Stimulation of lymphoid cells by components of BCG.

BCG was fractionated into a delipidated mycobacterial cell fraction (DMC) and lipid by exhaustive chloroform-methanol extraction. The effects of these fractions were tested on mouse spleen cells, nonadherent spleen cells (lymphocytes), thymus cells, and adherent spleen cells (macrophages) in vitro and were compared with effects of the whole bacilli and a methanol-extraction residue (MER). Tritiated thymidine incorporation into spleen cells, purified spleen lymphocytes, and thymus cells was measured as an indicator of activity on these cells; lymphocyte-activating factor (LAF) production was used to measure activation of macrophages. DMC and MER were at least equivalent to, and often exceeded, whole BCG in their stimulation of spleen cells and spleen lymphocytes. DMC was a poor thymic mitogen in contrast to MER, which was as strong as BCG in this regard. Lipid was far less effective a mitogen for all cells tested, and failed to augment the effectiveness of DMC on thymus cells when both were present in the incubation mixture. LAF production was significantly increased by whole BCG (18-fold above controls), whereas each fraction increased production threefold to sixfold. These in vitro results seemed to reflect the known in vivo activity of BCG and its components and suggest further antitumor applications.

Monoglycosyldiacylphenol-phthiocerol of Mycobacterium tuberculosis and Mycobacterium bovis.

The structure of a minor glycolipid of M. tuberculosis (strain Canetti) is shown to be 2-O-methyl-alpha-L-rhamnosyldiacylphenol-phthiocerol. A similar compound with non-methylated rhamnose as sugar moiety was also detected. In the course of this work, the structure of mycoside B from Mycobacterium bovis was reexamined, and was shown to be identical to that of the 2-O-methylrhamnosyldiacylphenol-phthiocerol of the Canetti strain, while it was described as a 2-O-methyl-beta-D-rhamnosyl derivative in the literature. This result is in agreement with the known close relationship between M. tuberculosis and M. bovis. Careful examination of chromatographic fractions containing the above mentioned lipids showed that the occurrence of mycoloyl residues in some phenol-phthiocerol glycolipids, postulated in the literature, was likely to be due to the presence of glycerol monomycolate contaminants.

Extraction and localization by electron microscopy of an immunosuppressor fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG).

BCG has been used all over the world to immunize against tuberculosis. Nevertheless in certain areas (South India) BCG vaccines failed to show any protective efficacy. Furthermore immunosuppressive cell populations have been reported in experimental mycobacterial infection in mice. The present work reports the localization and isolation of an immunosuppressor fraction from BCG. This lipid fraction called WDB inhibited the skin reactivity of delayed-type hypersensitivity (DTH) to the test antigen CEWA (crystalline egg white albumin) in guinea pigs and depressed the production of immune antibody to SRBC (sheep red blood cells) in mice. WDB is a glycolipid with an approximate mol.wt. of 62,000. By electron microscopy, WDB was located among the BCG extracellular metabolic products (ECMP) surrounding the BCG cell wall.

Purification and characterization of a tuberculin-active substance from Mycobacterium bovis BCG.

A new tuberculin-active substance, designated TAS-1D3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 is a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1D3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1D3 was about forty times more potent than that of purified protein derivative (PPD).

Characterization of methanol extraction residue (MER) from Bacillus Calmette-Guérin (BCG).

Analysis of methanol extraction residue (MER) from Bacillus Calmette-Guerin (BCG) was carried out to determine some specific chemical compositional characteristics. Samples of MER were found to contain approximately 40% protein and/or peptide, 3% soluble lipids, 17% bound lipids, 8% elemental nitrogen, and less than 2% mycolic acids. Amino acid analysis showed the presence of alanine, glycine and glutamic acid as the major amino acids. The data are reported in terms of the range found for each constituent over the samples analyzed. Somewhat consistent results were obtained between different MER preparations, but notable compositional variations were observed in samples of MER suspensions.

Local adoptive transfer of delayed-type hypersensitivity to tuberculin from M. bovis (BCG) infected mice.

Immunological properties of the cells mediating delayed-type hypersensitvity to tuberculin and genetic requirements of this reaction have been studied by the method of local adoptive transfer. Peritoneal cells from BCG-immunized mice transferred the reaction into unprimed recipients without any genetic restriction. In contrast, nonadherent peritoneal cells transferred the reaction only in H-2-compatible donor-recipient strain combinations. The use of H-2 recombinant strains has shown that delayed-type hypersensitivity to tuberculin in mice is restricted by the I-A locus. Monoclonal antibody against I-Ak beta abrogated (without complement) the transfer of the reaction to CBA recipients. The phenotype of nonadherent cells, transferring the reaction has been shown to be Thy-1+, Lyt-1+, 2-, L3T4+.

Rapid identification of Mycobacterium bovis by a thin-layer chromatographic technique.

Lipid analyses were performed on 107 isolates of mycobacteria, using thin-layer chromatography. A distinctive blue-gray spot was observed after spraying and charring at an Rf of 0.75 for 35 of 38 Mycobacterium bovis isolates examined. The spot, designated M bovis identifying lipid, was observed only with M bovis and was not seen on any other of 9 mycobacterial species examined. This procedure has a high rate of agreement (97.2%) with standard biochemical tests and can rapidly detect M bovis from naturally occurring infections. Although chemical and antigenic characteristics are not fully elucidated, the apparent specificity of this spot for M bovis represents a simple timesaving method for differentiating this species from other mycobacteria when compared with conventional methods presently used.

Identification of Mycobacterium bovis, using miniaturized thin-layer chromatography and morphologic characteristics.

Lipid analysis was performed on 204 isolates of mycobacteria, using miniaturized thin-layer chromatography (MTLC). A previously described lipid component was found in 92 (96.8%) of 95 isolates of Mycobacterium bovis and in 2 (1.8%) of 109 isolates of other mycobacteria. Using MTLC, an isolate could be identified as M bovis 3 to 4 weeks earlier than by use of conventional biochemical techniques. Examination of isolates for colonial and cellular morphologic characteristics was a useful adjunct in the identification of M bovis by use of MTLC.

[Identification of Mycobacteria and measurement of whole cell fatty acids using a gas chromatography computer system].

The technique of combination gas chromatography with computer used in measuring fatty acids of 28 species standard Mycobacteria was introduced in this study. The data of components of fatty acids in this genus and GC graphs with satisfaction were gained. Several peaks with good reproducibility were distinguished automatically, based on which, M. tuberculosis, M. bovis and other comment atypical Mycobacteria may be separated respectively. It was showed that this method have the characteristics of accurate, sensitive and rapid by means of the identification of clinical strains, also have certain practical value in clinical laboratory.

[Effect of thermal treatment on the structure and biological properties of tuberculin]. / Vliianie termicheskoi obrabotki na strukturu i biologicheskuiu aktivnost' tuberkulina.

The effect of heat treatment used in the course of tuberculin production by Mycobacteria tuberculosis cells on the structure and biological activity of tuberculin was studied. Methods of preparative and analytical gel-filtration were used. The largest quantity of the biologically active component was obtained after 2-hour heat treatment of the mycobacteria culture.