The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections.

A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid was not detected in bacteriologically negative tissue but lipoarabinomanan (LAM) and protein antigens were detected. Staining with LAM was strongest and gave least background. The transfer of this immunohistochemical technique to paraffin embedded material will allow examination of tissue with better morphology and from clinics without access to tissue freezing facilities.

Distribution of surface-exposed antigenic glycolipids in recent clinical isolates of Mycobacterium tuberculosis.

The distribution of surface-exposed antigenic glycolipids in seven recent clinical isolates of Mycobacterium tuberculosis was established. Thin-layer and liquid chromatographies revealed a uniformity in the glycolipid pattern. Chemical analysis of the individual glycolipids of a selected strain enabled the identification of glycolipids of serological interest in all the other clinical isolates. Phenolic glycolipid-Tb1 (PGL-Tb1) was lacking in all strains, but appreciable amounts of a partially deglycosylated version (PGL-Tb1D) were present in the seven isolates. Diacyltrehaloses (DATs) were detected in all strains, showing themselves to be major glycolipids. Lipooligosaccharides (LOS-II) were present in the seven strains studied though only in trace amounts. These results shed new light on the open debate on the distribution of these interesting glycolipids in typical clinical isolates of M. tuberculosis. In the search for a serological test for tuberculosis, and in accordance with our observations, we believe that PGL-Tb1 and LOS-II should not be the target molecules for serology and that it is worthwhile to continue investigating the value of DATs as antigens. We also believe that it would be of interest to undertake research to assess the usefulness of PGL-Tb1D as an antigen.

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.

Experimental leprosy in monkeys. II. Longitudinal serological observations in sooty mangabey monkeys.

In this study, 11 SMM were grouped and inoculated with differing doses of SMM-origin Mycobacterium leprae (ML) between 4.5 x 10(8) and 1 x 10(9) by either combined IV/IC routes or by IV or IC route alone. The combined route was the most effective in eliciting progressive, disseminated LL leprosy. In all, 6 of 7 SMM inoculated by the combined routes developed leprosy requiring treatment at some point. Only 1 of 4 inoculated by a single route developed persisting leprosy requiring chemotherapy. Either no disease or spontaneous regression of initial disease occurred in the other 3 animals inoculated by a single route. Doses in excess of 1 x 10(9) ML were more effective than lesser doses. An association was observed between the development of IgG anti-PGL-I ELISA OD values and resistance to leprosy and between IgM anti-PGL-I and leprosy progression or susceptibility. Serum PGL-I antigen levels, determined by dot ELISA, paralleled disease severity longitudinally. High positive OD values of anti-LAM IgG prior to ML inoculation were observed in the majority of leprosy-susceptible SMM in contrast to negative levels in more resistant animals. Anti-LAM IgG OD values exceeded the positive cut-off point after inoculation in 5 of 11 SMM; 3 of these 5 had concurrent detectable serum levels of PGL-I antigen.

Demonstration of PGL-I & LAM-B antigens in paraffin sections of leprosy skin lesions.

An investigation on the demonstration of PGL-I and LAM-B antigens in thirty-four paraffin embedded skin biopsies taken from leprosy patients who covered the whole spectrum of the disease and in four control specimens was carried out. Neither the PGL-I antigen nor the LAM-B antigen was demonstrated in the normal skin specimens that were used as negative control; and only the LAM-B antigen appeared in the tuberculosis specimens in which the PGL-I antigen was negative. The PGL-I antigen was demonstrated on thirty-three leprosy samples except one TT sample and the LAM-B antigen, on all samples by immunochemical staining technique. The antigens were identified as intracytoplasmic bacillary staining, in solitary, granular as well as debris patterns; and as soluble antigenic staining, in vacuolar or amorphous pattern. In LL and BL cases, the antigens were detected predominantly from macrophages and peripheral nerves in all five staining patterns; in BB cases, from macrophages mostly in the granular as well as debris patterns, from the nerves in the vacuolar pattern; while in TT and the majority of BT cases, they were mainly from nerve remnants inside the granuloma in the vacuolar or amorphous staining pattern. In addition, it is interesting to note that the immunochemical staining was able to differentiate the foamy change from the hydropic degeneration. We also found that the antigens distributed in arrector pili muscles and the walls of muscular vessels were obviously related to the unmyelinated nerve fibers innervating the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis.

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Structure of mycobacteria: recent developments in defining cell wall carbohydrates and proteins.

Work from this laboratory on the immunogens of Mycobacterium species has focused on those based on carbohydrates (with a view to the development of specific tools for the serodiagnosis of mycobacterioses) and on the cell-wall proteins, as a source of protective immunity and as a means of observing specific delayed-type hypersensitivity. Most mycobacteria are endowed with specific, highly antigenic glycolipids that are powerful for the serodiagnosis of individual mycobacterial infections: e.g., the phenolic glycolipids of Mycobacterium leprae and Mycobacterium bovis, the glycopeptidolipids of the Mycobacterium avium complex, and the acylated trehalose-containing lipooligosaccharides of species such as Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium malmoense. A search for analogous structures in Mycobacterium tuberculosis has revealed an antigenic diglycosyl diacylglycerol and the immunogenic phosphomannoinositides. Others have reported on the presence of a novel phenolic glycolipid in the Canetti strain of M. tuberculosis. The dominant carbohydrate-containing antigen of M. tuberculosis (responsible for the high-titer anti-arabinofuranosyl activity in tuberculous sera) is lipoarabinomannan, which has been purified in the native state from M. tuberculosis and shown to contain both phosphatidylinositol and phosphoinositol side-branches. The cell wall of M. tuberculosis--more precisely, the peptidoglycan skeleton--is a source of a few distinct, highly immunogenic protein antigens. The recognition, isolation, and characterization of these antigens will also be described.

Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle bacilli.

A family of major arabinose- and mannose-containing phosphorylated lipopolysaccharides was isolated from Mycobacterium leprae and Mycobacterium tuberculosis. The only antigenic member of the family, lipoarabinomannan (LAM)-B, was purified by anion exchange and gel filtration chromatography in detergent and recovered in large quantities (15 mg/g of bacteria). It yielded a broad diffuse band on polyacrylamide gel electrophoresis but appeared homogeneous by this criterion and gel filtration. Besides arabinose and mannose, it contained glycerol and a polyol phosphate and was acylated by lactate, succinate, palmitate, and 10-methyloctadecanoate. The phosphate was released by alkalinolysis and identified by thin layer chromatography and gas chromatography-mass spectrometry as myoinositol 1-phosphate. Thus, the group-specific "arabinomannan" of the genus Mycobacterium in the native state is acylated, contains the substituents of phosphatidylinositol, and is apparently membrane associated. LAM-B is one of the dominant immunogens of the leprosy bacillus reacting readily with antibodies from lepromatous leprosy patients and monoclonal antibodies in plate and nitrocellulose enzyme-linked immunosorbent assay and on electrophoretic immunoblots. It is immunologically cross-reactive with a like product from M. tuberculosis. LAM-B is clearly the pervasive "glycoprotein" antigen of the leprosy bacillus and may be the long sought lipoteichoic acid-like polymer of Mycobacterium with a role in cell wall physiology, macrophage recognition, and perhaps an involvement in cross-protective immunity.