Metabolism of the TMA group of antigens during the growth cycle of mycobacteria

The TMA (thermostable macromolecular antigens) group includes A60 of Mycobacterium tuberculosis and A7 of M. leprae, active components of tuberculin and lepromin. We have previously described the purification and composition of A60, and its ability to elicit immune reactions of humoral and cellular type. In the present work, the intracellular and extracellular distribution and composition of A60 have been traced, as a function of the replication cycle, in static surface cultures of M. bovis. In exponentially-growing mycobacteria, most A60 was present in the cytoplasm and had a high protein/polysaccharide ratio: this ratio, as well as the level of cytoplasmic A60, decreased after cessation of cell proliferation. The A60 fraction located within the cell wall increased during the stationary phase, but its protein/polysaccharide ratio underwent minor changes. A release of cellular polypeptides and polysaccharides into the extracellular fluid occurred during the declining and lysing phases: a fraction of it was represented by A60. This explains the practice of old tuberculin preparation by autoclaving filtrates of autolysed mycobacterial cultures. The pattern of an A60-like antigen in shaken homogeneous cultures of M. smegmatis was similar (most antigen present in cytoplasm during growth, increase of the wall fraction in stationary phase, and extracellular release during the declining phase)

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

Surface static cultures of Mycobacterium bovis BCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 6O (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29-45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60- and non-A60-proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins

Composition and Immunogenicity of the Polysaccharide Components of the Thermostable Macromolecular Antigen Group of Mycobacterial Antigens

The thermostable macromolecular antigen (TMA) group includes major components of the mycobacterial cell envelope and cytoplasm, which elicit humoral and cellular immune reactions, and seems to play important roles in infectious diseases. The best known member of this group, antigen A60 of Mycobacterium bovis BCG, was previously shown to contain three moieties of polysaccharides, free lipids, and polypeptides. In this work, the TMA polysaccharides of three pathogenic mycobacteria (M. avium, M. bovis and M. paratuberculosis) have been analyzed by coupled gas chromatography-mass spectrometry. In all cases the cores of the TMA complexes were represented by branched glucans of high molecular mass (about 10(6) daltons), for which structural models have been proposed. The immunogenicity of the polysaccharide components from the three TMA was verified with several immunological procedures (immunodiffusion and immunoelectrophoresis of the antigen, and immunoblotting of the corresponding electrofocused immunoglobulins). All tests tallied in showing a negligible immunogenicity of the glucans examined (inability to produce, upon injection, the synthesis of specific immunoglobulins), thus pointing to the protein moiety of TMA as the one responsible for the high immunoreactivity of the complexes

Circulating urotensin II levels in moderate to severe congestive heart failure: its relations with myocardial function and well established neurohormonal markers.

Urotensin II (UII) is a potent vasoactive cyclic peptide thought to play a role in myocardial hypertrophy and remodelling. We therefore determined UII plasma levels in congestive heart failure (CHF) patients and its relationship with the severity of the disease and well-established markers of left ventricular function. UII was significantly higher in CHF patients (n = 57) than in controls (n = 48) [geometric mean (pg/ml), 95% PI: 1.32 (0.67-2.59) versus 0.84 (0.31-1.61), p < 0.0001], was related to the functional class of the disease and correlated negatively with left ventricular ejection fraction (r = -0.316, P = 0.016). Furthermore, UII correlated significantly with Big-ET1 (r = 0.32, p = 0.03), BNP (r = 0.42, p = 0.005) but poorly with Nt-proANP (r = 0.28, p = 0.07). Our results suggest that UII could play a role in worsening the course of congestive heart failure and is associated with established markers of cardiovascular dysfunction

Accuracy of N-terminal-pro-atrial natriuretic peptide in patients admitted to emergency department.

OBJECTIVE: B-type natriuretic peptide (BNP) and N-terminal-pro-BNP (Nt-proBNP) are commonly used for the triage of patients in the emergency department (ED) with dyspnoea and/or chest pain. The aim of our study was to determine the accuracy of N-terminal-pro-ANP (Nt-proANP) in such patients. MATERIAL AND METHODS: Nt-proANP was measured by home-made radioimmunoassay in 137 ED patients admitted with cardiovascular and/or pulmonary disorders. BNP and Nt-pro-BNP were determined with automated assays. Final diagnosis was confirmed at discharge or after follow-up. RESULTS: Nt-proANP levels were significantly influenced by the diagnostic subgroups (ANOVA: p<0.001) and were [geometric mean (range)]: 19727 ng/L (5260-45200) in congestive heart failure (CHF, n=31), 6575 ng/L (1350-36000) in coronary artery disease (CAD, n=19), 5324 ng/L (1710-13150) in pulmonary embolism (PE, n=20), 5035 ng/L (1510-16600) in pulmonary diseases (PD, n=24) and 3001 ng/L (750-11860) in patients without cardiopulmonary diseases (n=43). Pairwise comparisons demonstrated that CHF patients had Nt-pro-ANP values higher than all other groups (p<0.05) and that patients without cardiopulmonary diseases had the lowest values (p<0.05). For diagnosis of CHF, the area under the ROC curve of Nt-proANP was 0.94 (95 % CI: 0.89-0.98) and was equivalent to Nt-proBNP (0.91; p=0.284) and BNP (0.93; p=0.572). CONCLUSIONS: The diagnostic accuracy of Nt-proANP was equivalent to BNP and Nt-proBNP in the present cohort of patients admitted to ED with dyspnoea and/or chest pain