Mycobacterial Glycolipid Research Articles

Mincle is a long sought receptor for mycobacterial cord factor

Mycobacterium tuberculosis is a leading killer worldwide, yet the adjuvancy of its cell wall has proven to be a valuable therapeutic tool for vaccination and immunotherapy. Much research effort has focused on the mycobacterial glycolipid trehalose-6,6’-dimycolate (TDM), a potent immunostimulant that is also known as cord factor. Now, the identification of the monocyte-inducible C-type lectin (Mincle) as an essential receptor for TDM provides new insight into the formation of the characteristic granulomas in tuberculosis and an avenue for rational adjuvant design.

Cooperation between a Coenzyme A-Independent Stand-Alone Initiation Module and an Iterative Type I Polyketide Synthase during Synthesis of Mycobacterial Phenolic Glycolipids

Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs play key roles in pathogenicity and host−pathogen interaction. Thus, elucidation of the PGL biosynthetic pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections. In this study, we report an investigation of the enzymatic requirements for the production of long-chain p-hydroxyphenylalkanoate intermediates of PGL biosynthesis. We demonstrate a functional cooperation between a coenzyme A-independent stand-alone didomain initiation module (FadD22) and a 6-domain reducing iterative type I polyketide synthase (Pks15/1) for production of p-hydroxyphenylalkanoate intermediates in in vitro and in vivo FadD22-Pks15/1 reconstituted systems. Our results suggest that Pks15/1 is an iterative type I polyketide synthase with a relaxed control of catalytic cycle iterations, a mechanistic property that explains the origin of a characteristic alkyl chain length variability seen in mycobacterial PGLs. The FadD22-Pks15/1 reconstituted systems lay an initial foundation for future efforts to unveil the mechanism of iterative catalysis control by which the structures of the final products of Pks15/1 are defined, and to scrutinize the functional partnerships of the FadD22-Pks15/1 system with downstream enzymes of the PGL biosynthetic pathway.

Identification of Mycobacterial α-Glucan As a Novel Ligand for DC-SIGN: Involvement of Mycobacterial Capsular Polysaccharides in Host Immune Modulation

Mycobacterium tuberculosis possesses a variety of immunomodulatory factors that influence the host immune response. When the bacillus encounters its target cell, the outermost components of its cell envelope are the first to interact. Mycobacteria, including M. tuberculosis, are surrounded by a loosely attached capsule that is mainly composed of proteins and polysaccharides. Although the chemical composition of the capsule is relatively well studied, its biological function is only poorly understood. The aim of this study was to further assess the functional role of the mycobacterial capsule by identifying host receptors that recognize its constituents. We focused on alpha-glucan, which is the dominant capsular polysaccharide. Here we demonstrate that M. tuberculosis alpha-glucan is a novel ligand for the C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). By using related glycogen structures, we show that recognition of alpha-glucans by DC-SIGN is a general feature and that the interaction is mediated by internal glucosyl residues. As for mannose-capped lipoarabinomannan, an abundant mycobacterial cell wall-associated glycolipid, binding of alpha-glucan to DC-SIGN stimulated the production of immunosuppressive IL-10 by LPS-activated monocyte-derived dendritic cells. By using specific inhibitors, we show that this IL-10 induction was DC-SIGN-dependent and also required acetylation of NF-kappaB. Finally, we demonstrate that purified M. tuberculosis alpha-glucan, in contrast to what has been reported for fungal alpha-glucan, was unable to activate TLR2.

Autophagy Gene Variant IRGM −261T Contributes to Protection from Tuberculosis Caused by Mycobacterium tuberculosis but Not by M. africanum Strains

The human immunity-related GTPase M (IRGM) has been shown to be critically involved in regulating autophagy as a means of disposing cytosolic cellular structures and of reducing the growth of intracellular pathogens in vitro. This includes Mycobacterium tuberculosis, which is in agreement with findings indicating that M. tuberculosis translocates from the phagolysosome into the cytosol of infected cells, where it becomes exposed to autophagy. To test whether IRGM plays a role in human infection, we studied IRGM gene variants in 2010 patients with pulmonary tuberculosis (TB) and 2346 unaffected controls. Mycobacterial clades were classified by spoligotyping, IS6110 fingerprinting and genotyping of the pks1/15 deletion. The IRGM genotype −261TT was negatively associated with TB caused by M. tuberculosis (OR 0.66, CI 0.52–0.84, Pnominal 0.0009, Pcorrected 0.0045) and not with TB caused by M. africanum or M. bovis (OR 0.95, CI 0.70–1.30. P 0.8). Further stratification for mycobacterial clades revealed that the protective effect applied only to M. tuberculosis strains with a damaged pks1/15 gene which is characteristic for the Euro-American (EUAM) subgroup of M. tuberculosis (OR 0.63, CI 0.49–0.81, Pnominal 0.0004, Pcorrected 0.0019). Our results, including those of luciferase reporter gene assays with the IRGM variants −261C and −261T, suggest a role for IRGM and autophagy in protection of humans against natural infection with M. tuberculosis EUAM clades. Moreover, they support in vitro findings indicating that TB lineages capable of producing a distinct mycobacterial phenolic glycolipid that occurs exclusively in strains with an intact pks1/15 gene inhibit innate immune responses in which IRGM contributes to the control of autophagy. Finally, they raise the possibility that the increased frequency of the IRGM −261TT genotype may have contributed to the establishment of M. africanum as a pathogen in the West African population.

Role of Phosphatidylinositol Mannosides in the Interaction between Mycobacteria and DC-SIGN

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM(6), which contains terminal alpha(1-->2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM(2) and PIM(4), respectively. To determine the role of PIM(6) in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM(6) (Delta pimE) was created, as well as a double knockout deficient in the production of both PIM(6) and the mannose caps on LAM (Delta pimE Delta capA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM(6) represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.

The mycobacterial glycolipid glucose monomycolate induces a memory T cell response comparable to a model protein antigen and no B cell response upon experimental vaccination of cattle

Glycolipids are presented to T cells by human group 1 CD1 proteins, but are not used as subunit vaccines yet. Experimental immunizations with pure mycobacterial glucose monomycolate (GMM) and keyhole limpet haemocyanin (KLH) in cattle, a species which, unlike mice, expresses group 1 CD1, showed that GMM was equally efficient as KLH in generating T cell responses in blood, but not in the draining lymph node. Also, KLH induced strong antibody responses whereas GMM did not. These data suggest that non-overlapping T cell populations are targeted and demonstrate the potential of glycolipids as a special class of subunit vaccine candidates.

A Liposome-Based Mycobacterial Vaccine Induces Potent Adult and Neonatal Multifunctional T Cells through the Exquisite Targeting of Dendritic Cells

BackgroundIn the search for more potent and safer tuberculosis vaccines, CAF01 was identified as a remarkable formulation. Based on cationic liposomes and including a synthetic mycobacterial glycolipid as TLR-independent immunomodulator, it induces strong and protective T helper-1 and T helper-17 adult murine responses to Ag85B-ESAT-6, a major mycobacterial fusion protein. Here, we assessed whether these properties extend to early life and how CAF01 mediates its adjuvant properties in vivo.Methods/FindingsFollowing adult or neonatal murine immunization, Ag85B-ESAT-6/CAF01 similarly reduced the post-challenge bacterial growth of M. bovis BCG, whereas no protection was observed using Alum as control. This protection was mediated by the induction of similarly strong Th1 and Th17 responses in both age groups. Multifunctional Th1 cells were already elicited after a single vaccine dose and persisted at high levels for at least 6 months even after neonatal priming. Unexpectedly, this potent adjuvanticity was not mediated by a massive targeting/activation of dendritic cells: in contrast, very few DCs in the draining lymph nodes were bearing the labeled antigen/adjuvant. The increased expression of the CD40 and CD86 activation markers was restricted to the minute portion of adjuvant-bearing DCs. However, vaccine-associated activated DCs were recovered several days after immunization.ConclusionThe potent adult and neonatal adjuvanticity of CAF01 is associated in vivo with an exquisite but prolonged DC uptake and activation, fulfilling the preclinical requirements for novel tuberculosis vaccines to be used in early life.

Synthetic Trehalose Glycolipids Confer Desiccation Resistance to Supported Lipid Monolayers

Lipid-derived desiccation resistance in membranes is a rare, unique ability previously observed only with trehalose dimycolate (TDM), an abundant mycobacterial glycolipid. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. The synthetic glycolipids consist of a simple trehalose disaccharide headgroup, similar to TDM, with hydrophobic tail groups of two 15- or 18-carbon chains. The synthetic trehalose glycolipids protected supported monolayers of phospholipids against dehydration even as minority components of the overall membrane, down to as little as 20 mol % trehalose glycolipid as assessed by assays of membrane fluidity. The dependence of the desiccation protection on the synthetic trehalose glycolipid fraction is nearly identical to that of TDM. The striking similarity of the desiccation resistance observed with TDM and the synthetic trehalose glycolipids, despite the variety of hydrophobic tail structures employed, suggests that interactions between the trehalose headgroup and surrounding molecules are the determining factor in dehydration protection.

11β-Hydroxysteroid Dehydrogenases Are Regulated during the Pulmonary Granulomatous Response to the Mycobacterial Glycolipid Trehalose-6,6′-Dimycolate

Objective: 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. Methods: 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. Results and Conclusion: IL-6, IL-1α and TNF-α 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 11β-hydroxysteroid dehydrogenases (11βHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11βHSD type 1 mRNA decreased 4-fold and 11βHSD type 2 (11βHSD2) 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 11βHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

Synthetic and Mycobacterial Trehalose Glycolipids Confer Dehydration Resistance to Supported Phospholipid Monolayers

We have recently demonstrated that the glycolipid trehalose dimycolate (TDM), a major outer membrane component of dehydration resistant Mycobacterium tuberculosis (MTb), can impart significant dehydration resistance to supported phospholipid membranes. We now report studies of other, related glycolipids both natural and synthetic that exhibit behavior similar to TDM, conferring protection from desiccation to membranes of which they are constituents. We examined solid-supported lipid monolayers, characterizing membrane integrity and two-dimensional fluidity before and after de- and re-hydration with fluorescence imaging and fluorescence recovery after photobleaching (FRAP). As with TDM, the degree of protection is dependent on the fraction of synthetic lipid in the monolayer and there is a distinct minimum fraction needed for protection by each glycolipid. We can control the synthetic lipid fluidity and minimum protecting fraction by designing and synthesizing lipids with particular hydrophobic chain lengths, saturation, and branching, thereby illuminating the role of molecular structure in biophysical function. The advent of these synthetic, protective glycolipids opens the door to the creation of lipid bilayers and liposomes since the relevant hydrophobic and hydrophilic domain sizes can be controlled. These new structures allow investigations of the physical origins of well-known mycobacaterial properties beyond dehydration resistance in controlled experimental contexts, such as the inhibition of membrane fusion.

Chemical Synthesis of All Phosphatidylinositol Mannoside (PIM) Glycans from Mycobacterium tuberculosis

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside (PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-limpet hemocyanin (KLH).

Trans-species activation of human T cells by rhesus macaque CD1b molecules

Despite crucial importance of non-human primates as a model of human infectious diseases, group 1 CD1 genes and proteins have been poorly characterized in these species. Here, we isolated CD1A, CD1B, and CD1C cDNAs from rhesus macaque lymph nodes that encoded full-length CD1 proteins recognized specifically by monoclonal antibodies to human CD1a, CD1b, and CD1c molecules, respectively. The monkey group 1 CD1 isoforms contained amino acid residues and motifs known to be critical for intramolecular disulfide bond formation, N-linked glycosylation, and endosomal trafficking as in human group 1 CD1 molecules. Notably, monkey CD1b molecules were capable of presenting a mycobacterial glycolipid to human CD1b-restricted T cells, providing direct evidence for their antigen presentation function. This also detects for the first time a trans-species crossreaction mediated by group 1 CD1 molecules. Taken together, these results underscore substantial conservation of the group 1 CD1 system between humans and rhesus macaque monkeys.

Mycobacterial Phenolic Glycolipid Inhibits Phagosome Maturation and Subverts the Pro‐inflammatory Cytokine Response

Inhibition of phagosome maturation is an important hallmark of mycobacterial pathogenesis. A variety of genomic, transcriptomic and proteomic approaches have been used to pin down the molecule responsible for this pathogenic principle. We in this study characterize a glycolipid of Mycobacterium marinum identified through a screen of mutants disabled in inhibiting phagosome maturation to be phenolphthiocerol diester (phenolic glycolipid, PGL). This molecule is sufficient to impart its ability to inhibit phagosome maturation onto other microbial cells and even inert beads that are used as model pathogens. In addition, it abrogates pro-inflammatory cytokine secretion induced by strong inducers such as heat-killed Mycobacterium bovis bacille Calmette-Guérin. This strong dual agonistic effect of PGL overrides pro-inflammatory and pro-lysosomal delivery impulses set not only by mycobacteria but also by other pathogens and thus provides convincing evidence that this molecule is a vital mycobacterial virulence factor.

Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans

In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-gamma+ T cells/10(5) PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.

Mycobacterial glycolipid trehalose 6,6′-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes

The granulomatous response is a characteristic histological feature of Mycobacterium tuberculosis infection responsible for organism containment. The development of cell-mediated immunity is essential for protection against disease, as well as being required for maintenance of the sequestering granulomatous response. Trehalose 6,6'-dimycolate (TDM; cord factor), a glycolipid associated with the cell wall of mycobacteria, is implicated as a key immunogenic component in M. tuberculosis infection. Models of TDM-induced hypersensitive granulomatous response have similar pathologies to that of active tuberculosis infection. Prior immunization (sensitization) of mice with TDM results in exacerbated histological damage, inflammation and lymphocytic infiltration upon subsequent TDM challenge. Adoptive transfer experiments were performed to ascertain the cell phenotype governing this response; CD4(+) cells were identified as critical for development of related pathology. Mice receiving CD4(+) cells from donor TDM-immunized mice demonstrated significantly increased production of Th1-type cytokines IFN-gamma and IL-12 within the lung upon subsequent TDM challenge. Control groups receiving naïve CD4(+) cells, or CD8(+) or CD19(+) cells isolated from TDM-immunized donors, did not exhibit an exacerbated response. The identified CD4(+) cells isolated from TDM-immunized mice produced significant amounts of IFN-gamma and IL-2 when exposed to TDM-pulsed macrophages in vitro. These experiments provide further evidence for involvement of a cell-mediated response in TDM-induced granuloma formation, which mimics pathological damage elicited during M. tuberculosis infection.

Mycobacterial Lipomannan Induces Granuloma Macrophage Fusion via a TLR2-Dependent, ADAM9- and β1 Integrin-Mediated Pathway

Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the beta(1) integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.

Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-α release

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6′-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6′-tetraacyl trehalose 2′-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-α) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1 h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-α release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-α release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.

Level of antibodies against mycobacterial glycolipid in the effusion for diagnosis of tuberculous pleural effusion

Diagnosing tuberculous pleural effusion (pTB) is often difficult because the culturing of tubercle bacilli results in a negative test in the majority of cases. Serological tests for the detection of antibodies to tuberculous glycolipid (TBGL) and lipoarabinomannan (LAM) have been introduced for the diagnosis of pulmonary tuberculosis. We examined the levels of these antibodies, adenosine deaminase (ADA) and interferon-gamma (IFN-gamma) in the pleural effusion and compared their diagnostic values in pTB. We studied 65 patients with pleural effusion. Of those, 19 patients were diagnosed as having pTB according to our broad case definition. The etiologies in the other 46 patients were malignant effusion, transdative effusion and miscellaneous diseases. Determiner TBGL antibody (D-TBGL-Ab) and MycoDot were used for the detection of anti-LAM and anti-TBGL antibodies, respectively, in the pleural effusion. The sensitivity of ADA was 78.9% (15/19) and the specificity 97.8% (45/46). The sensitivity of IFN-gamma was 84.2% (16/19) and the specificity 93.5% (43/46). The sensitivities of D-TBGL-Ab and MycoDot were both 52.6% (10/19) and their specificities were 95.7% (44/46) and 97.8% (45/46), respectively. When DTBGL-Ab (cutoff point: 2.0 U/ml) and ADA activity (cutoff point: 57 IU/l) were combined, the sensitivity was 94.7% (18/19) and the specificity 93.5% (43/46). In the diagnosis of pTB, D-TBGL-Ab and MycoDot each have low sensitivity but high specificity. When D-TBGL-Ab is used in combination with ADA, the sensitivity and specificity are both >90%. We conclude that D-TBGL-Ab and ADA in combination are useful in the diagnosis of pTB.

Interferon-γ independent formation of pulmonary granuloma in mice by injections with trehalose dimycolate (cord factor), lipoarabinomannan and phosphatidylinositol mannosides isolated fromMycobacterium tuberculosis

The mechanisms by which pulmonary granuloma formation is caused by administration of mycobacterial glycolipids such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM) were investigated. When peritoneal and alveolar macrophages were stimulated with TDM, LAM and PIM in vitro, TDM exhibited the strongest tumour necrosis factor (TNF)-inducing activity. Responsiveness of macrophages from mice defected Toll-like receptor 4 (TLR4) was much higher than that of the wild-type mice. Although PIM and LAM also had a significant activity, LAM rather than PIM stimulated higher TNF-alpha production by alveolar macrophage. When mycobacterial glycolipids were injected as water-in-oil-in-water emulsion into mice via the tail vein, development of pulmonary granuloma in response to glycolipids were related closely to their TNF-inducing activity and TDM exhibited the strongest activity. Granuloma formation was observed not only in mice lacking interleukin (IL)-12 signalling but also interferon (IFN)-gamma knock-out mice. Granuloma formation caused by glycolipids correlated with TNF-alpha levels in lungs. Administration of anti-TNF-alpha monoclonal antibody into TDM-injected IFN-gamma knock-out mice decreased in granuloma formation, suggesting that development of pulmonary granuloma by mycobacterial glycolipids such as TDM is due to IFN-gamma-independent and TNF-alpha-dependent pathway.

Influence of the Washing Buffer Composition on the Sensitivity of an Enzyme‐Linked Immunosorbent Assay Using Mycobacterial Glycolipids as Capture Antigens

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.