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Setting: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing., Background: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens., Methods: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples., Results: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU., Conclusion: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples., (© 2023 The Union.)

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. [ABSTRACT FROM AUTHOR]

Detection of cord factor may favor the definitive diagnosis and initiation of antituberculosis treatment. The aim of this study was to evaluate the interobserver concordance of mycobacterial smears stained with Ziehl-Neelsen technique for the presumptive identification of the Mycobacterium tuberculosis (M. tuberculosis). Three observers, healthcare students, conducted a blind reading of 30 slides stained with Ziehl-Neelsen and their results were compared to the results of an experient reader. Kappa coefficient with 95% interval was used to evaluate the concordance between the readings. No excellent interobserver concordance (α>0.75) was found. The interpretation and use of the cord factor for the presumptive identification of the M. tuberculosis is a technique that requires training for the interpretation of the variables of the smear stained with Ziehl-Neelsen technique and theoretical knowledge of the observer to deliver a correct result. [ABSTRACT FROM AUTHOR]

Within the Actinobacteria, the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus form the so-called CMNR group, also designated as mycolic acid-containing actinomycetes. Almost all members of this group are characterized by a mycolic acid layer, the mycomembrane, which covers the cell wall and is responsible for a high resistance of these bacteria against chemical and antibiotic stress. Furthermore, components of the mycomembrane are crucial for the interaction of bacteria with host cells. This review summarizes the current knowledge of mycolic acid synthesis and interaction with components of the immune system for the genus Corynebacterium with an emphasis on the pathogenic species Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans as well as the biotechnology workhorse Corynebacterium glutamicum. [ABSTRACT FROM AUTHOR]

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

For successful colonization of a host, pathogenic bacteria are equipped with a plethora of proteins and other components, often designated as virulence factors. However, many of these factors are involved in general adaptation to ecological niches rather than being specific for interaction of a bacterial pathogen with a distinct host. Therefore, a designation of these components as niche factors was proposed. While originally developed for different species colonizing the gastro-intestinal tract, in this communication the concept of niche factors is discussed in respect to a single genus, i.e. Corynebacterium species. [ABSTRACT FROM AUTHOR]

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

MCL (macrophage C-type lectin) and mincle (macrophage inducible C-type lectin) comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage associated and pathogen associated molecular patterns. In this review we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate (TDM) and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies.

The synthesis of an α′-mycolic acid of Mycobacterium smegmatis and other mycobacteria, containing a cis,cis -diene, and of the trehalose mono- and di-mycolates of this, and of a related α′-mycolic acid containing one cis -alkene, is reported. [ABSTRACT FROM AUTHOR]

The world’s most successful intracellular bacterial pathogen, Mycobacterium tuberculosis (MTB), survives inside macrophages by blocking phagosome maturation and establishes chronic infection characterized by the formation of granulomas. Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is the most abundant cell wall lipid of virulent mycobacteria, is sufficient to cause granuloma formation, and has long been known to be a major virulence factor of MTB. Recently, TDM has been shown to activate the Syk-Card9 signaling pathway in macrophages through binding to the C-type lectin receptor Mincle. The Mincle-Card9 pathway is required for activation of macrophages by TDM in vitro and for granuloma formation in vivo following injection of TDM. Whether this pathway is also exploited by MTB to reprogram the macrophage into a comfortable niche has not been explored yet. Several recent studies have investigated the phenotype of Mincle-deficient mice in mycobacterial infection, yielding divergent results in terms of a role for Mincle in host resistance. Here, we review these studies, discuss possible reasons for discrepant results and highlight open questions in the role of Mincle and other C-type lectin receptors in the infection biology of MTB.

Macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle) comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage-associated and pathogen-associated molecular patterns. In this review, we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies. [ABSTRACT FROM AUTHOR]

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Bacteriophages, the viruses of eubacteria, have developed unique mechanisms to interact with their host bacteria. They have been viewed as potential antibacterial therapeutics. Mycobacteriophage-derived compounds may interact with Mycobacterium tuberculosis (MTB) and/or its components, such as the cord factor, trehalose-6,6'-dimycolate (TDM), which is the most abundant glycolipid produced on the surface of MTB. TDM emulsion injected intravenously into mice induces lung immunopathology that mimics many aspects of MTB infection. Thus, TDM is an important target for anti-MTB agent development. On the basis of genomics information of mycobacteriophages, 200 peptides were synthesized. Their effects on MTB, their interactions with TDM, and anti-inflammatory activities were tested. One of them (PK34) showed MTB-killing activity with a minimal inhibitory concentration of 50 (Jig/ml and TDM-binding ability. In a mouse model, PK34 showed comparable ability to clear MTB as rifampin did in vivo. It also exerted strong activity to inhibit MTB or TDM-induced inflammation in vivo. PK34 significantly inhibited inflammatory cytokines secretions by inactivating MAPK and PKB signals while it maintained certain proinflammatory cytokine production. It is possible to prospect for TDM-binding and/or anti-MTB peptides by mining the mycobacteriophages genome. In addition to its direct MTB-killing ability, PK34 might be a useful adjunct in the treatment of granulomatous inflammation occurring during myco-bacterial infection or a template for developing antituberculosis (TB) agents because of its immunoregulative effects. As a TDM-binding peptide, PK34 may be a promising tool to study TDM's interactions with corresponding receptors and signal pathways. [ABSTRACT FROM AUTHOR]

The world's most successful intracellular bacterial pathogen, Mycobacterium tuberculosis (MTB), survives inside macrophages by blocking phagosome maturation and establishes chronic infection characterized by the formation of granulomas. Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is the most abundant cell wall lipid of virulent mycobacteria, is sufficient to cause granuloma formation, and has long been known to be a major virulence factor of MTB. Recently, TDM has been shown to activate the Syk-Card9 signaling pathway in macrophages through binding to the C-type lectin receptor Mincle. The Mincle-Card9 pathway is required for activation of macrophages by TDM in vitro and for granuloma formation in vivo following injection of TDM. Whether this pathway is also exploited by MTB to reprogram the macrophage into a comfortable niche has not been explored yet. Several recent studies have investigated the phenotype of Mincle-deficient mice in mycobacterial infection, yielding divergent results in terms of a role for Mincle in host resistance. Here, we review these studies, discuss possible reasons for discrepant results and highlight open questions in the role of Mincle and other C-type lectin receptors in the infection biology of MTB. [ABSTRACT FROM AUTHOR]

The cell wall of the pathogenic bacterium Rhodococcus equi ( R. equi) contains abundant trehalose monomycolate (TMM) and trehalose dimycolate (TDM), the glycolipids bearing mycolic acids. Here, we describe multiple-stage (MS) linear ion-trap (LIT) mass spectrometric approaches toward structural characterization of TMM and TDM desorbed as [M + Alk] (Alk = Na, Li) and as [M + X] (X = CHCO, HCO) ions by electrospray ionization (ESI). Upon MS ( n = 2, 3, 4) on the [M + Alk] or the [M + X] adduct ions of TMM and TDM, abundant structurally informative fragment ions are readily available, permitting fast assignment of the length of the meromycolate chain and of the α-branch on the mycolyl residues. In this way, structures of TMM and TDM isolated from pathogenic R. equi strain 103 can be determined. Our results indicate that the major TMM and TDM molecules possess 6, and/or 6'-mycolyl groups that consist of mainly C14 and C16 α-branches with meromycolate branches ranging from C18 to C28, similar to the structures of the unbound mycolic acids found in the cell envelope. Up to 60 isobaric isomers varying in chain length of the α-branch and of the meromycolate backbone were observed for some of the TDM species in the mixture. This mass spectrometric approach provides a direct method that affords identification of various TMM and TDM isomers in a mixture of which the complexity of this lipid class has not been previously reported using other analytical methods. [ABSTRACT FROM AUTHOR]

Abstract: The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6′-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6′-dimycolate (TDM) is synthesized from two molecules of trehalose-6′-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K m and K cat were determined with values of 129.6±8.1µM and 65.4±4.1min−1, respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z′ factor of 0.67–0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope. [Copyright &y& Elsevier]

Abstract: Lung surfactant is a lipoprotein mixture synthesized by the type II alveolar cells in the lungs and lines the air–aqueous interface. It is essential for stabilizing the pulmonary alveoli. Deficiency or dysfunction of the surfactant due to chemicals or toxins results in alveolar collapse and if untreated leads to respiratory distress. In pulmonary tuberculosis, Mycobacterium tuberculosis bacteria reside in the alveoli, in close physical proximity with the alveolar surfactant. The surface active mycobacterial cell wall lipids may easily detach from bacterial cell wall and interact with surfactant components altering their surface activity. In this study, effects of mycolic acid and cord factor (the abundant surface lipids of mycobacterial cell wall) on the surface activity of modified bovine surfactant extract (Survanta™) were characterized in vitro using the Wilhelmy surface balance at physiological temperature (37°C) and pH 7.4. Inhibitory effects of mycobacterial cell wall lipids on the terminal airway surfactant were evaluated using a capillary surfactometer. Addition of graded amounts of mycolic acid and cord factor significantly increased the minimum surface tension of Survanta™ monolayer from <1mN/m to ∼11–20mN/m. Presence of mycobacterial cell wall lipids decreased the rate and magnitude of Survanta™ adsorption and fluidized the surfactant monolayer. Capillary surfactometer study exhibited 100% capillary closure on addition of mycobacterial cell wall lipids to the modified bovine surfactant extract. AFM imaging revealed alteration of surface topography and presence of aggregates on addition of mycobacterial cell wall lipids to Survanta™ monolayer. This study suggests a biophysical inhibition of lung surfactant which may play a role in the pathogenesis of pulmonary tuberculosis. [Copyright &y& Elsevier]

Abstract: Trehalose 6,6′-dimycolate (TDM) is the most abundant lipid extracted from Mycobacterium tuberculosis (MTB). TDM promotes MTB survival by decreasing phagosomal acidification and phagolysosomal fusion in macrophages. Delipidation of MTB using petroleum ether removes TDM and decreases MTB survival within host cells. TDM reconstituted onto MTB restores its virulent wild-type characteristics. We investigated the role of TDM in regulating surface marker expression in MTB-infected macrophages. Macrophages were infected with wild-type, delipidated, and TDM-reconstituted MTB for 24h and measured for changes in surface marker expression. TDM on MTB was found to specifically target MHCII, CD1d, CD40, CD80 and CD86. Both wild-type and TDM-reconstituted MTB suppressed or induced no change in expression of these surface markers, whereas delipidated MTB increased expression of the same markers. MTB-infected macrophages were also overlaid with MHCII-restricted T cell hybridomas which recognize Antigen 85B. Macrophages infected by wild-type and TDM-reconstituted MTB did not present antigen as well as delipidated MTB-infected macrophages. The evidence shown furthers supports the notion that TDM present on MTB promotes its survival and persistence in host macrophages. [Copyright &y& Elsevier]

Abstract: 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°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 (SCMmax). 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 SCMmax from 160 to 40mN/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. [Copyright &y& Elsevier]

Despite an available vaccine and effective antibiotics, Mycobacterium tuberculosis is still the causative agent of almost 2 million deaths every year. The cell wall of M. tuberculosis is composed of sugars and lipids of exotic structure, many of which contribute to its pathogenicity. The majority of the enzymes responsible for building this structure are essential. However, they share very little homology with well-characterized enzymes, which makes their identification in the genome difficult. Despite this, our knowledge of the structure of the cell wall of M. tuberculosis is fairly complete and an increasing number of genes have been identified that are involved in its biosynthesis. By contrast, data concerning regulation of the expression of these genes and control of the cell wall composition are restricted. This review summarizes current information on the genetics of cell wall biosynthesis in M. tuberculosis, incorporating available data on gene organization and regulation. [ABSTRACT FROM AUTHOR]

Summary: Setting: M. tuberculosis (MTB) lose virulence during prolonged culture on artificial media. This loss of virulence is associated with a change in colony morphology. Several studies suggested that trehalose 6,6′ dimycolate (TDM or cord factor), contributes to colony morphology. Objective: To investigate the role of TDM in colony morphology of MTB using clinical isolates selected to have colony morphology typical of virulent or attenuated organisms. Design: Use immunohistochemical and physical chemical methods to assess the presence and distribution of TDM in rapidly growing pellicles of MTB. Results: TDM forms an insoluble crystalline monolayer at the air–water interfaces that is more rigid than that formed by any other biologic amphiphile and is strong enough to support a spreading pellicle of MTB. The surface of young pellicles of the isolate with virulent morphology displayed the regular linear pattern characteristic of monolayers of TDM. TDM was also identified in the open spaces of pellicles of MTB by immunohistochemistry. MTB with morphology of attenuated organisms had neither of these properties. Conclusion: These data suggest that the characteristic morphology of colonies of virulent MTB is due to TDM released from the surface of the organisms. [Copyright &y& Elsevier]

Abstract: 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 1h 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. [Copyright &y& Elsevier]

Abstract: We aimed to reveal the regulatory function of macrophage scavenger receptor-A (MSR-A) in proinflammatory cytokine production by macrophages stimulated with mycobacterial cord factor (CF). By the culture with CF, MSR-A (+/+) alveolar macrophages and Kupffer cells produced TNF-α/MIP-1α in a time- and dose-dependent manner. However, the amounts of cytokines produced by them were much less compared to those produced by MSR-A (−/−) macrophages. Consistent with this, treatment of MSR-A (+/+) macrophages with anti-MSR-A antibody increased TNF-α production. Binding of CF to MSR-A was demonstrated by measuring the binding affinity. These results indicate that CF binds MSR-A, and MSR-A down-regulates TNF-α/MIP-1α production by activated macrophages, suggesting the role of this receptor in suppression of excessive inflammatory responses during mycobacterial infection. [Copyright &y& Elsevier]

Abstract: Low dose aerosol infection of C57BL/6 mice with a clinical strain of Mycobacterium tuberculosis (UTE 0335 R) induced intragranulomatous necrosis in pulmonary granulomas (INPG) at week 9 postinfection. Infection of different knockout (KO) mouse strains with UTE 0335 R induced INPG in all strains and established two histopathological patterns. The first pattern was seen in SCID mice and in mice with deleted α/β T receptor, TNF R1, IL-12, IFN-γ, or iNOS genes, and showed a massive INPG with a high granulomatous infiltration of the lung, a large and homogeneous eosinophilic necrosis full of acid-fast bacilli, with marked karyorrhexis, coarse basophilic necrosis, and surrounded by patches delimited by partially conserved alveolar septum full of PMNs. The second pattern was seen in mice with deleted IL-1 R1, IL-6, IL-10, CD4, CD8 or γ/δ T cell receptor genes, and showed more discrete lesions with predominant homogeneous eosinophilic necrosis with few bacilli and surrounded by a well-defined lymphocyte-based ring. Local expression of IFN-γ, iNOS, TNF and RANTES showed no significant differences between these mouse strains generating a discrete INPG. Mouse strains showing a massive INPG showed higher, lower or equal expression values compared to the control strain. In conclusion, the severity of the INPG pattern correlated with pulmonary CFU counts, irrespective of the genetic absence or the infection-induced levels of cytokine mediators. [Copyright &y& Elsevier]

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 (TBSBSA). 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 anticord 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. [ABSTRACT FROM AUTHOR]

Cystic periventricular leukomalacia (cPVL), the principal ischemic brain injury in premature infants, is characterized by necrosis of the white matter in the periventricular region and the major neuropathology for spastic motor deficits in cerebral palsy or epilepsy. Recent reports strongly suggest that the brain injury associated with cPVL may have already occurred in utero. In this study we searched retrospectively for possible clinical situations related to cPVL to facilitate assessment of optimal management. A total of 201 babies born at gestational ages from 24 to 33 weeks were entered into the study (1992–1997) and examined for involvement of 18 factors in cPVL retrospectively. And psychomotor development was examined at least until 18 months of corrected age. Among 201 premature babies 35 cases were diagnosed as cPVL later developed spastic diplegia. There are 23 cases of preeclampsia, no infant suffering from cPVL. In the univariate analysis, exposure to antenatal indomethacin, cord length ≥40 cm, and a low Apgar score were significantly associated with a 2–3 risk increased of cPVL occurrence, while antenatal magnesium sulfate reduced the risk. Chorioamnionitis was positively correlated with the risk, but did not reach statistical significance. In the multivariate analysis we found the statistical significance in exposure to antenatal indomethacin, a low Apgar score, and antenatal magnesium sulfate. Our results suggested that preeclampsia and antenatal exposure of magnesium sulfate reduced the risk while antenatal exposure of indomethacin and low Apgar score associated with the occurrence of cPVL. These findings support a growing consensus that cPVL is often the result of maternal and fetal factors as well as antenatal treatment. [Copyright &y& Elsevier]

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 1H-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. [Copyright &y& Elsevier]