Levamisole augmentation of lymphocyte hyporesponsiveness to phytohaemagglutinin in patients with pulmonary tuberculosis.

Levamisole has been shown to augment the in vitro responsiveness of lymphocytes to PHA stimulation. The response of lymphocytes of 14 (42%) of 33 patients with pulmonary tuberculosis was augmented by levamisole. Augmentation was observed in 9 (64%) of 14 PHA hyporesponsives lymphocytes and in 5 (26%) of 19 normo-responsive lymphocytes.     

Progression of pulmonary tuberculosis and efficiency of bacillus Calmette-Guérin vaccination are genetically controlled via a common sst1-mediated mechanism of innate immunity

Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1(S) macrophages responded to stimulation with IFN-gamma in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4(+) T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1(S) animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Chromosome analyses in man in the course of chemoprophylaxis against tuberculosis and of antituberculosis chemotherapy with isoniazid

Summary Cytogenetic analyses were carried out in lymphocytes of 37 persons before and during a primary chemoprophylaxis or a preventive chemotherapy with isoniazid as weil as of 30 patients suffering from active pulmonary tuberculosis before and during a chemotherapy with isoniazid in combination with two other antimycobacterial drugs. The results of the four participating research groups consistently reveal no indication for a chromosome damaging activity of isoniazid, of its metabolites in the human organism and of the administered drug regimens.     

Natural antimycobacterial metabolites: current status

Over the years the introduction of very effective drugs has revolutionized the treatment of tuberculosis. In recent years, however, emerging multiple drug resistance has become a major threat and thus calls for an urgent search for new and effective treatments for this deadly disease. This review is complementary to earlier reviews and covers more recent reports of naturally occurring compounds, and in some cases synthetic analogs, largely from plants, fungi and marine organisms that demonstrate significant activity in the in vitro bioassays against Mycobacterium tuberculosis, and other mycobacterial species. Included also are traditional medicinal uses of specific plants when utilized to treat tuberculosis and other pulmonary diseases.     

The potential of azole antifungals against latent/persistent tuberculosis

The aim of the present study was to evaluate the chemotherapeutic potential of econazole against latent tuberculosis. The activity of econazole and clotrimazole was tested against the latent bacilli (Mycobacterium tuberculosis H(37)Rv) developed by nutrient starvation under in vitro conditions and by drugs under in vivo conditions. The latent bacteria developed under in vitro latent conditions were acid-fast negative, nonreplicating, resistant to conventional antitubercular drugs and showed low respiration rates. Econazole as well as clotrimazole were found to have strong antimycobacterial potential against latent Mycobacterium tuberculosis under in vitro conditions as seen by reductions in colony-forming units. Further, econazole prevented the formation of drug-induced latency and significantly reduced bacterial burden from lungs and spleens of latent tuberculosis-infected mice. We conclude that azole drugs bear significant therapeutic potential against latent tuberculosis.     

Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection

Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.     

Monocyte cytokine secretion in patients with pulmonary tuberculosis differs from that of healthy infected subjects and correlates with clinical manifestations

Cell-mediated immunity, leading to Mycobacterium tuberculosis (Mtb)-constraining granuloma formation, is the major component of host defense against tuberculosis and is regulated by the balance of cytokines secreted mostly by mononuclear phagocytes and lymphocytes. To better understand the role of monocytes in the regulation of the immune response against pulmonary tuberculosis, we examined IL-10, IL-12 and TNF-alpha release by monocytes from healthy purified protein derivative (PPD) reactors and pulmonary tuberculosis patients with or without systemic reactions (e.g., fever, weight loss, asthenia). Our study shows that, probably as a result of in vivo priming by circulating antigens, monocytes from patients, especially those with systemic manifestations, have a biased ex vivo cytokine secretion, with high IL-10 and TNF-alpha but low IL-12, in contrast with PPD reactors. Higher spontaneous IL-10 and TNF-alpha release persisted when monocytes were co-cultured with autologous lymphocytes. Challenge of patients' monocytes with a virulent Mtb strain led to a further enhancement of IL-10 and TNF-alpha, but not of IL-12. When lymphocytes were added to these cultures, IL-10 and TNF-alpha elevation persisted and, in the patients with a systemic reaction, both IL-12 and IFN-gamma were significantly reduced compared to PPD reactors. Intragroup comparisons revealed that in the patients with systemic reactions, the lymphocyte-monocyte interaction resulted in a positive feedback for IL-10 secretion, while in the patients without systemic reaction and PPD reactors, the feedback was positive for IL-12 secretion. Thus, in tuberculosis, there appears to exist a relationship between the immunological findings and the distinct clinical manifestations.     

Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG

Background

In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production.

Methodology/Principal Findings

To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli.

Conclusions/Significance

We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG''s ability to protect against pulmonary TB.     

Human immunoregulating (suppressing?) CD4+, CD25+ T lymphocytes ex vivo and in vitro,in the normal state and in pathology

Among T lymphocytes CD4+, the subpopulation of cells carrying the alpha-chain of the receptor of interleukin-2 (CD25) and designated as regulating T cells (Treg) has recently been marked out. Such cells produce mainly suppressing effect in the immune system and have been identified not only in experimental animals, but also in humans. The prolonged experience of the evaluation of such cells has been summarized with the cells evaluated in the peripheral blood and in culture, after their stimulation in vitro, lymphocytes obtained from healthy persons and patients with some diseases leading to the development of immunodeficient states (bronchial asthma in the state of exacerbation, newly diagnosed pulmonary tuberculosis, chronic pyelonephritis). In all cases an increased circulation of T cells CD4+, CD25(4), which may be indicative of their immunosuppressing action was found. The correlation between the high level of these cells ex vivo and a decrease in the proliferative activity of T cells in vitro is noted. The level and functional properties of the subpopulation of T lymphocytes CD4+, CD25+ are among the most informative criteria of the functioning of me immune system in the normal state and in immunopathology. Search for medicinal preparations modulating the function of human regulating T lymphocytes is necessary.     

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.     

Up-regulation of VCAM-1 and differential expansion of beta integrin-expressing T lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis infection

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of beta1high/beta7-/low, and to a lesser extent beta7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while alpha4 integrin was predominantly expressed on beta1high/beta7-/low cells, alphaE integrin was primarily associated with beta7high. The majority of activated/memory T cells recruited during infection expressed high levels of beta1 integrin and undetectable or low levels of beta7 integrin. These T cells were capable of producing IFN-gamma, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of beta1high, beta7-, and beta7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to alpha4 or alpha4beta7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.     

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.     

Inactivation of CD4+ CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load

Mycobacterium tuberculosis uses numerous mechanisms to avoid elimination by the infected host. In this study, we investigated the possibility whether, similar to other pathogens, M. tuberculosis exploits natural CD4+ CD25+ T-regulatory cells (Treg) to suppress the effector function of responding host lymphocytes, thus enhancing its survival. During a Mycobacterium bovis bacille calmette guerin (BCG) pulmonary infection, we observed a 2.8-fold increase in forkhead box P3 (Foxp3+) CD25+ Treg in the lung. To inactivate the Treg in vivo, an mAb was given against CD25 (PC61) 3 days before a pulmonary infection with BCG or M. tuberculosis. Following PC61 treatment, we observed significantly decreased CD25 expression on CD4+ T lymphocytes for at least 23 days in the blood, spleen and lung when compared with the control mice. To determine whether Treg inactivation affected the protective antimycobacterial immune response, we measured cytokine production by flow cytometry. We observed small, but significant increases in the percentages of both IFN-gamma-producing and IL-2-producing CD4+ cells from the spleen and the IL-2-producing CD4+ cells from the lungs of PC61-treated BCG-infected mice compared with the infected control mice. Despite this, there was neither a difference between the lung bacterial burdens of PC61-treated mice and control mice, measured until day 44 postinfection, nor was there an effect on infection-induced lung pathology. Together, these data imply that the absence of natural Treg early after infection results in a small increase in cytokine production, but this does not alter the course of either M. tuberculosis or BCG infections. This contrasts with the important role that natural Treg play in the pathogenesis of many other intracellular infectious organisms.     

T cell cytokine patterns in active tuberculosis patients in a northeastern area of Romania

Developing countries like Romania have a high incidence of tuberculosis. Literature data suggest that people in these countries have an important Th2-type immune background which prevents a protective Th1 response of the host against Mycobacterium tuberculosis. Our study is the first attempt in Romania to identify cytokine patterns in active tuberculosis. The study included 15 patients with active pulmonary tuberculosis and 11 healthy volunteers. Peripheral blood mononuclear cells (PBMC), stained with carboxyfluoresceine-diacetate-succinimidylester (CFSE), were incubated for 7 days with purified protein derivate (PPD) or with medium alone. Intracellular synthesis of IFNgamma and IL-4 in proliferated (CFSE(low)) T cells was detected by flowcytometry. The results showed that both Th1 (IFNgamma) and Th2 (IL-4) cytokines are produced in response to in vitro PPD-stimulation of PBMC from pulmonary tuberculosis patients and healthy controls. Moreover, the proportion between IFNgamma and IL-4 is tilted in favour of IL-4 in PPD-activated (CD3+ CFSE(low)) cells from healthy persons, who did not develop active tuberculosis during the two-year study time interval. This predominance of Th2 effectors points to the need to further investigate the role of IL-4 in the M. tuberculosis infection.     

Natural killer cells in patients with pulmonary tuberculosis]

In the existent literature the number of works devoted to the subpopulation of natural killer cells (NK) is not significant. The purpose of the present study was to determine the NK content in the blood of pulmonary tuberculosis patients; to establish correlation of the level of their content with the content of the previously studied T-lymphocyte subpopulations; to determine the intensity of the fluorescence of NK, CD3+, CD4+, CD8+ lymphocytes. The data were obtained on the significant increase in the NK mean level in tuberculosis patients (20.37 +/- 1.74) as compared with that in healthy subjects (12.77 +/- 2.56). The NK fluorescence intensity (56.33 +/- 2.28) conditioned by the Fc-receptor expression intensity is significantly lower than the analogous index in healthy volunteers (82.4 +/- 7.69). The NK level in the blood of tuberculosis patients correlates with the content of CD3+, CD8+ lymphocytes as well as with the CD4+/CD8+ index.     

Human NK cells positively regulate gammadelta T cells in response to Mycobacterium tuberculosis

The decrease in NK cell activity and the loss of gammadelta T cells in active pulmonary tuberculosis patients have been reported. In this study, we observed that the proliferating response of gammadelta T cells to the heat-treated Ags of Mycobacterium tuberculosis from different individuals was noted to be dependent on the content or function of NK cells in PBMC in a population study. We also found that NK cells were directly rapidly activated by the heat-treated Ags from M. tuberculosis (H37Ra) in vitro; in turn, the activated NK cells improved gammadelta T cell proliferation both by CD54-mediated cell-cell contact through the forming immune synapse and by soluble factors TNF-alpha, GM-CSF, and IL-12, but not IFN-gamma. Our results demonstrated that an interaction between NK cells and gammadelta T cells existed in antituberculosis immunity. Up-regulating the function of NK cells might be beneficial to the prevention and control of pulmonary tuberculosis.     

Compartmentalization of a CD4+ T lymphocyte subpopulation in tuberculous pleuritis

The study of T lymphocytes from pleural fluid and tissue of patients with tuberculous pleuritis provides an opportunity to evaluate the human immune response to infection at the site of disease activity. Therefore, we investigated the phenotype and function of CD4+ pleural fluid cells from patients with tuberculous pleuritis. Pleural fluid was enriched with CD4+CDw29+ T lymphocytes, which are thought to represent "memory" T cells. Immunoperoxidase staining of pleural tissue confirmed the predominance of CD4+CDw29+ T lymphocytes at the site of disease activity. CD4+ subpopulations were evaluated for their ability to contribute to a cell-mediated immune response against Mycobacterium tuberculosis by assaying immune function in vitro. Pleural fluid-derived CD4+CDw29+ cells, but not CD4+CDw29- lymphocytes, proliferated vigorously and produced high levels of IFN-gamma when stimulated with purified protein derivative of M. tuberculosis. CD4+CDw29+ clones produced IFN-gamma specifically in response to purified protein derivative of M. tuberculosis but not to an irrelevant Ag, tetanus toxoid. IFN-gamma levels were markedly elevated in pleural fluid, compared to peripheral blood, suggesting production of this lymphokine in vivo at the site of tissue inflammation. The sum of these data indicate that, in tuberculous pleuritis, CD4+CDw29+ cells are concentrated at the site of disease activity, produce IFN-gamma and are likely to play an important role in the local human cell-mediated immune response to M. tuberculosis.     

Large intestine dysbacteriosis and therapy efficacy in patients with respiratory tract tuberculosis in sanatoria]

Long-term chemotherapy of tuberculosis leads to dysbacteriosis of the large intestine, that significantly decreases tolerance of tuberculosis drugs, provokes persistence of tuberculosis intoxication and retards involution of tuberculosis process in the lungs. Recovery of the bifidoflora and lactoflora due to the use of an original sour-milk drink developed by the authors was stated in 65 and 55% of the patients respectively. Moreover, it promoted higher tolerance of the chemotherapeutics since its composition includes amino acids, vitamins B, C, D and E, enzymes and microelements. It should be indicated that the drink was used simultaneously with the chemotherapy and did not require its discontinuation. Therefore, it is recommended that the scheme of the treatment of patients with pulmonary tuberculosis in sanatoria should include corrigating agents and in particular probiotics containing live bifido- and lactobacteria having no contraindications and side effects, and providing elimination of intestinal dysbacteriosis.     

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.