Absence of IgM and IgG antibody response to the three components of the Mycobacterium bovis BCG 85 complex antigen after BCG vaccination.
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Adult↗
Biomedical subjects
K Huygen
Publications and source records attributed to K Huygen.
Role of the TH1-TH2 balance in the development of autoimmunity in rats.
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Animals↗
Effects of chemotherapy on antibody levels directed against PGL-I and 85A and 85B protein antigens in lepromatous patients.
IgG antibodies against antigens 85A and 85B from Mycobacterium bovis BCG, IgM antibodies against phenolic glycolipid-I (PGL-I) and circulating PGL-I antigen were measured in the serum of 11 patients with lepromatous leprosy receiving multidrug therapy (MDT). Before treatment, 6 patients were reactive to antigen 85A, 10 patients to antigen 85B, and 11 patients to PGL-I; circulating PGL-I was detected in the sera of all of them. After 2 years of MDT PGL-I antigen could no longer be detected in all of the patients, except for two who were not compliant with treatment. IgG antibodies directed against the 85A and 85B antigens and IgM antibodies against the PGL-I antigen also decreased significantly during treatment but more slowly. The determination of circulating PGL-I antigen remains the most appropriate tool for monitoring lepromatous leprosy under MDT.
Antibodies, Bacterial↗
The humoral immune response after BCG vaccination: an immunoblotting study using two purified antigens.
The specific IgG response induced by BCG vaccination was investigated in 27 adults using two purified BCG antigens in a dot immunoblotting assay. The reflectance values increased significantly after vaccination when P64 but not when P32 was used as the antigen. The values measured after vaccination were compared with those obtained when testing sera from patients with tuberculosis. We observed that the level of anti-P64 IgG reached after BCG immunization was high enough to preclude a serological differential diagnosis with tuberculosis.
Adult↗
Isoelectrophoretic characterization of protein antigens present in mycobacterial culture filtrates and recognized by monoclonal antibodies directed against the Mycobacterium bovis BCG antigen 85 complex.
Nine monoclonal antibodies (MoAbs) were produced against Mycobacterium bovis BCG antigen 85 complex. Using isoelectric focusing combined with Western (immunoblot) blot analysis, antigenically related proteins could be identified in culture filtrates from M. tuberculosis, M. bovis, M. Kansasii, M. avium, M. xenopi, M. gordonae, M. fortuitum, M. phlei and M. smegmatis. Most of the MoAbs were found to be broadly cross-reactive between the various mycobacterial species, albeit some minor differences were observed. These MoAbs reacted generally, in each species, with different components. One MoAb (VID1-14) was found to be specifically directed only against antigen 85B from M. bovis, M. tuberculosis and M. kansasii.
Animals↗
Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins.
Although demonstration of delayed hypersensitivity to purified protein derivative of tuberculin (PPD) is an important element in the diagnosis of infection with Mycobacterium tuberculosis, many patients with tuberculosis are anergic. Several possible mechanisms for this specific lack of response have been described. We have now uncovered an additional one. T-cell fibronectin (FN), a lymphokine secreted by activated T cells, is closely associated with the initiation of delayed hypersensitivity reactions. Mycobacterial antigen 85 (Ag85) proteins have been shown to bind to plasma FN. The ability of Ag85 to bind to T-cell FN and modulate expression of delayed hypersensitivity was therefore studied. Purified Ag85 proteins from M. tuberculosis, Mycobacterium bovis BCG, or Mycobacterium kansasii bound to T-cell FN, fibroblast FN, and plasma FN in vitro. Purified 65-kDa heat shock protein (hsp65) from M. bovis BCG did not bind to any FN. Ag85, but not hsp65, inhibited the ability of T-cell FN to agglutinate monocytes in vitro in a dose-dependent manner. In vivo, mixtures of PPD or dinitrophenyl-ovalbumin and purified M. tuberculosis or M. bovis BCG Ag85 proteins elicited significantly smaller delayed hypersensitivity inflammatory reactions in sensitized guinea pigs than did PPD or dinitrophenyl-ovalbumin alone. Purified hsp65 did not inhibit expression of delayed hypersensitivity to PPD or dinitrophenyl-ovalbumin. We suggest that Ag85 proteins could inhibit in vivo expression of delayed hypersensitivity during mycobacterial infections because of their interaction with T-cell FN.
Agglutination↗
Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.
Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.
Animals↗
Humoral responses against the 85A and 85B antigens of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.
Immunoglobulin G antibodies against the 85A and 85B components of the Mycobacterium bovis BCG antigen 85 complex separated by isoelectric focusing were investigated in serum samples from 129 patients representing the major forms of leprosy, 111 tuberculous patients, and 153 healthy subjects. For both of the antigens, a higher degree of staining was observed for lepromatous leprosy patients and patients with active tuberculosis than for the other groups. Because sera from some healthy subjects recognized the 85A antigen, we suggest that antigen 85B is the most useful component of the antigen 85 complex for the serodiagnosis of the multibacillary forms of leprosy or of the active forms of tuberculosis.
Antibodies, Bacterial↗
An isoelectric focusing method for the study of the humoral response against the antigen 85 complex of Mycobacterium bovis BCG in the different forms of leprosy.
Isoelectric focusing was used to separate the three components of the antigen 85 complex of Mycobacterium bovis BCG. Antibody responses of leprosy patients against each Mycobacterium bovis BCG. Antibody responses of leprosy patients against each component were quantitated by densitometric analysis of immunoblot assays. The 85A component was recognized by 40% (8/20) of the lepromin positive and negative healthy subjects, by 76% (19/25) of the tuberculoid and by 96% (24/25) of the lepromatous leprosy sera. In contrast, the 85B component was not stained by the control sera, nor by the tuberculoid leprosy sera but by 64% (16/25) of the lepromatous leprosy sera. The results suggest that antigen 85B contains one or several epitopes that are specifically recognized by sera of lepromatous leprosy patients only.
Antibodies, Bacterial↗
Interleukin-6 production in Mycobacterium bovis BCG-infected mice.
Mycobacterium bovis BCG and its subcellular components (bacterial extract, culture filtrate, purified protein derivative, and muramyl dipeptide MDP) are potent in vitro IL-6 inducers in spleen cell cultures from uninfected and BCG-infected BALB/c mice. Both plastic adherent and nonadherent spleen cells are capable of producing IL-6. Athymic nude mice produce more IL-6 than euthymic mice, suggesting that monocyte/macrophages are the main IL-6-producing cells in response to BCG. Finally, IL-6 production seems to be controlled to some extent by T lymphocytes, as down-regulation of CD4+ cells resulted in a marked increase in IL-6 production. Interferon-gamma does not seem to be involved in this regulation.
Animals↗
T cell response to purified filtrate antigen 85 from Mycobacterium bovis Bacilli Calmette-Guérin (BCG) in leprosy patients.
T cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from 25 healthy controls and 39 leprosy patients were tested against BCG-bacilli and culture filtrate. Mycobacterium leprae and purified antigen 85 (the major secreted 30-32 kD protein antigen) from M. bovis strain BCG. In lepromin negative healthy controls, blastogenesis was low to M. leprae and completely negative to antigen 85. IFN-gamma levels were very low, close to detection limits. In all lepromin positive controls, significant proliferation and IFN-gamma secretion was found in response to M. leprae and antigen 85. In the group of lepromatous leprosy (LL) patients, 25/29 of patients (with either positive (13) or negative (12) lymphoproliferative response to BCG) were unreactive to M. leprae or to antigen 85. Four LL patients with positive T cell response to BCG responded with detectable lymphoproliferative response and IFN-gamma secretion to antigen 85. All tuberculoid (TT) leprosy patients responded to BCG, M. leprae and antigen 85. Hence, T cells from leprosy patients and controls demonstrate a marked parallelism of responsiveness towards whole M. leprae and purified antigen 85 from M. bovis BCG, suggesting strong cross-reactivity between the two species and underlining the biological importance of such secreted antigens.
Antigens, Bacterial↗
Humoral immune response of tuberculous patients against the three components of the Mycobacterium bovis BCG 85 complex separated by isoelectric focusing.
An isoelectric-focusing technique followed by Western blot (immunoblot) analysis was used to investigate the immunoglobulin G response of tuberculous patients against each of the three components of the Mycobacterium bovis BCG antigen 85 complex. The 85A component was stained by the tuberculous as well as the non-tuberculous sera. In contrast, the 85B and the 85C proteins of the complex were not stained by the control sera but were stained by 20 of 28 tuberculous serum samples.
Adult↗
Antibody levels to whole culture filtrate antigens and to purified P32 during treatment of smear-positive tuberculosis.
Antimycobacterial IgG levels were measured repeatedly during treatment in 12 patients with moderate or severe pulmonary tuberculous disease using a dot immunobinding assay. We used reflectance densitometry equipment to quantify the immunoperoxidase staining and a Mycobacterium bovis BCG culture filtrate and the purified P32 protein as antigens. Antibody response against whole culture filtrate and P32 antigen increased after a three-month period of treatment. After this antibiotherapy was completed, the estimated amount of antibodies directed against the P32 decreased while those against the whole culture filtrate remained at the same level. A serologic test using P32 as the antigen seems to allow a better discrimination between active and healed tuberculosis.
Antibodies, Bacterial↗
Poly I:C activated macrophages are tumoricidal for TNF-alpha-resistant 3LL tumor cells.
Successive coculture of Lewis lung carcinoma (3LL) cells with T cell-derived lymphokines and LPS-activated macrophages has led to the acquisition of 3LL tumor variants (macrophage-resistant 3LL tumor variants (3LL-R)), manifesting a highly reduced sensitivity to the cytotoxic potential of T cell-derived lymphokines and LPS-activated macrophages and TNF-alpha. However, when 3LL-R cells are cocultured with Poly I:C-activated macrophages or with conditioned medium derived from these effector cells a significant lysis is observed. TNF-alpha participates in the cytolytic process of Poly I:C-activated macrophages as anti-TNF-alpha antibodies abolish the cytotoxic effect of these effector cells. In addition, class I IFN is involved because IFN-alpha and IFN-beta act synergistically on TNF-alpha mediated lysis of 3LL-R cells within 18 h. Moreover, anticlass I IFN antibodies abolish the cytolytic capacity of Poly I:C-activated macrophages. Hence, Poly I:C-induced macrophage-mediated cytolysis of 3LL-R cells may result from 1) the induction of macrophages by Poly I:C to secrete high amounts of TNF-alpha and class I IFN and 2) a synergism between IFN-alpha/IFN-beta and TNF-alpha on lysis of 3LL-R cells. This synergism does not result from a class I IFN-mediated enhancement of TNF-alpha receptor expression on 3LL-R cells. Therefore, the sensitivity of 3LL-R cells to TNF-alpha-mediated lysis in the presence of class I IFN is most probably regulated at the post-TNF-alpha receptor level. Furthermore, treatment of mice with Poly I:C strongly reduces the metastatic capacity of 3LL-R tumor cells, suggesting the participation of macrophages in the eradication of the established metastasis. Hence, TNF-alpha-resistant 3LL-R tumor cells may serve as a useful tool for the detection of alternative macrophage-related cytotoxins leading to the destruction of neoplastic cells both in vitro and in vivo.
Animals↗
Antibody repertoire against culture filtrate antigens in wild house mice infected with Mycobacterium bovis BCG.
Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 10(6) variable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4, 6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.
Alleles↗
Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG.
Two susceptible (Bcgs) mouse strains, BALB/c and C57BL/6, were compared by Western blot (immunoblot) analysis for their immunoglobulin G response to 14-day-old BCG culture filtrate (CF) following intravenous infection with live Mycobacterium bovis BCG. The two strains demonstrated a completely different antibody repertoire. BALB/c antibodies were directed against a wide range of CF antigens between 20 and about 100 kilodaltons (kDa), with a preferential recognition of the 65-kDa heat shock protein and the 32-kDa fibronectin-binding protein. C57BL/6 sera, on the other hand, showed a much more restricted antibody pattern, almost exclusively directed against three antigens with estimated molecular sizes of 37, 38, and 40 kDa. Whereas the 37- and 38-kDa antigens were also recognized by BALB/c mice, the 40-kDa antigen was very intensely stained by C57BL/6 sera only. F1 mice had the restricted antibody pattern of C57BL/6 after one injection of BCG and had a hybrid BALB/c-C57BL/6 phenotype following a boost injection of BCG 2 months after the initial infection. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments suggests that a gene in the K-IA region of the H-2 locus is associated with the preferential recognition of certain CF antigens. Inoculation with the same dose of killed BCG failed to elicit an antibody response to these filtrate antigens.
Animals↗
The 32 kDa protein antigen of M. bovis B.C.G. and M. tuberculosis H37Rv.
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Antibodies, Bacterial↗
Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis.
We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140- or 125-kDa beta-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, M. Weckx, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promoter sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG alpha-antigen (K. Matsuo, R. Yamaguchi, A. Yamazaki, H. Tasaka, and T. Yamada, J. Bacteriol. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B- and T-cell epitope regions on the 32-kDa antigen.
Amino Acid Sequence↗