cord-factors and Tuberculosis--Pulmonary

The primary and secondary tuberculosis features two completely different pathogenesis.At present,the pathogenesis of primary tuberculosis has been clear,whereas that of secondary tuberculosis remains unclear.In order to decipher the mechanism of secondary infection of

Topics: Coinfection; Cord Factors; Humans; Mycobacterium tuberculosis; Tuberculosis; Tuberculosis, Pulmonary

Topics: Bacterial Infections; Carcinogens; Chronic Disease; Cord Factors; Cytokines; DNA Damage; Humans; Inflammation Mediators; Lung Neoplasms; Mycobacterium tuberculosis; Neoplasms; Oxidative Stress; Risk; Time Factors; Tuberculosis, Pulmonary

Mycobacterium tuberculosis (MTB) is a pathogen that infects and kills millions yearly. The mycobacterium's cell wall glycolipid trehalose 6,6'-dimycolate (TDM) has been used historically to model MTB induced inflammation and granuloma formation. Alterations to the model can significantly influence the induced pathology. One such method incorporates intraperitoneal pre-exposure, after which the intravenous injection of TDM generates pathological damage effectively mimicking the hypercoagulation, thrombus formation, and tissue remodeling apparent in lungs of infected individuals. The purpose of these experiments is to examine the histological inflammation involved in the TDM mouse model that induces development of the hemorrhagic response. TDM induced lungs of C57BL/6 mice to undergo granulomatous inflammation. Further histological examination of the peak response demonstrated tissue remodeling consistent with hypercoagulation. The observed vascular occlusion indicates that obstruction likely occurs due to subendothelial localized activity leading to restriction of blood vessel lumens. Trichrome staining revealed that associated damage in the hypercoagulation model is consistent with intra endothelial cell accumulation of innate cells, bordered by collagen deposition in the underlying parenchyma. Overall, the hypercoagulation model represents a comparative pathological instrument for understanding mechanisms underlying development of hemorrhage and vascular occlusion seen during MTB infection.

Topics: Animals; Blood Coagulation; Cord Factors; Disease Models, Animal; Endothelium, Vascular; Female; Granuloma, Respiratory Tract; Lung; Mice, Inbred C57BL; Mycobacterium tuberculosis; Pneumonia; Tuberculosis, Pulmonary; Vascular Remodeling

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.

Topics: Acyltransferases; Adolescent; Adult; Antibodies, Bacterial; Antibody Specificity; Antigens, Bacterial; Bacterial Proteins; Cord Factors; Dithiothreitol; Enzyme-Linked Immunosorbent Assay; Humans; Immunoassay; Immunoglobulin G; Maleimides; Middle Aged; Mycobacterium tuberculosis; Polymerase Chain Reaction; Serologic Tests; Tuberculosis; Tuberculosis, Pulmonary; Young Adult

The role of macrophage-inducible C-type lectin (Mincle) in anti-inflammatory responses has not yet been fully characterized. Herein, we show that engagement of Mincle by trehalose-dimycolate or mycobacteria promotes IL-10 production in macrophages, which causes down-regulation of IL-12p40 secretion. Thus, Mincle mediates both pro- as well as anti-inflammatory responses.

Topics: Animals; Cells, Cultured; Cord Factors; Gene Expression Regulation; Humans; Immunity, Innate; Interleukin-10; Interleukin-12 Subunit p40; Lectins, C-Type; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacteriaceae; Mycobacterium bovis; Toll-Like Receptor 2; Tuberculosis, Pulmonary

Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients.

Topics: Antigens, Bacterial; Cord Factors; Immunoassay; Polymerase Chain Reaction; Tuberculosis; Tuberculosis, Pulmonary

The mechanisms involved in interactions between Mycobacterium tuberculosis and host innate immune cells determine outcome. Antigen-presenting cells, including macrophages and dendritic cells, express many pattern recognition receptors to identify pathogen-associated molecular patterns, thereby initiating an immune response. A major mycobacterial virulence factor, trehalose-6',6-dimycolate, is recognised by the macrophage-inducible C-type lectin, Mincle, which leads to the activation of the Syk-Card9 signalling pathway in macrophages. Mincle is encoded by CLEC4E, and we investigated polymorphisms in this gene to assess its role in tuberculosis susceptibility. Four tagging single nucleotide polymorphisms (SNPs) (rs10841845, rs10841847, rs10841856 and rs4620776) were genotyped using TaqMan(®) SNP assays in 416 tuberculosis cases and 405 healthy controls. Logistic regression models were used for analysis. No association was detected with any of the SNPs analysed. This research highlights tuberculosis disease complexity where recognition proteins which specifically bind mycobacterial glycolipids cannot be conclusively associated with the disease in genetic studies.

Topics: Adult; Case-Control Studies; Cord Factors; Female; Genetic Predisposition to Disease; Genotype; Humans; Lectins, C-Type; Male; Middle Aged; Mycobacterium tuberculosis; Polymorphism, Single Nucleotide; Receptors, Immunologic; Tuberculosis, Pulmonary; Young Adult

Trehalose 6,6'dimycolate (TDM) is a glycolipid found in nearly pure form on the surface of virulent Mycobacterium tuberculosis (MTB). This manuscript investigated the production of TDM, growth rate and colony morphology of multiple strains of MTB, each of which had been isolated from both pulmonary (sputum) and extrapulmonary sites of multiple patients. Since sputum contains MTB primarily from cavities and extrapulmonary biopsies are typically granulomas, this provided an opportunity to compare the behavior of single strains of MTB that had been isolated from cavities and granulomas. The results demonstrated that MTB isolated from pulmonary sites produced more TDM (3.23 ± 1.75 μg TDM/mg MTB), grew more rapidly as thin spreading pellicles, demonstrated early cording, and climbed culture well walls. In contrast, extrapulmonary isolates produced less TDM (1.42 ± 0.58 μg TDM/mg MTB) (p < 0.001) and grew as discrete patches with little tendency to spread or climb. Both Beijing pulmonary isolates and the non-Beijing pulmonary isolates produced significantly more TDM (1.64 ± 0.46 μg TDM/mg MTB) and grew faster than the Beijing and non-Beijing extrapulmonary isolates (1.14 ± 0.63 μg TDM/mg MTB) (p < 0.001 and p < 0.005 respectively). These results indicate that MTB from pulmonary sites (cavities) grows faster and produces more TDM than strains isolated from extrapulmonary sites (granulomas). This report suggests a critical role for TDM in cavitary TB.

Topics: Biopsy; Cord Factors; Granuloma; Humans; Mycobacterium tuberculosis; Sputum; Time Factors; Tuberculosis, Pulmonary; Virulence

World Health Organization had estimated 9.4 million tuberculosis cases on 2009, with 1.7 million of deaths as consequence of treatment and diagnosis failures. Improving diagnostic methods for the rapid and timely detection of tuberculosis patients is critical to control the disease. The aim of this study was evaluating the accuracy of the cord factor detection on the solid medium Middlebrook 7H11 thin layer agar compared to the Lowenstein Jensen medium for the rapid tuberculosis diagnosis.. Patients with suspected tuberculosis were enrolled and their sputum samples were processed for direct smear and culture on Lowenstein Jensen and BACTEC MGIT 960, from which positive tubes were subcultured on Middlebrook 7H11 thin layer agar. Statistical analysis was performed comparing culture results from Lowenstein Jensen and the thin layer agar, and their corresponding average times for detecting Mycobacterium tuberculosis. The performance of cord factor detection was evaluated determining its sensitivity, specificity, positive and negative predictive value.. 111 out of 260 patients were positive for M. tuberculosis by Lowenstein Jensen medium with an average time ± standard deviation for its detection of 22.3 ± 8.5 days. 115 patients were positive by the MGIT system identifying the cord factor by the Middlebrook 7H11 thin layer agar which average time ± standard deviation was 5.5 ± 2.6 days.. The cord factor detection by Middlebrook 7H11 thin layer agar allows early and accurate tuberculosis diagnosis during an average time of 5 days, making this rapid diagnosis particularly important in patients with negative sputum smear.

Topics: Bacteriological Techniques; Cord Factors; False Negative Reactions; Humans; Mycobacterium tuberculosis; Predictive Value of Tests; Sensitivity and Specificity; Sputum; Tuberculosis, Pulmonary

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.

Topics: Adjuvants, Immunologic; Animals; Antigens, Bacterial; Bacterial Load; Cord Factors; Disease Models, Animal; Drug Carriers; Female; Humans; Lung; Mice; Mice, Inbred BALB C; Microspheres; Mycobacterium tuberculosis; Tuberculosis Vaccines; Tuberculosis, Pulmonary; Vaccines, DNA

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.

Topics: Adjuvants, Immunologic; Animals; CD11b Antigen; CD18 Antigens; Cord Factors; Gene Expression Regulation; Granuloma, Respiratory Tract; Lectins, C-Type; Lung; Membrane Proteins; Mice; Mice, Knockout; Mycobacterium tuberculosis; Neutrophil Infiltration; Neutrophils; Pneumonia; Protein Kinases; Signal Transduction; Toll-Like Receptor 2; Tuberculosis, Pulmonary

Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown.. One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6'-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology.. IL-6, IL-1alpha and TNF-alpha mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11beta-hydroxysteroid dehydrogenases (11betaHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11betaHSD type 1 mRNA decreased 4-fold and 11betaHSD type 2 (11betaHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11betaHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 1; 11-beta-Hydroxysteroid Dehydrogenase Type 2; Animals; Cord Factors; Corticosterone; Cytokines; Disease Models, Animal; Down-Regulation; Female; Granuloma, Respiratory Tract; Immune Tolerance; Lung; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; RNA, Messenger; Tuberculosis, Pulmonary; Up-Regulation

Although the incidence of pulmonary tuberculosis is decreasing, the number of immunocompetent patients with Mycobacterium avium complex (MAC) pulmonary disease is steadily increasing. Therefore, albeit not contagious, MAC pulmonary disease needs to be diagnosed rapidly and accurately. We examined the serodiagnostic contributions of serum immunoglobulin G antibody titers against the species-specific and -common mycobacterial lipid antigens in the diagnosis of MAC pulmonary disease.. Serum samples were obtained from 65 patients with MAC pulmonary disease, 15 patients with suspected MAC disease, 25 patients with pulmonary tuberculosis, 10 patients with Mycobacterium kansasii disease, and 100 healthy volunteers (control subjects). We measured the serum immunoglobulin G antibody titers against trehalose monomycolate (TMM-M) and apolar-glycopeptidolipid (GPL), lipid antigens extracted from MAC.. In patients with MAC pulmonary disease, the antibody titers against TMM-M and apolar-GPL were significantly higher than those in the other patient groups or in the control subjects. By receiver operator characteristic curve analysis, an optical density of 0.27, corresponding to the optimal cutoff antibody titer against TMM-M, was associated with a sensitivity of 89.2% and a specificity of 97.0%, and an optical density of 0.33, corresponding to the optimal cutoff antibody titer against apolar-GPL, was associated with a sensitivity of 89.2% and a specificity of 94.0%.. Measurements of antibody titers against TMM-M and apolar-GPL would be useful in the diagnosis of MAC pulmonary disease and in the differential diagnosis of mycobacterial pulmonary disease.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Bacterial; Antigens, Bacterial; Cord Factors; Female; Glycolipids; Humans; Immunoglobulin G; Lipids; Male; Middle Aged; Mycobacterium avium Complex; Tuberculosis, Pulmonary; Young Adult

Molecular interactions between mycobacterial cell wall lipid, cord factor (CF) and the abundant surfactant lipid, dipalmitoylphosphatidylcholine (DPPC) were investigated using Langmuir monolayers at physiological temperatures (37 degrees C). Surface topography of the films was visualized by atomic force microscopy (AFM). Thermodynamic behavior of the mixed monolayers was evaluated by investigating the molecular area excess, excess Gibbs free energy of mixing and maximum compressibility modulus (SCM(max)). Cord factor formed immiscible and thermodynamically unstable monolayers with DPPC. Monolayer presence of cord factor altered the physical state of DPPC monolayers from liquid condensed to liquid expanded with the lowering of SCM(max) from 160 to 40 mN/m, respectively. AFM imaging exhibited smooth homogenous surface topography of DPPC films which in the presence of cord factor was markedly altered with the appearance of aggregates and increased surface roughness. The results highlight the capacity of cord factor to disturb DPPC monolayer organization and structure. Interfacial presence of cord factor results in DPPC monolayer fluidization. Lung surfactant function is attributed to its ability to form well packed low compressibility films. Such molecular interactions suggest a dysfunction of lung surfactant in pulmonary tuberculosis due to surfactant monolayer fluidization.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cord Factors; Humans; Microscopy, Atomic Force; Pulmonary Surfactants; Tuberculosis, Pulmonary; Unilamellar Liposomes

Disease due to the Mycobacterium avium complex (MAC) is one of the most important opportunistic pulmonary infections. Since the clinical features of MAC pulmonary disease and tuberculosis (TB) resemble each other, and the former is often difficult to treat with chemotherapy, early differential diagnosis is desirable. The humoral immune responses to both diseases were compared by a unique multiple-antigen ELISA using mycobacterial species-common and species-specific lipid antigens, including glycopeptidolipid (GPL)-core. The results were assessed for two patient groups hospitalized and diagnosed clinically as having TB or MAC pulmonary disease. Diverse IgG antibody responsiveness was demonstrated against five lipid antigens: (1) monoacyl phosphatidylinositol dimannoside (Ac-PIM2), (2) cord factor (trehalose 6,6'-dimycolate) (TDM-T) and (3) trehalose monomycolate from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) (TMM-T), and (4) trehalose monomycolate (TMM-M) and (5) GPL-core from MAC. Anti-GPL-core IgG antibody was critical, and detected only in the primary and the secondary MAC diseases with high positivity, up to 88.4 %. However, IgG antibodies against Ac-PIM2, TDM-T and TMM-T were elevated in both TB and MAC patients. Anti-TMM-M IgG antibody was also elevated in MAC disease preferentially, with a positive rate of 89.9 %, and therefore, it was also useful for the diagnosis of the disease. IgG antibody levels were increased at the early stages of the disease and declined in parallel to the decrease of bacterial burden to near the normal healthy control level, when the anti-mycobacterial chemotherapy was completed successfully. Unexpectedly, about 25 % of hospitalized TB patient sera were anti-GPL-core IgG antibody positive, although the specificity of GPL-core was sufficiently high (95.8 % negative in healthy controls), suggesting that a considerable number of cases of latent co-infection with MAC may exist in TB patients. Taken together, the combination of multiple-antigen ELISA using mycobacterial lipids, including GPL-core and TMM-M, gives good discrimination between healthy controls and sera from patients with TB or MAC disease, although for accurate diagnosis of TB more specific antigen(s) are needed.

Topics: Antibodies, Bacterial; Antigens, Bacterial; Cord Factors; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Lipids; Mycobacterium avium Complex; Mycobacterium tuberculosis; Phosphatidylinositols; Serologic Tests; Species Specificity; Tuberculosis, Pulmonary

Cord formation of Mycobacterium tuberculosis complex is very uncommon in smear specimen prepared directly from sputum, although such a finding is well known in solid or liquid media and has recently been evaluated as a rapid method for presumptive identification in special liquid media (BACTEC or MGIT). We examined 308 (Mycobacterium tuberculosis 271 and Nontuberculous mycobacteria 37) positive smear specimens prepared directly from sputum in our hospital. These specimens all showed a "modified Gaffky scale" as +2 or more and this cord formation was found in four cases (five specimens). Each of these specimens was from a patient with severe lung tuberculosis showing cavity formation and each patient was complicated severe diabetes mellitus. The morphology of cord formation on smear specimens prepared directly from sputum was similar to that in liquid or solid media, and consequently the relevant bacilli were identified as Mycobacterium tuberculosis complex by PCR examination. In this study, we assessed "cord formation" in smear specimen prepared directly from sputum as a more rapid presumptive identification of Mycobacterium tuberculosis complex based on microscopic morphology, as well as cord formation in liquid or solid media.

Topics: Aged; Bacteriological Techniques; Cord Factors; Diabetes Complications; Humans; Male; Middle Aged; Mycobacterium tuberculosis; Sputum; Tuberculosis, Pulmonary

Trehalose 6,6'-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas.

Topics: Adipose Tissue; Animals; Cord Factors; Granuloma; Lipid Metabolism; Lung; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Necrosis; Peritoneum; Tuberculosis, Pulmonary

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

Topics: Animals; Cell Line; Colony Count, Microbial; Cord Factors; Cyclopropanes; Immunity, Innate; Interleukin-6; Macrophage Activation; Macrophages; Methyltransferases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Mycobacterium tuberculosis; Tuberculosis, Pulmonary; Tumor Necrosis Factor-alpha

Immunogenic glycolipids from the cell wall of Mycobacterium tuberculosis are potential capture antigens in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnostis of tuberculosis. Typically, washing steps in ELISAs are performed with buffers containing a detergent. However, Tween-20, the most commonly added detergent, was reported to be able to remove the coating of certain glycolipid antigens from microtitre wells. In order to determine the influence of the washing buffer composition on the results, we measured serum immunoglobulin G (IgG) against three mycobacterial glycolipids by ELISA, conducting three separate experiments with three different buffers: Tris-buffered saline (TBS), TBS plus 0.02% Tween-20 (TBS-Tween), or TBS plus 0.3% bovine serum albumin (TBS-BSA). The capture antigens applied were lipoarabinomannan with the basic arabinose-containing motif (AraLAM), the mannose-capped version of lipoarabinomannan (ManLAM), and trehalose-6,6'-dimycolate (cord factor). All ELISAs achieved acceptable specificities around 95%. The sensitivities, however, varied widely, depending upon the sort of washing buffer used. In 38 patients with sputum smear-positive pulmonary tuberculosis and control groups of 79 patients with non-tuberculosis lung disease and 92 healthy volunteers, the anti-cord factor ELISA achieved 100%, 31.6%, and 60.5% with TBS, TBS-Tween, and TBS-BSA, respectively. Corresponding sensitivity values for AraLAM were 39.5%, 26.3%, and 23.7%, and for ManLAM 94.7%, 65.8%, and 55.3%. We conclude that Tween-20 or BSA should be omitted from the washing buffer in ELISAs, when the capture antigen is of lipid nature.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antibodies; Antigens, Bacterial; Buffers; Cattle; Child; Cord Factors; Enzyme-Linked Immunosorbent Assay; Female; Glycolipids; Humans; Lipopolysaccharides; Male; Middle Aged; Mycobacterium tuberculosis; Polysorbates; Respiratory Tract Diseases; ROC Curve; Sensitivity and Specificity; Serum Albumin, Bovine; Tuberculosis, Pulmonary

Mycobacterial O-acyltrehaloses have been described as highly specific and sensitive reagents for tuberculosis immunodiagnosis. An O-acyltrehalose-containing lipid fraction from the rapidly growing Mycobacterium fortuitum was found to include additional antigens, which presented high cross-reactivity with sera from tuberculosis-infected patients. Based on a combination of selective chemical degradations, thin-layer-chromatography analyses and (1)H-nuclear magnetic resonance spectroscopy, the antigenic by-product was identified as 6,6'-dimycoloyl trehalose, the so-called cord factor. The lipid was purified and tested in ELISA for pulmonary tuberculosis serodiagnosis. Sensitivity and specificity of the test were found to be 66.6-74.1% and 95.2-99.0%, respectively, showing a slightly higher efficiency as compared to the ELISA performed using 6,6'-dimycoloyl trehalose from Mycobacterium tuberculosis. No cross-reactivity was found with sera from Nocardia-infected individuals.

Topics: Antibodies, Bacterial; Antigens, Bacterial; Chromatography, Thin Layer; Cord Factors; Enzyme-Linked Immunosorbent Assay; Humans; Magnetic Resonance Spectroscopy; Mycobacterium fortuitum; Mycobacterium tuberculosis; Tuberculosis, Pulmonary

The persistence of tuberculosis within pulmonary granulomatous lesions is a complex phenomenon, with bacterial survival occurring in a focal region of high immune activity. In part, the survival of the organism may be linked to the ability of the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor) to inhibit fusion events between phospholipid vesicles inside the host macrophage. At the same time, TDM contributes to macrophage activation and a cascade of events required for initiation and maintenance of granulomatous responses. This allows increased sequestration of organisms and further survival and persistence within host tissues. Bacterial viability, macrophage cytokine and chemokine response, and intracellular trafficking were investigated in Mycobacterium tuberculosis from which TDM had been removed. Removal of surface lipids led to enhanced trafficking of organisms to acidic compartments; reconstitution of delipidated organisms with either pure TDM or the petroleum ether extract containing crude surface lipids restored normal responses. Use of TDM-coated polystyrene beads demonstrated that TDM can mediate intracellular trafficking events, as well as influence macrophage production of pro-inflammatory molecules. Thus, the presence of TDM may be an important determinant for successful infection and survival of M. tuberculosis within macrophages.

Topics: Animals; Cell Line; Cord Factors; Cytokines; Inflammation Mediators; Macrophage Activation; Macrophages; Mice; Mycobacterium tuberculosis; Tuberculosis, Pulmonary; Virulence

Mice treated with viable Mycobacterium tuberculosis with no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidated M. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-DL-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-12, IL-10, gamma interferon (IFN-gamma), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-gamma, IL-6, TNF-alpha, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.

Topics: Animals; Cells, Cultured; Cord Factors; Cytokines; Drug Carriers; Inflammation; Lactic Acid; Leukocytes; Leukocytes, Mononuclear; Lung; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Microspheres; Mycobacterium tuberculosis; Neutrophil Infiltration; Neutrophils; Nitric Oxide; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Tuberculosis, Pulmonary

The detection of anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in active (smear-and/or culture-positive) and inactive (smear-and culture-negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium-infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human-type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC-infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti-cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (alpha-, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti-cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized alpha- and keto mycolic acids weakly. Pre-absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti-cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.

Topics: Antibodies, Bacterial; Antigens, Bacterial; Cord Factors; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycolipids; Humans; Immunoglobulin G; Mycobacterium avium Complex; Mycobacterium avium-intracellulare Infection; Mycobacterium tuberculosis; Mycolic Acids; Tuberculosis, Pulmonary

Enzyme-linked immunosorbent assay (ELISA) was used to examine serum antibody levels of mycobacterial antigen in 92 patients with active tuberculosis, 36 with cured tuberculosis, 45 with nontuberculous mycobacteriosis, and as 31 with other diseases. Glycolipid fraction containing mainly cord factor (trehalose-6,6'-mycolate) from Mycobacterium tuberculosis H37Rv were used as ELISA antigen. Overall positive rates of the ELISA tests in the patients with active pulmonary tuberculosis, those with nontuberculous mycobacteriosis, and those with other diseases were 67.4%, 75.6%, and 6.5%, respectively. Patients with tuberculosis and those with nontuberculous mycobacteriosis differed from the control group (p < 0.0001). Higher positive rates were correlated with bacterial loads (smear-positive vs smear-negative, p < 0.01) and with chest roenrgenographic findings (far advanced or other cavitary vs noncavitary, p < 0.01). Because 18 of 36 smear-negative patients (50%) had positive results, we believe that the ELISA test with this antigen can be useful for diagnosis of M. tuberculosis, especially in patients with smear-negative.

Topics: Antibodies, Bacterial; Antigens, Bacterial; Cord Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Male; Middle Aged; Serologic Tests; Tuberculosis, Pulmonary

We report the development of an EIA specific for antituberculosis antibody in human serum for the clinical evaluation of tuberculosis. We developed a TLC immunostaining method to detect specific antigens for antibodies in the serum of patients with tuberculosis. The detected specific antigens, TDM and specific gylcolipid fraction, were individually purified from M. tuberculosis H37Rv by column chromatography. The two purified fractions were mixed and the mixture, termed TBGL antigen, was applied to an enzyme immunoassay suitable for the measurement of antituberculosis antibodies in serum. This EIA meets all the requirements of routine clinical assay in terms of sensitivity (detection limit: 0.125 U/ml), reproducibility (total CV : 3.3-6.0%), accuracy (recovery: 96-105%), simplicity and rapidity (< 2.5 h). Clinical validation of the assay was confirmed by the measurement of the anti-tuberculosis antibody in the serum of normal subjects and patients with pulmonary tuberculosis. The EIA tested in this study showed a high serodiagnostic discriminating power (90% sensitivity and 98% specificity).

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Bacterial; Antigen Presentation; Antigens, Bacterial; Cord Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Mycobacterium tuberculosis; Reproducibility of Results; Sensitivity and Specificity; Serologic Tests; Tuberculosis, Pulmonary

Kyowa Medex Co., Ltd. developed the kit for the sero-diagnosis of tuberculosis, which detects IgG antibodies against tuberculous glycolipids antigen containing cord factor (TBGL antigen) prepared from M. tuberculosis using the enzyme-linked immunosorbent assay technique. We evaluated the kit using clinical specimens and the results are as follows: 1) In total, 34 out of 39 cases (87.2%) with active pulmonary tuberculosis showed positive anti-TBGL antibody. 2) Patients with cavity, patients with extensive lesions and patients excreting large amount of acid fast bacilli tended to show high positivity rates. 3) The antibody titers increased in 7 out of 11 cases after starting the antituberculous chemotherapy. 4) The use of the antibody is unsuitable for the determination of the activity of tuberculosis since the antibody titers only slightly decreased even after chemotherapy for two years. 5) Two out of four nontuberculous mycobacteriosis cases showed high antibody titers 6) All three AIDS patients with tuberculosis showed low antibody titers. 7) The antibody was negative in almost all healthy controls showing a positive PPD skin test after vaccination with BCG, and it was therefore assumed that the antibody titer is not increased by BCG vaccination. 8) The antibody titers of the staff members working in the tuberculosis wards were not high compared with those of staff members working in the other wards.

Topics: Adult; AIDS-Related Opportunistic Infections; Antibodies, Bacterial; BCG Vaccine; Cord Factors; Enzyme-Linked Immunosorbent Assay; Evaluation Studies as Topic; Female; Humans; Male; Middle Aged; Reagent Kits, Diagnostic; Serologic Tests; Tuberculosis; Tuberculosis, Osteoarticular; Tuberculosis, Pulmonary; Vaccination

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.

Topics: Animals; Arachidonic Acid; Calcium; Carbohydrate Sequence; Cord Factors; Diglycerides; Enzyme Activation; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Phospholipids; Protein Kinase C; Sensitivity and Specificity; Tuberculosis, Pulmonary; Tumor Necrosis Factor-alpha

Immunoglobulin G (IgG) antibodies against purified cord factor (trehalose-6,6'-dimycolate) prepared from Mycobacterium tuberculosis H37Rv were determined by enzyme-linked immunosorbent assay (ELISA), and its diagnostic usefulness was evaluated. Serum specimens from 65 patients with active pulmonary tuberculosis, 58 patients with inactive pulmonary tuberculosis, 36 patients with diseases other than tuberculosis, and 66 healthy adults were examined. Patients with active pulmonary tuberculosis showed significantly higher titers of IgG antibodies against cord factor than did other groups (p < 0.001). The antibody titer greater than 0.29 in absorption difference (492 to 630 nm) of 160-times diluted serum was set as positive in ELISA. For patients with active and untreated pulmonary tuberculosis, the ELISA had a sensitivity of 81% and a specificity of 96%. From these results, it was concluded that the detection of IgG antibodies against cord factor is useful for serodiagnosis of active pulmonary tuberculosis. It was also indicated that the anticord factor antibody titers decline to the normal level as a result of antituberculosis chemotherapy.

Topics: Adult; Aged; Antibodies, Bacterial; Antigens, Bacterial; Antitubercular Agents; Cord Factors; Drug Therapy, Combination; Enzyme-Linked Immunosorbent Assay; Evaluation Studies as Topic; Female; Humans; Male; Middle Aged; Mycobacterium tuberculosis; Serologic Tests; Tuberculosis, Pulmonary

Detection of IgG antibodies against purified cord factor (trehalose-6, 6'-dimycolate) prepared from Mycobacterium tuberculosis H37Rv was carried out by the method of enzyme-linked immunosorbent assay (ELISA) and its diagnostic usefulness was also evaluated in this study. Sera from 65 patients with active pulmonary tuberculosis, 58 patients with inactive pulmonary tuberculosis, 36 patients with diseases other than tuberculosis and 66 healthy adults were examined. Patients with active pulmonary tuberculosis showed significantly higher titers of IgG antibodies against cord factor than other groups (p < 0.001). Patients with inactive pulmonary tuberculosis also showed significantly higher titers of IgG antibodies against cord factor than patients with diseases other than tuberculosis and healthy adults (p < 0.001). An antibody titers of greater than 0.29 were established as a positive ELISA test. For patients with active pulmonary tuberculosis, the ELISA had a sensitivity of 85% and a specificity of 96%. From these results, it is concluded that the detection of IgG antibodies against cord factor is useful for the serodiagnosis of active or inactive pulmonary tuberculosis.

Topics: Adult; Aged; Cord Factors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Mycobacterium tuberculosis; Serologic Tests; Tuberculosis, Pulmonary

Factors contributing to protection against experimental tuberculosis have been studied with refined and well characterized fractions from mycobacteria and with certain unrelated antigens. Mice were vaccinated intravenously with various combinations of materials presented on minute oil droplets in saline emulsions and were later challenged by aerosol. The minimal composition of an effective vaccine was P3 (a trehalose mycolate similar to cord factor) plus an antigen, which could be tuberculoprotein, or a low-molecular-weight tuberculin-active peptide, or unrelated antigen such as bovine serum albumin or bacterial endotoxin. Development of a hypersensitivity granuloma in the lungs appeared to be essential to protection in this laboratory model.

Topics: Animals; Bacteria; Bacterial Proteins; BCG Vaccine; Cell Migration Inhibition; Cord Factors; Granuloma; Hypersensitivity, Delayed; Lung Diseases; Macrophages; Male; Mice; Mycobacterium tuberculosis; Peptides; Serum Albumin, Bovine; Tuberculin; Tuberculosis, Pulmonary

A water-soluble mycobacterial glycopeptide was obtained in large quantities from the culture supernatant fluid of M. tuberculosis strain DT. This glycopeptide was strongly adjuvant-active when injected, in a water-in-oil emulsion contianing ovalbumin, into guinea-pigs. In addition, it was devoid of cord factor toxicity in mice, polyarthritogenic activity in rats and cavity stimulating activity in rabbit lungs.

Topics: Adjuvants, Immunologic; Animals; Arthritis; Cord Factors; Cornea; Glycopeptides; Glycoproteins; Guinea Pigs; Hypersensitivity, Delayed; Immunoglobulin G; Mycobacterium tuberculosis; Ovalbumin; Precipitin Tests; Rabbits; Rats; Rats, Inbred Lew; Skin Tests; Solubility; Tuberculosis, Pulmonary

Topics: Antibodies; Antigens; Complement System Proteins; Cord Factors; Flocculation Tests; Humans; Mycobacterium tuberculosis; Serologic Tests; Tuberculosis; Tuberculosis, Pulmonary