Subcellular localization of Mycobacterium leprae-specific phenolic glycolipid (PGL-I) antigen in human leprosy lesions and in M. leprae isolated from armadillo liver.

Phenolic glycolipid (PGL-I), an antigen specific to Mycobacterium leprae, was localized subcellularly in M. leprae residing in human skin, in M. leprae isolated from armadillo liver ('isolated M. leprae') and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. Ultracryosections and Araldite sections of comparable specimens were used for comparison of localization results. A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathin sections. K4M-immunogold methods gave very satisfactory quantitative gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosections and the qualititatively superior Araldite sections, but new sites were also localized. Cell walls in human M. leprae and in isolated M. leprae possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated M. leprae cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprae-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. leprae, and (2) intra- and extracellularly in epidermal areas where basal cells harboured M. leprae in untreated multibacillary patients.

Immunogold labeling method for Mycobacterium leprae-specific phenolic glycolipid in glutaraldehyde-osmium-fixed and Araldite-embedded leprosy lesions.

Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-specific antigen currently used for serodiagnosis of preclinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Araldite-embedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching of plastic sections with H2O2, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal antibodies to PGL-I. Gold labeling was seen predominantly on cell walls of M. leprae, in vacuolar spaces of bacillated phagolysosomes, and occasionally on the cytoplasm and cell membrane of M. leprae. Host macrophage cytoplasm was labeled very infrequently. This technique allows studies on possibly persisting antigenic PGL-I in multibacillary leprosy patients during or after multidrug therapy. The method may also prove useful for subcellular localization of specific bacterial lipids in other mycobacterial diseases, including tuberculosis.

In situ locations of Mycobacterium leprae-specific antigens. Immunoelectronoptical studies.

Lipid or protein antigen sites in Mycobacterium leprae proper and in M. leprae -infected human or armadillo tissues were investigated by immunogold-electron microscopy. Simultaneous preservation of immunogenicity of antigens and conservation of ultrastructural details of M. leprae and host cells was aimed at by subjecting organisms and tissues, prior to immunolabelling, to differing fixation, embedding and ultramicrotomy techniques. The M. leprae-specificity of monoclonal antibodies (MoAbs) utilized in the study was tested first. Hereto, ultracryosections of M. leprae, M. tuberculosis and M. nonchromogenicum suspended in gelatin were employed. MoAb anti-phenolic glycolipid I (PGL I) and MoAb anti-36 kD were found to be specific for M. leprae. MoAb anti-65 kD also labelled the cytoplasm of M. tuberculosis. After incubation with MoAb anti-lipid MAIS, employed as control MoAb, no gold labelling of leprosy bacilli or host cells was seen. PGL-I immunogenicity was still present after "hard" fixation of M. leprae and host cells in glutaraldehyde-OsO4 and after Araldite embedding. This enabled the qualitative demonstration of PGL-I inside the cell wall and capsular area of M. leprae and in vacuoles of bacillated phagolysosomes of macrophages in Araldite-embedded human skin biopsies and armadillo liver parenchymal cells. Sites of 65 kD and, to a lesser extent, of 36 kD protein antigens in M. leprae were demonstrable only in ultracryosections of non-fixed organisms and not in Araldite sections. Results are discussed and recommendations for future investigations on M. leprae antigen sites are presented.