Leprosy immunopathogenesis and vaccine development.

The truly effective immunity against intracellular parasites, including mycobacteria, is mediated by monocyte/macrophages, and in the immunologically responding (resistant) host these phagocytes need minimal antigenic stimulus, specific or non-specific, to become activated and be microbicidal. T-cell mediated delayed hypersensitivity (DTH) causes tissue damage and destruction, which is particularly unwelcome in leprosy because of its nerve-damaging potential. Gamma interferon (INF-gamma), the terminal lymphokine of a DTH response, promotes mycobacterial survival and growth. There are T-cells (TH1 subtypes) that produce DH response either independent of, or, only partly dependent on INF-gamma; this type of DH peaking at 24 hours appears similar to the Jones-Mote type rather than to the tuberculin type of DTH peaking at 48-72 hours and is devoid of the necrotic component of tuberculin type of DTH. M. leprae antigens normally elicit this Jones-Mote type of DH. Suppressor T-cells are associated with a protective immune response, while helper T-cells mediating DTH are harmful. In view of this immunobiology, it would appear that pathogenic mycobacteria that generate a tuberculin type DTH response should not be used as immunogens in leprosy.

Growth of Mycobacterium leprae in a redox system: III. Evidence of growth at low temperature (psychrophilia) and, further refinement of growth medium.

Considerable growth enhancement, largely as non-acid fast, slender and long rods has been seen when incubated at 10 degrees C. Concentration of some of the media constituents have been reduced that has improved the quantum of growth. A remarkable proneness to physical disintegration of the grown bacilli has been seen and its significance discussed. Also, the possible immunogenic advantage of non-AF M. leprae has been discussed. The question of identification is still not solved, and work is in progress.

Growth of Mycobacterium leprae in a redox system: II. Further improvements in the system and growth efficiency.

Improvement of the Redox System for growth of M. leprae as brought about by modification in the concentration and mode of preparation of individual media constituents, and by addition of newer substances, is being reported. A structural modification in the construction of the Thunberg's tubes and flasks that are used as culture vessels, has been introduced for ease of handling. Vitamin E (alpha-tocopherol) has been found to be useful. Concentrations of Liposomes and Gelatin in the medium could be reduced by at least five folds, considerably easing thereby smearing and harvesting of cultures. Dimercaptopropanol British Anti-lewisite or BAL) has been used, but its usefulness or otherwise is yet to be determined conclusively. The basis of intracellular parasitism of M. leprae has been discussed.

Growth of Mycobacterium leprae in a redox system.

Mycobacteria recovered from human lepromatous nodules and presumably M. leprae, have been grown in a medium that ensured a minimal oxygen tension at initiation of growth, and an increasing availability of oxygen as bacillary growth increased requiring marginal increments in oxygen tension. This physicochemical environment was achieved by the addition of strong biological reductants in the medium, and a combination of partial vacuum and alkaline pyrogallol in the culture vessel. In addition, n-tetradecane, a straight chain hydrocarbon, and lipids like Cholesterol and Lecithin, all three substances mixed in the aqueous medium as liposomes, were added and found to be useful. Menadione, or Vitamin K3, added to the medium considerably improved growth efficiency. Growth occurred initially as non-acid fast coccoids and bacilli that gradually changed to acid-fast bacilli and globi, and cell-wall deficient, spherical L-form elements. Appearance of growth in any form was perceptible within 1 to 2 weeks and optimal growth as acid fast bacilli took upto 3 months. Both the acid fast and non-acid fast bacilli could not be grown in conventional media, but the non-acid fast coccoids could be readily isolated from these cultures in a specially enriched liquid medium. The problem of harvesting of the growth free of lipid-hydrocarbon substances has still not been solved, as also an optimum oxidation-reduction potential. The growth is transferable.

Skin test response to M. leprae precursor coccoid organisms in a high leprosy endemic area as compared to lepromin.

The paper describes the results on several hundred skin tests on humans using antigens from leprosy derived non-acid fast coccoid organisms, and a mycobacterium that grew out of one of the coccoid organisms. Early skin test response to the cytoplasmic fraction of one of the leprosy derived, non-acid fast, coccoid organism was very highly correlated with standard lepromin.