Application of a viability-staining method for Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad.

Mycobacterium leprae cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to M. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of M. leprae with the course of athymic mouse foot pad infection to optimize the provision of viable M. leprae as a research reagent. The data show that in untreated suspensions of M. leprae there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of M. leprae with ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity of M. leprae declines more than the extent of membrane damage at 37 degrees C within 72 h, but that they are not significantly affected at 33 degrees C. Irradiation at 10(4) Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by which M. leprae is killed. This staining method helped us predict reliably that the smaller M. leprae-infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.

Nerve lesions induced by macrophage activation.

The neuropathies associated with infectious processes, including leprosy, retroviral infections and Chagas' disease, represent the largest group of neuropathies in the world. Segmental demyelination and axonal degeneration of nerve fibres are associated with inflammatory infiltrates which contain a large number of mononuclear phagocytes. In order to learn more about the role played by macrophage activation in the nerve lesions observed in inflammatory neuropathies, we have performed a morphological study of nerves injected with products of activation of macrophages including proteolytic enzymes and cytokines (tumour necrosis factor and alpha beta-interferon). We have also studied the effects on nerve fibres of macrophages activated by ingestion of proteose-peptone, a foreign protein, and in the course of a delayed-type hypersensitivity (DTH) reaction. We have found that proteases and urokinase were potent demyelinating agents and that activated macrophages were also able to induce significant demyelination of neighbouring fibres. In contrast, injection of TNF alpha induced more severe nerve lesions consisting of axonal degeneration of the majority of nerve fibres. We thus conclude that infected macrophages which penetrate the endoneurium and macrophages activated in a DTH reaction can both cause neuropathy.