Platelets direct monocyte differentiation into epithelioid-like multinucleated giant foam cells with suppressive capacity upon mycobacterial stimulation.

BACKGROUND: Epithelioid, foam, and multinucleated giant cells (MNGCs) are characteristics of tuberculosis granulomas, yet the precise genesis and functions of these transformed macrophages are unclear. We evaluated the role of platelets as drivers of macrophage transformation in mycobacterial infection. METHODS: We employed flow cytometry and microscopy to assess cellular phenotype and phagocytosis. Immune assays allowed quantification of cytokines and chemokines, whereas gene microarray technology was applied to estimate global transcriptome alterations. Immunohistochemical investigations of tuberculosis granulomas substantiated our findings at the site of infection. RESULTS: Monocytes differentiated in presence of platelets (MP-Macs) acquired a foamy, epithelioid appearance and gave rise to MNGCs (MP-MNGCs). MP-Macs up-regulated activation markers, phagocytosed mycobacteria, and released abundant interleukin 10. Upon extended culture, MP-Macs shared transcriptional features with epithelioid cells and M2 macrophages and up-regulated CXCL5 transcripts. In line with this, CXCL5 concentrations were significantly increased in airways of active tuberculosis patients. The platelet-specific CD42b antigen was detected in MP-Macs, likewise in macrophages, MNGCs, and epithelioid cells within tuberculosis granulomas, along with the platelet aggregation-inducing factor PDPN. CONCLUSIONS: Platelets drive macrophage differentiation into MNGCs with characteristics of epithelioid, foam, and giant cells observed in tuberculosis granulomas. Our data define platelets as novel participants in tuberculosis pathogenesis.

The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis modulates the survival of intracellular Mycobacterium tuberculosis and autophagy in macrophages.

Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis (ATB) diagnosis. Therefore, we identified significantly dysregulated circRNAs in ATB patients and healthy controls (HC) and explored their molecular mechanism. We found that hsa_circ_0002371 was significantly up-regulated in PBMCs of ATB patients and Mycobacterium tuberculosis H37Rv- or Mycobacterium bovis bacillus Calmette Guerin (BCG)-infected THP-1 cells. Functional experiments demonstrated that hsa_circ_0002371 inhibited autophagy in BCG-infected THP-1 cells and promoted intracellular BCG survival rate. In terms of mechanism, hsa_circ_0002371 facilitated the expression of hsa-miR-502-5p, as shown by bioinformatics and dual-luciferase reporter gene analysis, respectively. Notably, hsa-miR-502-5p inhibited autophagy via suppressing autophagy related 16 like 1 (ATG16L1) in BCG-infected macrophages and thus promoting intracellular BCG growth. In summation, hsa_circ_0002371 increased the suppression of hsa-miR-502-5p on ATG16L1 and inhibited autophagy to promote Mtb growth in macrophages. In Conclusion, our data suggested that hsa_circ_0002371 was significantly up-regulated in the PBMCs of ATB patients compared with HC. The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis promoted the survival of intracellular Mtb and inhibited autophagy in macrophages. Our findings suggested hsa_circ_0002371 could act as a potential diagnostic biomarker and therapeutic target.

Clinical value of ELISA-MPT64 for the diagnosis of tuberculous pleurisy.

Tuberculous pleurisy is one of the common extrapulmonary tuberculosis diseases. However, the diagnosis of tuberculous pleurisy still lacks a useful and effective tool, mainly due to paucity of Mycobacterium tuberculosis organisms in pleural effusion. Previous studies have confirmed that the MPT64 protein is highly specific and is secreted only by M. tuberculosis (MTB) complex. Therefore, in this study, we developed ELISA based on recombinantly expressed MPT64 in combination with rabbit polyclonal antibodies. The ELISA-MPT64 method was validated using MTB strains and tested against clinical samples. Nested PCR, Löwenstein-Jensen (L-J) culture and smear microscopy were employed as the comparative tools for assessing the performance of the assay. Our results demonstrate that the newly established ELISA-MPT64 technique is a rapid and useful tool for the diagnosis of tuberculous pleurisy.

[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64].

OBJECTIVE: To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. METHODS: The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. RESULTS: A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. CONCLUSION: Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.

[Expression and purification of CFP32 of Mycobacterium tuberculosis and its serodiagnostic analysis].

OBJECTIVE: To establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity. METHODS: Rv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA. RESULTS: Recombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively. CONCLUSION: The recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

A novel recombinant BCG-expressing pro-apoptotic protein BAX enhances Th1 protective immune responses in mice.

One-third of the world's population is infected with Mycobacterium tuberculosis (MTB). The protective efficacy of bacille Calmette Guérin (BCG) vaccine against tuberculosis (TB) in adults is highly controversial even though the BCG vaccine has been available for more than 90 years. Because BCG is effective against infantile tuberculosis meningitis and miliary tuberculosis in young children and provides cost-effective prevention from tuberculosis for developing countries, it would be desirable to modify the existing BCG vaccine to provide more comprehensive protection. In our study, we constructed a novel recombinant BCG strain expressing pro-apoptotic BAX (rBCG::BAX) and demonstrated that it significantly induced the apoptosis of macrophages infected with rBCG::BAX both in vitro and in vivo. In addition, it significantly enhanced Ag85B-specific IFN-γ enzyme-linked immunospot responses, IFN-γ secretion, IL-2 secretion and the ratio of Ag85B-specific IgG2b/IgG1, and it significantly decreased Ag85B-specific IL-4. Furthermore, it presumably facilitated antigen presentation by inducing a significant up-regulation in the expression of MHC-II and B7.1 (CD80) co-stimulatory molecules on macrophages. In conclusion, these results suggest that the rBCG::BAX strain elicited predominantly a Th1 protective immune responses and might be a potential tuberculosis vaccine candidate for further study.

A rapid loop-mediated isothermal amplification assay targeting hspX for the detection of Mycobacterium tuberculosis complex.

A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study.

BACKGROUND: Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. METHODS AND FINDINGS: The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). CONCLUSIONS: The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.