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.

Serologic and immunocytochemical analysis of the Mycobacterium avium cell envelope.

Whole cell sonicates of Mycobacterium avium ATCC 15769, and subcellular fractions (CYT, cytosol; CM, cytoplasmic membrane; CWS, delipidated cell wall skeleton; OL, native lipids; and SDS, sodium dodecylsulfate extract of whole cells) were injected into rabbits to produce corresponding antisera. Immunoelectrophoretic analysis and immunochemical observations using electron microscopy showed that few of the antigens synthetized intracellularly were exported and located in the bacterial outer layers, and that the outer layers contained wall specific antigens possibly in situ assembled.

The use of subcellular components of BCG for studying host-Mycobacterium interaction in relation to leprosy

Mycobacterium bovis-BCG was sonified and centrifuged at 90,000g for 2h to obtain pellet (P90) and supernatant (S90), and bacilli broken by chilled X-press were fractionated into cell wall (CW), plasma membrane and cytosol. Rabbits were immunized with P90, S90 and whole sonified-broken BCG. The antigenic make-up of these antisera. These analysed by cross-immunoelectrophoresis using the antisera. These subcellular preparations were also used for detecting circulating antibodies in the sera of 45 leprosy patients by Ouchterlony´s technique and immunoelectrophoresis. Although there were many antigenic determinants common to more than one fraction, some components were found in only one fraction. Furthermore, different leprosy patients showed different patterns of antibody response to the antigens in the different fractions. In addition, these fractions were also used to assess cell-mediated immunity in mice sensitized with lyophilized whole BCG as well as with irradiated Mycobacterium. it was found that BCG, intraperitoneally injected, engedered different specifically antigen-sensitized populations of lymphocytes in the spleen and lymph node. Moreover, a certain degree of antigenic cross-reactivity was observed between BCG and M. leprae, possibly at the T-cell level. In vitro experiments suggested that the CW stimulated B cells, indicating a mitogenic ctivity leading to polyclonal activation. Finally, in vitro priming experiments established that these subcellular fractions could sensitize human peripheral lymphocytes, and upon secondary culture all primed cells were able to respond to homologous preparations but not always to heterologous preparations, thus offering a means to distinguish antigens of interest from those antigenically less complicated fractions at the T-cell level.