concanavalin-a and Tuberculosis--Pulmonary

Interferon-gamma release assays (IGRAs) are blood tests used to measure the amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune function in IGRAs.. This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests.. The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the highest and PHA was the lowest amount. The median values of three mitogens were in the following order: PWM≥Con A≥ positive control>>PHA-P>>negative control. PWM and PHA were heat stable, while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that calcium supplementation improved the PWM-induced production of IFN-γ.. Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a potential indicator of cytokine production in the diagnostic as well as research settings, and calcium supplementation improved stimulation.

Topics: Adult; Aged; Cations; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Female; Hot Temperature; Humans; Interferon-gamma; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocytes; Tuberculosis, Pulmonary; Young Adult

Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin-labelled mycobacterial cell wall proteins revealed five Concanavalin A-reactive proteins that bind macrophages. Among these proteins, we identified PstS-1, a 38-kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS-1 binds the mannose receptor. We purified PstS-1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS-1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m-periodate and α-D-mannosidase. Gas chromatography revealed that purified PstS-1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS-1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre-incubation of cells with GlcNAc, mannan and α-methyl mannoside. The interaction of PstS-1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.

Topics: Acetylglucosamine; Acyltransferases; Adhesins, Bacterial; alpha-Mannosidase; Animals; Antigens, Bacterial; ATP-Binding Cassette Transporters; Bacterial Adhesion; Bacterial Proteins; Cell Line, Tumor; Cell Wall; Concanavalin A; Immunoprecipitation; Lectins, C-Type; Macrophages; Mannans; Mannose; Mannose Receptor; Mannose-Binding Lectins; Membrane Proteins; Methylmannosides; Mice; Mycobacterium tuberculosis; Periodic Acid; Phagocytosis; Protein Binding; Receptors, Cell Surface; Tuberculosis, Pulmonary

This work investigated the effect of previous Mycobacterium avium exposure on the protective ability of the DNA vaccine pVAXhsp65 against inflammation in the pulmonary parenchyma. BALB/c mice were presensitized with heat-killed M. avium and then immunized with three doses of pVAXhsp65 prior to challenge with Mycobacterium tuberculosis. M. avium sensitization induced high levels of spontaneous IL-5 production that were concomitant with a positive delayed-type hypersensitivity reaction; antigen-specific IFN-γ production was also observed upon splenocyte stimulation. Prior exposure to M. avium resulted in altered cytokine and antibody production induced by immunization with pVAXhsp65; instead of a Th1 response, vaccinated mice previously exposed to M. avium developed a strong Th2 response. This switch to a Th2 response coincided with the loss of the anti-inflammatory effect of pVAXhsp65 vaccination previously observed in the pulmonary parenchyma of mice infected with M. tuberculosis. These results suggest that exposure to environmental mycobacteria can modulate immune responses induced by mycobacterial vaccines other than bacillus Calmette-Guérin.

Topics: Animals; Bacterial Proteins; Chaperonin 60; Concanavalin A; Female; Hypersensitivity, Delayed; Immunity, Cellular; Immunity, Humoral; Immunoglobulin G; Interferon-gamma; Interleukin-5; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mycobacterium avium; Mycobacterium tuberculosis; Spleen; T-Lymphocytes; Tuberculosis, Pulmonary; Vaccination; Vaccines, DNA

Cytokine messenger RNA (mRNA) expression was investigated in the spleen and lung digest cells of bacillus Calmette-Guérin (BCG)-vaccinated and non-vaccinated guinea pigs following low-dose, pulmonary exposure to virulent Mycobacterium tuberculosis. After purified protein derivative (PPD) stimulation, the levels of lung cell interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and spleen cell interleukin-12 (IL-12) p40 mRNAs were significantly increased in the non-vaccinated M. tuberculosis-infected guinea pigs compared to the BCG-vaccinated guinea pigs. In contrast, the expression of anti-inflammatory transforming growth factor-beta and IL-10 mRNAs was significantly enhanced in the spleens of BCG-vaccinated animals. Despite the presence of protective cytokine mRNA expression, the non-vaccinated guinea pigs had significantly higher lung and spleen bacterial burdens. In contrast, BCG-vaccinated guinea pigs controlled the bacterial multiplication in their lungs and spleens, indicating that both protective as well as anti-inflammatory cytokine responses are associated with a reduction in bacteria. In addition, lung digest cells from non-vaccinated guinea pigs contained a significantly higher percentage of neutrophils, CD3(+) and CD8(+) T cells, while the percentage of macrophages was increased in the BCG-vaccinated animals. Total and purified lung digest T cells co-cultured with lung macrophages (LMøs) proliferated poorly after PPD stimulation in both non-vaccinated and BCG-vaccinated animals while robust proliferation to PPD was observed when T cells were co-cultured with peritoneal macrophages (PMøs). Macrophages within the lung compartment appear to regulate the response of T cells irrespective of the vaccination status in guinea pigs. Taken together, our results suggest that type I cytokine mRNA expression is not associated with vaccine-induced protection in the low-dose guinea pig model of tuberculosis.

Topics: Animals; BCG Vaccine; Cell Proliferation; Concanavalin A; Disease Models, Animal; Guinea Pigs; Interferon-gamma; Interleukin-10; Interleukin-12 Subunit p40; Lung; Macrophages, Alveolar; Macrophages, Peritoneal; Mitogens; Mycobacterium tuberculosis; RNA, Messenger; Spleen; Stem Cells; T-Lymphocytes; Transcription, Genetic; Transforming Growth Factor beta; Tuberculin; Tuberculosis, Pulmonary; Tumor Necrosis Factor-alpha; Vaccination

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.

Topics: Animals; Antigens, Bacterial; Cell Division; Cells, Cultured; Concanavalin A; Female; Flow Cytometry; In Vitro Techniques; Mice; Mice, Inbred BALB C; Mycobacterium fortuitum; Mycobacterium tuberculosis; T-Lymphocytes; Trehalose; Tuberculosis, Pulmonary

Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients.. To elucidate the role of HLA-DR genes/gene products on lymphocyte responses to Mycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease.. Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects.. Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 micrograms/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects.. The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response against M. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects.

Topics: Adult; Concanavalin A; Female; HLA-DR Antigens; Humans; Immunity, Cellular; Interferon-alpha; Lymphocyte Activation; Lymphocytes; Male; Mycobacterium tuberculosis; Tuberculosis, Pulmonary

The relationship between alveolar macrophages (AM) and lymphocytes may be important in the early establishment of infection with Mycobacterium tuberculosis. AM in several species have been shown to suppress lymphoproliferation by producing inhibitors that include nitric oxide (NO).. To study this phenomenon in the guinea pig, the mitogen-induced proliferation of splenic lymphocytes was quantified under various conditions of co-culture with resident AM.. Guinea pig AM consistently and profoundly suppressed proliferation in the co-cultures at AM:lymphocyte ratios of 1:4 or greater. The inclusion of a NO synthesis inhibitor, N-monomethyl-L arginine (NMMA), in the co-culture medium did not influence the suppression of Con A-induced lymphoproliferation by resident guinea pig AM. No nitrite could be detected in supernatant fluids of co-cultured AM and splenocytes. Attempts to stimulate guinea pig AM with LPS in combination with recombinant murine and human IFN-gamma, infection with live Listeria monocytogenes, or incubation with the supernatants from ConA-activated guinea pig lymphocytes failed to generate NO metabolites. The addition of catalase or indomethacin to the Con A-induced AM-splenocyte co-cultures, to inhibit hydrogen peroxide (H2O2) or prostaglandin E2 (PGE2), respectively, did not counteract the suppression mediated by AM. Cell contact was necessary for the co-cultures to generate their inhibitory effects on lymphoproliferation, however, the suppression was actually mediated, at least in part, by soluble factors produced in the co-cultures.. These results suggest that resident alveolar macrophages suppress lymphocyte proliferation in the guinea pig, but that the effect is not mediated by NO, PGE2 or H2O2. The failure to demonstrate NO synthesis under a variety of stimulatory conditions, which resulted in macrophage activation, suggests that the guinea pig is similar to the human in that regard.

Topics: Analysis of Variance; Animals; Arginine; Catalase; Coculture Techniques; Concanavalin A; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Inhibitors; Female; Guinea Pigs; Hydrogen Peroxide; Immune Tolerance; Indomethacin; Lipopolysaccharides; Lymphocyte Activation; Macrophage Activation; Macrophages, Alveolar; Macrophages, Peritoneal; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Tuberculosis, Pulmonary

The relationship between Mycobacterium tuberculosis 50- to 55-kDa protein and Mycobacterium bovis BCG 45- to 47-kDa antigen was examined by using immunological and biochemical criteria. Reciprocal cross-reactivity with a rabbit polyclonal antiserum against the M. bovis BCG protein and with a monoclonal antibody raised against the M. tuberculosis antigen was observed. The epitope recognized by this antibody was apparently present only in proteins of M. tuberculosis and M. bovis BCG among the 11 mycobacterial species tested. The amino-terminal sequences and total amino acid contents of these proteins showed strong similarities. Both antigens are glycoproteins as assessed by binding of concanavalin A, labeling of carbohydrate moieties with biotin-hydrazide, and digestion of carbohydrates with jack bean alpha-D-mannosidase, which produced a reduction of the molecular weights of the proteins and totally eliminated concanavalin A binding. Both M. tuberculosis and M. bovis BCG proteins are secreted, since they were found mainly in the culture medium. Analysis of M. tuberculosis 50- to 55-kDa antigen by two-dimensional gel electrophoresis showed at least seven different components, as previously described for the M. bovis BCG antigen. Solid-phase immunoassays showed that the purified M. tuberculosis 50- to 55-kDa protein was recognized by serum specimens from 70% of individuals with pulmonary tuberculosis from a total of 77 Mexican patients examined.

Topics: Amino Acid Sequence; Amino Acids; Animals; Antigens, Bacterial; Concanavalin A; Cross Reactions; Electrophoresis, Gel, Two-Dimensional; Female; Glycoproteins; Humans; Mannosidases; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Molecular Weight; Mycobacterium bovis; Mycobacterium tuberculosis; Tuberculosis, Pulmonary

Functional activity of T-lymphocyte suppressors (spontaneous and Con-A induced) has been studied in 17 patients with destructive pulmonary tuberculosis. Patients with ++fibrous-cavernous pulmonary tuberculosis were found to have characteristic disturbances of the regulatory lymphocyte function and a high index of theophylline-resistant E-RFC/theophylline-sensitive E-RFC in the presence of a general reduction of the immunity T-system indices. A higher balance of the regulatory lymphocytes was seen in patients with infiltrative tuberculosis in various disorders of the immunologic parameters.

Topics: Adult; Concanavalin A; Female; Humans; In Vitro Techniques; Leukocyte Count; Lymphocyte Activation; Lymphopenia; Male; Middle Aged; Rosette Formation; T-Lymphocytes; Tuberculosis, Pulmonary

In vitro proliferative response of lung cells from mice infected with Mycobacterium tuberculosis H37Rv against PPD and Con A was studied. It was shown that the infected lung contained immune T cells, but their response in vitro was totally inhibited by plastic and nylon wool adherent suppressor cells. The whole population of lung cells from infected, but not intact mice, efficiently suppressed the proliferative response of immune lymph node cells against various antigens (non-specific suppression). The inhibition of response again depended on the presence of plastic adherent lung cells. Our data suggest that at least two suppressor pathways are induced in the course of tuberculosis infection: one being specific for mycobacterial antigens and other non-specific. Both types of suppressor pathways depend on the plastic adherent lung cells from tuberculosis lesion.

Topics: Animals; Antigens, Bacterial; Cell Adhesion; Cell Division; Cells, Cultured; Concanavalin A; Immune Tolerance; In Vitro Techniques; Lung; Lymph Nodes; Mice; Mice, Inbred CBA; Mycobacterium tuberculosis; T-Lymphocytes; T-Lymphocytes, Regulatory; Tuberculin; Tuberculosis, Pulmonary

In Mycobacterium tuberculosis culture filtrates, three concanavalin A (ConA)-binding bands of 55, 50 and 38 kilodaltons (kD) were identified by labelling blotted proteins with a ConA-peroxidase conjugate. Binding was inhibited by the competitor sugar alpha-methyl mannoside and by reduction with sodium m-periodate. Bands of 55, 50 and 38 kD stained with Coomasie blue were sensitive to digestion with proteases, thus indicating that they are proteins. Glycoproteins were isolated by lectin affinity chromatography or by elution from nitrocellulose membranes. On the isolated form, the 55-50 kD doublet glycoprotein was 65.4% protein and 34.6% sugar. The purified 38 kD molecule was 74.3% protein and 25.7% carbohydrate. By immunoblot, antibodies against mycobacterial glycoproteins were demonstrated in immunized rabbits and in patients with pulmonary tuberculosis, but not in healthy individuals. Treatment with sodium m-periodate abolished binding of rabbit antibodies to the 38 kD glycoprotein. Reactivity of the 55-50 kD doublet glycoprotein was not altered by reduction. By immunoblot with monoclonal antibodies TB71 and TB72, a carbohydrate-dependent and a carbohydrate-independent epitope could be identified on the 38 kD glycoprotein.

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Chromatography, Affinity; Concanavalin A; Epitopes; Glycoproteins; Humans; Molecular Weight; Mycobacterium tuberculosis; Rabbits; Tuberculosis, Pulmonary

Topics: Adolescent; Adult; Concanavalin A; Female; Humans; Male; Middle Aged; T-Lymphocytes, Regulatory; Tuberculosis, Pulmonary

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).

Topics: Adult; Concanavalin A; Female; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-2; Leukocytes; Male; Middle Aged; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocytes; Tuberculosis, Pulmonary

The percentage of total and stable E-rosette forming lymphocytes (tE-RFC and sE-RFC, respectively) were determined in the peripheral blood of patients with either active non-chronic, active chronic or inactive pulmonary tuberculosis as well as in healthy controls, both skin test positive and negative. Patients had low percentages of tE-RFC but increased values of sE-RFC when compared with controls. Lymphocytes cultured for 48 hr with or without 10 micrograms/ml of Con A exhibited decreased capacity to form total E-rosettes. Mitogen stimulation resulted in a very significant increase in the percentage of sE-RFC from control individuals. Although lymphocytes from tuberculous patients were able to respond to Con A stimulation, this response was significantly diminished compared to the controls. Incubation of the cells in the presence of various concentrations of PPD did not affect the formation of total E-rosettes by normal cells nor by lymphocytes obtained from tuberculous patients. Nevertheless, PPD induced a significant increase in the percentage of sE-RFC from normal tuberculin positive controls whereas lymphocytes from the same group incubated without antigen or cells from tuberculin negative controls cultured in the presence of 50 micrograms/ml of PPD did not form sE-RFC, indicating that the response might be antigen specific. Even though lymphocytes from patients with active tuberculosis formed higher numbers of stable E-rosettes than controls, they did not respond to antigen with significant increases of sE-RFC. In all the experiments it was not possible to demonstrate differences between the 3 groups of patients with various stages of the infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Concanavalin A; Erythrocytes; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphocytes; Rosette Formation; Tuberculin; Tuberculin Test; Tuberculosis, Pulmonary

Lymphocytes of tuberculous patients activated in vitro with purified protein derivative of tuberculin (PPD) were examined for rosette formation with human autologous erythrocytes. The percentage of the rosette-forming cell (auto-RFC) was increased when pleural fluid lymphocytes of patients with tuberculous pleurisy and peripheral blood lymphocytes (PBL) from patients with tuberculosis or from tuberculin skin test positive healthy individuals were stimulated with PPD in vitro, whereas no increase of auto-RFC was observed in PBL from tuberculin skin test negative donors. Increased numbers of auto-RFC were shown to form rosettes with sheep erythrocytes but have no IgG Fc receptors on their surfaces. It was also shown that adherent cells were required for the PPD-induced increase of auto-RFC. Depletion of PPD-induced auto-RFC by the density gradient sedimentation technique led to a significant decrease in the degree of the proliferative response to gradient sedimentation technique let to a significant decrease in the degree of the proliferative response to PPD and the number of the auto-RFC after stimulation with PPD. These findings strongly suggest that PPD-induced auto-RFC represents the consequence of an immunological interaction between sensitized T lymphocytes and relevant antigen PPD, and reflects the PPD responsiveness of tuberculosis at the T cell level.

Topics: Cell Adhesion; Cell Separation; Concanavalin A; Erythrocytes; Humans; Immunoglobulin Fc Fragments; Lymphocyte Activation; Lymphocytes; Receptors, Fc; Rosette Formation; T-Lymphocytes; Tuberculin; Tuberculosis, Pleural; Tuberculosis, Pulmonary

To estimate the potential adverse consequences of rifampin therapy on cell-mediated immunity in tuberculosis, we measured in vitro lymphocyte responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, and in vitro and in vivo responses to purified protein derivative tuberculin. Thirty-seven patients treated with therapeutic combinations containing rifampin were compared with 13 persons who had never received the drug. After initial improvement, responses to phytohemagglutinin became depressed in patients receiving rifampin for periods of 4 to 24 months. No significant changes were noted in lymphocyte responses to concanavalin A or pokeweed mitogen. In vitro and in vivo responses to purified protein derivative tuberculin were not altered. Because a favorable therapeutic outcome was achieved in all subjects, we concluded that rifampin does not have clinically significant immunosuppressive activity.

Topics: Animals; Concanavalin A; Humans; Immunity, Cellular; In Vitro Techniques; Lectins; Lymphocytes; Male; Middle Aged; Mitogens; Rifampin; Tuberculin; Tuberculosis, Pulmonary