and nondialyzable. Proliferation induced by some mycobacteria and concanavalin A could also be blocked by the factor. Interestingly, blastogenic response by a few other antigens and phytohemagglutinin could not be inhibited by the suppressor factor. Mononuclear cells pretreated with such lysate from lepromatous macrophages for 24 h could induce suppressive activity in the cells in vitro in an autologous system. Treatment of these cells with carbonyl iron after the induction phase, to remove phagocytic cells, did not abolish their suppressive activity. The lepromatous macrophage lysate also generated suppressive activity in a T-lymphocyte-enriched population of normal subjects. These studies are interpreted to indicate that immunosuppression in lepromatous patients is produced by both macrophages and T lymphocytes. The exact phase in which either of these cells acts as a suppressor may be different. Specific suppression by macrophages to M. leprae can be an early event, and nonspecific suppression by T lymphocytes may be a later event in the course of lepromatous leprosy.

Organized nerve cultures of dorsal root ganglia from neonatal mice were infected with Mycobacterium leprae, the causative agent of leprosy. A significant multiplication of the acid-fast bacilli was observed within the Schwann cell component of the culture. The growth of these bacilli was sensitive to antileprosy drugs and was not observed directly in bacteriological media. These organisms were brightly stained with the monoclonal antibody to phenolic glycolipid-I, a M. leprae-specific marker. The antigenic, pathogenic, and biochemical characteristics of this mycobacterium are under investigation.

Three cases of partial defect of the helix reconstructed by an advancement of the chondrocutaneous helical flap are presented. The operative technique employed in these cases is described. The principle underlying the operative procedure is that of advancement of the adjacent, intact helical margin as a flap based on a wide postauricular skin pedicle. The defect is in fact transferred to the extensile lobule. The reconstructed helix is more consistent with the architecture of the normal ear than when repair has been accomplished by some of the other methods proposed. Safety is combined with economy of tissue and time.

THE inability to culture Mycobacterium leprae in vitro has been a great obstacle to the progress of research in leprosy. This obstacle has been in part overcome by the introduction of the morphological index for the assessment of the viability of M. leprae, although the index is relatively crude. Biochemical studies1,2have revealed the specificity of the enzyme o-diphenoloxidase in relation to M. leprae as opposed to other bacteria included in the genus of mycobacteria. o-Diphenoloxidase oxidises 3,4-dihydroxyphenylalanine (DOPA) to quinone.