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

Boddingius, J.; Dijkman, H.

doi:10.1177/37.4.2926124

Cited by 14 publications

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“…T h e nature of this capsule has been chemically defined in two species, namely M. lepraemurium (Draper & Rees, 1973) and M . leprae (Boddingius & Dijkman, 1989;Hunter & Brennan, 1983), where it has been shown t o contain species-specific glycolipids surrounding the pathogens inside their host cells. In the case of M. tuberculosis, although speciesspecific glycolipids have been characterized in some strains (Daffk et al, 1987(Daffk et al, , 1991a, the nature of its capsule remains unknown, as these lipids were not detected in some of the virulent tubercle bacilli (Daffk e t al., 1987,1991a, b).…”

Section: A O R T a L O -M A G N E A N D O T H E R Smentioning

confidence: 99%

Molecular composition of the outermost capsular material of the tubercle bacillus

et al. 1995

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To gain insight into the pathogenesis of tuberculosis, a molecular definition of the tubercle bacillus cell envelope, which is involved in the early stages of the infection, is required. The cell-surface-exposed material of the pathogen was isolated by mechanical means and chemically analysed. It was shown by scanning electron microscopy that the method used for extracting the surfacecovering material preserves the integrity of the bacilli. Surprisingly, in view of the current opinion, only small amounts of lipids ( 1 4 % ) were present. Polysaccharides and proteins were the main components of the material. The polysaccharides were neutral and lipid-free D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 120,13 and 4 kDa, respectively. Based on NMR spectroscopy and conventional chemical analyses, the major structural motifs of the purified polysaccharides were established as being identical to those of the polysaccharides we previously isolated from the culture filtrate of the tubercle bacillus. lmmunocytochemical studies showed that these compounds were not only surface-located but were also present in the inner capsular compartment. The major protein constituents exhibited the same mobilities on SDS-PAGE as those of the culture filtrate of the tubercle bacillus and readily reacted with the monoclonal antibodies directed against these molecules. These proteins included the 19 and 38 kDa lipoproteins, the 30/31 kDa fibronectin-binding proteins and the 40 kDa L-alanine dehydrogenase. These findings suggest that the culture filtrate material represents part of the capsule which, in an in wivo context, could contribute to the electron transparent zone surrounding the tubercle bacillus. The 24 kDa (MPBTT64) protein was found to be a secreted protein, as it was detected almost exclusively in the culture filtrate. Taken together, the data give a new insight into the surface-exposed compounds of the tubercle bacillus and may explain part of the nature and limitation of the host immunity towards the pathogen.

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Section: A O R T a L O -M A G N E A N D O T H E R Smentioning

confidence: 99%

Molecular composition of the outermost capsular material of the tubercle bacillus

et al. 1995

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“…leprae were isolated from infected armadillo liver following standardized WHO/IMMLEP procedures (World Health Organization, 1980 M. leprae-gelatin blocks. Blocks containing 5 x 106 M. leprae per ml gelatin (5 %, w/v) were prepared as described previously (Boddingius & Dijkman, 1989a).…”

Section: Methodsmentioning

confidence: 99%

“…Neither the surface location of PGL-I in M. leprae, indicated by immunofluorescence (Young et al, 1984), nor possibly persisting pockets of extrabacterial PGL-I antigen in infected tissues (Boddingius & Dijkman, 1989a), have yet been investigated in detail in a quantitatively satisfactory way. The localization of either peribacterial (surface) or intrabacterial (nonsurface) PGL-I, and of small amounts of extrabacterial PGL-I, all require immuno-electron microscopic methods.…”

Section: Introductionmentioning

confidence: 99%

“…However, the method was considered merely qualitative in character, and tissue embedding in Lowicryl was thought to be essential for any quantitative evaluation of PGL-I locations (Boddingius & Dijkman, 1989a).…”

Section: Introductionmentioning

confidence: 99%

“…The first two steps were partially carried out by us in pilot studies on 36 kDa and 65 kDa protein antigens (Boddingius & Dijkman, 1989b). All three steps were partially realized in studies of PGL-I using hydrophobic Araldite resin as an embedding medium (Boddingius & Dijkman, 1989a).…”

Section: Introductionmentioning

confidence: 99%

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

Boddingius

Dijkman²

1990

Journal of General Microbiology

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Phenolic glycolipid (PGL-I), an antigen specik to Mycubacterium leprae, was localized subcellularly in M. Zeprae residing in human skin, in M. leprae isolated from armadillo liver ('isolated M Ceprae)) 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. U l t r a c r y d o n s 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 ultrathh sections. K4M-immunogold methods gave very satisfactory quantitive gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosectiom and the qualititatively superior Araldite sections, but new sites were also localized. Cell walls in human M. Zeprae and in isolated M. leprcre 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 A4 leprue cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprue-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. Zeprcre, and (2) htra-and extracellularly in epidermal areas where basal cells harboured M. kpme in untreated multibacillary patients.

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The structure of the mycobacterial cell envelope is not yet understood

Draper¹

1991

Research in Microbiology

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

Cited by 14 publications

References 0 publications

Molecular composition of the outermost capsular material of the tubercle bacillus

Molecular composition of the outermost capsular material of the tubercle bacillus

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

The structure of the mycobacterial cell envelope is not yet understood

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