Bone marrow-derived macrophages were stained with a variety of monoclonal antibodies (mAb) with s... more Bone marrow-derived macrophages were stained with a variety of monoclonal antibodies (mAb) with specificity for the mycobacterial heat shock protein (hsp) 60 or with specific rabbit antiserum raised against mycobacterial hsp 60. The mAb ML30 as well as the specific antiserum brightly stained bone marrow-derived macrophages whereas all the other mAb as well as normal rabbit antiserum gave no or negligible reactivity. Surface expression of the shared epitope is observed by day 3 of in vitro cultivation and markedly increased by interferon-gamma stimulation on day 9. Although hsp 60 is thought to be restricted to the mitochondrial compartment, antibody responses to this molecule have been implicated in autoimmunity. Our finding that bone marrow-derived macrophages express a cross-reactive epitope recognized by an mAb with specificity for amino acids 275 to 295 of the mycobacterial hsp 60 is consistent with such a role.
Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 1996
One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtain... more One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtained from 121 patients in the UK. To compare serotyping and genetic analysis for species identification of MAC isolates from patients with and without the acquired immunodeficiency syndrome (AIDS). Clinical MAC isolates were cultured and analyzed by serotyping, the commercially available Accuprobe kit, hybridization with genes coding for the 19 kDa and 38 kDa antigens of M. tuberculosis and fingerprinting with the pMB22 probe derived from M. paratuberculosis. Species classification on the basis of genetic analysis was similar to serovar typing, with only exceptional discrepancies. Serovar prevalence was different in the two groups of patients, and different from those reported in other countries. MAC isolates from AIDS patients were exclusively M. avium, whereas patients without AIDS had MAC infections with M. avium and M. intracellulare in about equal proportion. M. intracellulare clinica...
The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who ... more The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who had negative tuberculin purified protein derivative (PPD) skin tests at diagnosis. Polyclonal CD4 T cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4 cell lines from the disease site responded to PPD and to the 16- and 38-kDa proteins and derived epitopes in vitro. Four months after chemotherapy, the patients became responsive to PPD. The proliferative response to PPD and to the 16- or 38-kDa proteins and their derived peptides decreased in CD4 T cell lines from the disease site and increased in lines from the peripheral blood. These results indicate that CD4 T cells recognizing a vast array of M. tuberculosis epitopes are compartmentalized at the site of disease in anergic patients but appear in peripheral blood after chemotherapy.
The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children a... more The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children affected by active tuberculosis (TB) and in healthy children who tested positive (PPD+) or negative (PPD-) for purified protein derivative. PPD+ healthy and diseased children responded equally well to PPD in vitro. In contrast, only 18% of PPD+ TB patients responded to peptide p38G derived from the 38-kDa protein of Mycobacterium tuberculosis. Analysis of the whole gammadelta T cell population and of its Vgamma9/Vdelta2 subset showed similar frequencies in PPD+ children with TB and in healthy PPD+ and PPD- children. Vgamma9/Vdelta2 cells from children with TB responded to 5 different phosphoantigens similarly to those from healthy PPD+ children, but healthy PPD- children responded very poorly. Chemotherapy had contrasting effects on the tested lymphocyte population, represented by increase of alphabeta and decline of Vgamma9/Vdelta2 T cell responses. T cell responses in childhood TB may be similar to those in adult TB.
There is evidence in natural human disease and experimental infection in mice that host genetic f... more There is evidence in natural human disease and experimental infection in mice that host genetic factors influence susceptibility to infection with Mycobacterium tuberculosis and the progress of the disease. In mouse models, both H-2 and non-H-2 genes have been implicated. In this study, four inbred strains of mice (Balb/b, Balb/k, B10, B10.BR), selected for combinations of two different H-2 haplotypes on two different non-H-2 backgrounds, were inoculated with M. tuberculosis, strain H37Rv, by intraperitoneal injection. The histological features of the granulomatous inflammatory response in the liver and lungs were investigated during the first 18 weeks of the infection. Granuloma fraction, mean granuloma area, bacillary load, and the density of acid-fast bacilli within granulomata were measured. Animals of all four strains showed the same general pattern of infection with an early, and later self-limiting, infection of the liver and delayed onset, but progressive, infection of the lung. The non-H-2 related genetic background appears to influence the morphology of the granulomatous inflammatory response. In comparison, H-2 differences appeared to be small and inconsistent.
Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tubercul... more Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.
Identification of antigenic determinants of the polar immune response in leprosy may illuminate b... more Identification of antigenic determinants of the polar immune response in leprosy may illuminate both protection and pathogenesis. Thirty subjects were studied (22 with polar disease and 8 healthy controls who were heavily exposed but disease-free) by assaying the proliferative, interferon (IFN)-gamma, and antibody responses to recombinant antigens of Mycobacterium leprae (10, 28, 36, and 65 kDa). The 10-kDa antigen elicited IFN-gamma production from all tuberculoid (TT) and borderline tuberculoid (BT) patients but little from controls, lepromatous (LL), or borderline lepromatous (BL) patients (P<.05). Production of 65-kDa-specific IFN-gamma was higher in TT/BT than in controls or LL/BL patients (P<.006). All subjects produced 65-kDa-specific antibody, but it was higher in LL/BL patients than in healthy controls, whose responses were higher than in TT/BT subjects (P=.035). The 36-kDa antibody responses were selectively increased in LL/BL subjects (P<.02). The intermediate phenotype of the controls suggests that M. leprae-specific production of IFN-gamma may contribute to pathology and to protection in leprosy.
The T cell repertoire of 59 patients with untreated tuberculosis was compared with that of 46 bac... more The T cell repertoire of 59 patients with untreated tuberculosis was compared with that of 46 bacille Calmette-Guérin-vaccinated controls by assaying the proliferative responses to six permissively recognized peptides from the 16-, 19-, and 38-kDa molecules of Mycobacterium tuberculosis. A trend from higher to lower reactivity following this order: vaccinated controls > lymph node disease > localized extrapulmonary > pulmonary > pleural was seen for 4 of the peptides (P < .03). The decreased response of blood lymphocytes from patients with pleural tuberculosis was partially accounted for by sequestration of peptide-responsive cells within the pleural fluid. Chemotherapy "reversed" the depressed proliferative responses of patients with pulmonary and pleural tuberculosis depending on the peptide origin, being greatest for peptides of 16 kDa, transient for those of 19 kDa, and least for those of 38 kDa. These data demonstrate antigen specificity in the decreased responsiveness of patients with tuberculosis.
We report in this study that the nature of the injected immunogen or infection of mice can change... more We report in this study that the nature of the injected immunogen or infection of mice can change the specificity of antibodies against different epitopes of the mycobacterial 38 kDa antigen. Following immunization with a selection of 14 peptides, only p65-83 and p285-304 resulted in antibodies binding to the whole antigen, whereas the recombinant antigen induced antibodies to p201-220 and p230-249, which are non-immunogenic following peptide immunization. Killed tubercle bacilli stimulated antibodies exclusively against the N-terminal 1-20 peptide, whilst infection with live tubercle bacilli led to antibodies recognizing non-continuous epitopes, demonstrable by binding to the 38 kDa protein, but to none of the tested peptides. The most striking finding is that protein immunization failed to induce antibodies to p65-83, although this epitope is immunodominant for T cells, irrespective of the mode of sensitization. Since this peptide contains non-overlapping B (69-75) and T (75-81) cell stimulatory cores, it appears that T help dedicated to the adjacent B epitope following peptide immunization may have shifted in favour of B cells reacting either with distantly located linear epitopes p1-20 and p201-220 following immunization with the whole protein, or to conformational epitopes following tuberculous infection of mice. The mechanisms which can deviate T help from one to another B cell specificity could involve regulation of antigen processing, liable to the nature of the immunogen. The described results suggesting distinct functional pairing between T and B epitopes, but with limited fidelity, may have general implications for the design of subunit vaccines.
Bone marrow-derived macrophages were stained with a variety of monoclonal antibodies (mAb) with s... more Bone marrow-derived macrophages were stained with a variety of monoclonal antibodies (mAb) with specificity for the mycobacterial heat shock protein (hsp) 60 or with specific rabbit antiserum raised against mycobacterial hsp 60. The mAb ML30 as well as the specific antiserum brightly stained bone marrow-derived macrophages whereas all the other mAb as well as normal rabbit antiserum gave no or negligible reactivity. Surface expression of the shared epitope is observed by day 3 of in vitro cultivation and markedly increased by interferon-gamma stimulation on day 9. Although hsp 60 is thought to be restricted to the mitochondrial compartment, antibody responses to this molecule have been implicated in autoimmunity. Our finding that bone marrow-derived macrophages express a cross-reactive epitope recognized by an mAb with specificity for amino acids 275 to 295 of the mycobacterial hsp 60 is consistent with such a role.
Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 1996
One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtain... more One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtained from 121 patients in the UK. To compare serotyping and genetic analysis for species identification of MAC isolates from patients with and without the acquired immunodeficiency syndrome (AIDS). Clinical MAC isolates were cultured and analyzed by serotyping, the commercially available Accuprobe kit, hybridization with genes coding for the 19 kDa and 38 kDa antigens of M. tuberculosis and fingerprinting with the pMB22 probe derived from M. paratuberculosis. Species classification on the basis of genetic analysis was similar to serovar typing, with only exceptional discrepancies. Serovar prevalence was different in the two groups of patients, and different from those reported in other countries. MAC isolates from AIDS patients were exclusively M. avium, whereas patients without AIDS had MAC infections with M. avium and M. intracellulare in about equal proportion. M. intracellulare clinica...
The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who ... more The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who had negative tuberculin purified protein derivative (PPD) skin tests at diagnosis. Polyclonal CD4 T cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4 cell lines from the disease site responded to PPD and to the 16- and 38-kDa proteins and derived epitopes in vitro. Four months after chemotherapy, the patients became responsive to PPD. The proliferative response to PPD and to the 16- or 38-kDa proteins and their derived peptides decreased in CD4 T cell lines from the disease site and increased in lines from the peripheral blood. These results indicate that CD4 T cells recognizing a vast array of M. tuberculosis epitopes are compartmentalized at the site of disease in anergic patients but appear in peripheral blood after chemotherapy.
The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children a... more The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children affected by active tuberculosis (TB) and in healthy children who tested positive (PPD+) or negative (PPD-) for purified protein derivative. PPD+ healthy and diseased children responded equally well to PPD in vitro. In contrast, only 18% of PPD+ TB patients responded to peptide p38G derived from the 38-kDa protein of Mycobacterium tuberculosis. Analysis of the whole gammadelta T cell population and of its Vgamma9/Vdelta2 subset showed similar frequencies in PPD+ children with TB and in healthy PPD+ and PPD- children. Vgamma9/Vdelta2 cells from children with TB responded to 5 different phosphoantigens similarly to those from healthy PPD+ children, but healthy PPD- children responded very poorly. Chemotherapy had contrasting effects on the tested lymphocyte population, represented by increase of alphabeta and decline of Vgamma9/Vdelta2 T cell responses. T cell responses in childhood TB may be similar to those in adult TB.
There is evidence in natural human disease and experimental infection in mice that host genetic f... more There is evidence in natural human disease and experimental infection in mice that host genetic factors influence susceptibility to infection with Mycobacterium tuberculosis and the progress of the disease. In mouse models, both H-2 and non-H-2 genes have been implicated. In this study, four inbred strains of mice (Balb/b, Balb/k, B10, B10.BR), selected for combinations of two different H-2 haplotypes on two different non-H-2 backgrounds, were inoculated with M. tuberculosis, strain H37Rv, by intraperitoneal injection. The histological features of the granulomatous inflammatory response in the liver and lungs were investigated during the first 18 weeks of the infection. Granuloma fraction, mean granuloma area, bacillary load, and the density of acid-fast bacilli within granulomata were measured. Animals of all four strains showed the same general pattern of infection with an early, and later self-limiting, infection of the liver and delayed onset, but progressive, infection of the lung. The non-H-2 related genetic background appears to influence the morphology of the granulomatous inflammatory response. In comparison, H-2 differences appeared to be small and inconsistent.
Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tubercul... more Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.
Identification of antigenic determinants of the polar immune response in leprosy may illuminate b... more Identification of antigenic determinants of the polar immune response in leprosy may illuminate both protection and pathogenesis. Thirty subjects were studied (22 with polar disease and 8 healthy controls who were heavily exposed but disease-free) by assaying the proliferative, interferon (IFN)-gamma, and antibody responses to recombinant antigens of Mycobacterium leprae (10, 28, 36, and 65 kDa). The 10-kDa antigen elicited IFN-gamma production from all tuberculoid (TT) and borderline tuberculoid (BT) patients but little from controls, lepromatous (LL), or borderline lepromatous (BL) patients (P<.05). Production of 65-kDa-specific IFN-gamma was higher in TT/BT than in controls or LL/BL patients (P<.006). All subjects produced 65-kDa-specific antibody, but it was higher in LL/BL patients than in healthy controls, whose responses were higher than in TT/BT subjects (P=.035). The 36-kDa antibody responses were selectively increased in LL/BL subjects (P<.02). The intermediate phenotype of the controls suggests that M. leprae-specific production of IFN-gamma may contribute to pathology and to protection in leprosy.
The T cell repertoire of 59 patients with untreated tuberculosis was compared with that of 46 bac... more The T cell repertoire of 59 patients with untreated tuberculosis was compared with that of 46 bacille Calmette-Guérin-vaccinated controls by assaying the proliferative responses to six permissively recognized peptides from the 16-, 19-, and 38-kDa molecules of Mycobacterium tuberculosis. A trend from higher to lower reactivity following this order: vaccinated controls > lymph node disease > localized extrapulmonary > pulmonary > pleural was seen for 4 of the peptides (P < .03). The decreased response of blood lymphocytes from patients with pleural tuberculosis was partially accounted for by sequestration of peptide-responsive cells within the pleural fluid. Chemotherapy "reversed" the depressed proliferative responses of patients with pulmonary and pleural tuberculosis depending on the peptide origin, being greatest for peptides of 16 kDa, transient for those of 19 kDa, and least for those of 38 kDa. These data demonstrate antigen specificity in the decreased responsiveness of patients with tuberculosis.
We report in this study that the nature of the injected immunogen or infection of mice can change... more We report in this study that the nature of the injected immunogen or infection of mice can change the specificity of antibodies against different epitopes of the mycobacterial 38 kDa antigen. Following immunization with a selection of 14 peptides, only p65-83 and p285-304 resulted in antibodies binding to the whole antigen, whereas the recombinant antigen induced antibodies to p201-220 and p230-249, which are non-immunogenic following peptide immunization. Killed tubercle bacilli stimulated antibodies exclusively against the N-terminal 1-20 peptide, whilst infection with live tubercle bacilli led to antibodies recognizing non-continuous epitopes, demonstrable by binding to the 38 kDa protein, but to none of the tested peptides. The most striking finding is that protein immunization failed to induce antibodies to p65-83, although this epitope is immunodominant for T cells, irrespective of the mode of sensitization. Since this peptide contains non-overlapping B (69-75) and T (75-81) cell stimulatory cores, it appears that T help dedicated to the adjacent B epitope following peptide immunization may have shifted in favour of B cells reacting either with distantly located linear epitopes p1-20 and p201-220 following immunization with the whole protein, or to conformational epitopes following tuberculous infection of mice. The mechanisms which can deviate T help from one to another B cell specificity could involve regulation of antigen processing, liable to the nature of the immunogen. The described results suggesting distinct functional pairing between T and B epitopes, but with limited fidelity, may have general implications for the design of subunit vaccines.
Uploads