Objectives miR-207 has been identified as being expressed in natural killer (NK) cell exosomes that play a role in disease progression; however, to date, there are no studies specifically linking miR-207 to tuberculosis (TB). Methods Bioinformatics methods employed for prediction, followed by a dual luciferase reporter assay to determine whether lysosome-associated membrane protein 2 (LAMP2) is targeted by miR-207. The experiments were divided into four groups using the liposome transfection method (OP-LAMP2 group: co-transfected with miR-207 mimics and LAMP2 overexpression plasmid; EP group: co-transfected with mimics NC and null-loaded plasmid; siLAMP2 group: transfected with siLAMP2; and siLAMP2-NC group: transfected with siLAMP2-NC). TB infection was modeled using H37Ra-infected Ana-1 cells. The impact of LAMP2 on intracellular mycobacterial load and clearance of extracellular residual mycobacteria were assessed by tuberculosis colony-forming unit counting. Flow cytometry was used to assess the total apoptosis rate. Real-time fluorescent quantitative PCR was conducted to determine the relative expression of LAMP2, apoptosis genes, pyroptosis genes, and autophagy genes. Western blot analysis was performed to measure the relative expression of LAMP2 proteins, apoptosis proteins, pyroptosis proteins, and autophagy proteins. Results Dual luciferase reporter assay test showed that there was a targeting relationship between LAMP2 and miR-207. The transfection model was successfully constructed under real-time fluorescent quantitative PCR and Western blot statistical analysis, and microscopic observation. The infection model was successfully established under microscopic observation. Colony forming unit counting revealed that the number of colonies in the OP-LAMP2 group was lower than that in the EP group, while the number of colonies in the siLAMP2 group was higher than that in the siLAMP2-NC group. Flow cytometry assay revealed that the total apoptosis in OP-LAMP2 group was lower than that in EP group, and the total apoptosis in siLAMP2 group was higher than that in siLAMP2-NC group. Real-time fluorescence quantitative PCR and Western blot analysis revealed that the relative expression of apoptosis and pyroptosis-related proteins and genes in the control group was lower in the OP-LAMP2 group compared to the EP group, and higher in the siLAMP2 group compared to the siLAMP2-NC group. Real-time fluorescence quantitative PCR detected that the relative expression of autophagy positively regulated genes Microtubule-associated protein 1 light chain 3(LC3)and Beclin1 in the OP-LAMP2 group was higher in the OP-LAMP2 group compared to the EP group, and lower in the siLAMP2 group compared to the siLAMP2-NC group, while the relative expression of negatively regulated autophagy genes followed the opposite trend to that of autophagy positively regulated genes. The relative expression of autophagy-related proteins was consistent with the trend of autophagy genes. Conclusions miR-207 enhances macrophage apoptosis, cellular pyroptosis and inhibits autophagy, promoting survival of Mycobacterium tuberculosis by targeting the autophagy-related protein LAMP2, thus offering a novel therapeutic direction for tuberculosis.
Lysosomal-Associated Membrane Protein 2/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Autophagy/genetics* ; Humans ; Macrophages/metabolism* ; Apoptosis/genetics* ; Tuberculosis/metabolism* ; Cell Line ; Pyroptosis/genetics*

Objective To explore the effect of miR-582-5p on Mycobacterium tuberculosis (Mtb)-infected macrophages by regulating dual specificity phosphatase 1 (DUSP1). Methods THP-1 macrophages were divided into six groups: control group, Mtb group, inhibitor-NC group, miR-582-5p inhibitor group, miR-582-5p inhibitor+si-NC group, and miR-582-5p inhibitor+si-DUSP1 group. QRT-PCR was applied to detect the gene expression of miR-582-5p and DUSP1 in cells. ELISA kit was used to detect the levels of interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). CCK-8 method was applied to detect cell proliferation. Flow cytometry was applied to detect cell apoptosis rate. Western blot analysis was used to measure the protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X (BAX), and cleaved-caspase 3 (c-caspase-3) in cells. In addition, the target relationship between miR-582-5p and DUSP1 was verified. Results Compared with the control group, the expression of miR-582-5p, levels of IFN-γ, IL-6, TNF-α, IL-1β, bacterial load and OD450 values (24 h, 48 h), and the protein expression of Bcl2 in macrophages were higher in the Mtb group, while the mRNA expression of DUSP1, apoptosis rate, and the protein expression levels of c-caspase-3, BAX and DUSP1 were lower. Compared with the Mtb group and the inhibitor-NC group, the above-mentioned indicators in the miR-582-5p inhibitor group were partially reversed. Down-regulation of DUSP1 expression partially reversed the inhibitory effect of down-regulation of miR-582-5p expression on Mtb-infected macrophages. Conclusion Inhibiting the expression of miR-582-5p can up-regulate DUSP1, thereby inhibiting the proliferation and inflammatory response of Mtb-infected macrophages and promoting cell apoptosis.
Humans ; Macrophages/metabolism* ; Dual Specificity Phosphatase 1/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Tuberculosis/microbiology* ; Apoptosis/genetics* ; THP-1 Cells ; Cell Proliferation/genetics* ; Interferon-gamma/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics*

OBJECTIVES: This study aimed to investigate the impact of foam macrophages (FMs) on the intracellular survival of Mycobacterium tuberculosis (MTB) and identify the molecular mechanisms influencing MTB survival. METHODS: An in vitro FM model was established using oleic acid induction. Transcriptomic and metabolomic analyses were conducted to identify the key molecular pathways involved in FM-mediated MTB survival. RESULTS: Induced FMs effectively restricted MTB survival. Transcriptomic and metabolomic profiling revealed distinct changes in gene and metabolite expression in FMs during MTB infection compared with normal macrophages. Integrated analyses identified significant alterations in the cyclic adenosine monophosphate (cAMP) signaling pathway, indicating that its activation contributes to the FM-mediated restriction of MTB survival. CONCLUSIONS FMs inhibit MTB survival. The cAMP signaling pathway is a key contributor. These findings enhance the understanding of the role of FMs in tuberculosis progression, suggest potential targets for host-directed therapies, and offer new directions for developing diagnostic and therapeutic strategies against tuberculosis.
Mycobacterium tuberculosis/physiology* ; Transcriptome ; Metabolomics ; Foam Cells/microbiology* ; Humans ; Metabolome ; Tuberculosis/microbiology* ; Gene Expression Profiling

DNA double-strand breaks represent a common type of serious DNA damage in living organisms, causing instability of the genome and leading to cell death. Homologous recombination and non-homologous end-joining (NHEJ) are the two main ways to repair DNA double-strand breaks. The core components involved in the NHEJ pathway are highly conserved in both yeast and humans. A few bacteria such as Mycobacterium, Pseudomonas aeruginosa, and Bacillus subtilis also have the NHEJ mechanism. NHEJ plays a key role in the double strand repair of Mycobacterium in latency. This paper summarizes the mechanism and important components of NHEJ in Mycobacterium, introduces the application of NHEJ in gene editing, and reviews the research progress of the NHEJ pathway in Mycobacterium. We hope to bring new insights into the molecular mechanism and provide clues for the application of NHEJ in Mycobacterium.
DNA End-Joining Repair/physiology* ; Gene Editing/methods* ; Mycobacterium/physiology* ; DNA Breaks, Double-Stranded ; Humans

Objective The aim of this study was to evaluate the diagnostic performance of T-SPOT.TB for tuberculous lymphadenitis. Methods Suspected tuberculous lymphadenitis patients between September 2010 and September 2018 who had both peripheral blood T-SPOT.TB test and lymph node biopsy were retrospectively enrolled in this study. The cutoff value of T-SPOT.TB test for peripheral blood was set as 24 spot forming cell (SFC)/10 ⁶ periphreral blood monocyte cell (PBMC) according to the instruction of testing kits. The gold standard for diagnosis of TBL was the combination of microbiology results, histopathology results and patient's response to anti-TB treatment. Diagnostic efficacy of T-SPOT.TB was evaluated, including sensitivity, specificity, accuracy, predictive values, and likelihood ratio. Results Among 91 patients who met the inclusion criteria, we excluded 8 cases with incomplete clinical information and 6 cases who lost to follow-up. According to the gold standard, there were 37 cases of true TBL (9 confirmed TBL and 28 probable TBL), 30 cases of non-TBL, and 10 cases of clinically indeterminate diagnosis who were excluded from the final analyses. The T-SPOT.TB tests yielded 43 cases of positive response and 24 cases of negative response. The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR) of peripheral blood T-SPOT.TB for diagnosing TBL were 89.2%, 66.7%, 79.1%, 76.7%, 83.3%, 2.68 and 0.16, respectively. The number of SFCs of T-SPOT.TB in TBL patients [432(134-1264)/10 ⁶ PBMCs] was higher than that in non-TBL patients [0 (0-30) /10 ⁶PBMCs] with a significant difference (Z=-5.306, P <0.001). Conclusion T-SPOT.TB is a rapid and simple diagnostic test for TBL with a high sensitivity and negative predictive value.
Adolescent ; Adult ; Aged ; Female ; Humans ; Interferon-gamma Release Tests ; Male ; Middle Aged ; Mycobacterium tuberculosis/physiology* ; T-Lymphocytes/immunology* ; Tuberculosis, Lymph Node/diagnosis* ; Young Adult

The objective of this prospective study of the risks of treatment failure in patients with drug-susceptible pulmonary tuberculosis (PTB) was to provide reference data to help develop a disease control strategy. Participants were recruited in eight provinces of China from October 2008 to December 2010. A total of 1447 patients with drug-susceptible PTB and older than 15 years of age were enrolled. Demographic characteristics, bacteriological test results, and patient outcome, i.e., cure or treatment failure were recorded and compared using the chi-square or Fisher's exact tests. Multivariate logistic regression was used to identify factors associated with risk of treatment failure. Of the 1447 patients who were enrolled, 1349 patients (93.2%) were successfully treated and 98 (6.8%) failed treatment. Failure was significantly associated with age 365 years [odds ratio (OR)=2.522, 95% confidence interval (CI): (1.097-5.801)], retreatment [OR=2.365, 95% CI: (1.276-4.381)], missed medicine [OR=1.836, 95% CI: (1.020-3.306)], treatment not observed [OR=1.879 95% CI: (1.105-3.195)], and positive culture result after the first [OR=1.971, 95% CI: (1.080-3.597)] and second month [OR=4.659, 95% CI: (2.590-8.382)]. The risk factors associated with treatment failure were age 365 years, retreatment, missed medication, treatment not observed, and positive culture at the end of month 1 or month 2. These risk factors should be monitored during treatment and interventions carried out to reduce or prevent treatment failure and optimize treatment success.
Adolescent ; Adult ; Aged ; Antitubercular Agents ; therapeutic use ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; drug effects ; physiology ; Prospective Studies ; Retreatment ; Risk Factors ; Treatment Failure ; Tuberculosis, Multidrug-Resistant ; drug therapy ; epidemiology ; microbiology ; Tuberculosis, Pulmonary ; drug therapy ; epidemiology ; microbiology ; Young Adult

Lung disease caused by nontuberculous mycobacteria (NTM) represents an increasing proportion of all mycobacterial diseases. We investigated recent occurrences of NTM and evaluated the clinical significance of NTM isolates from 752 respiratory specimens collected from patients at National Health Insurance Service Ilsan Hospital between January 2007 and May 2011. Specimens were incubated on solid and liquid media (BACTEC MGIT 960, BD, USA) for 6-8 weeks, and PCR and reverse blot hybridization were performed (REBA Myco-ID, Molecules & Diagnostics, Korea). Clinical features of the patients were reviewed through medical records. The most frequently isolated organism was Mycobacterium avium (46.7%), followed by M. intracellulare (14.8%), M. fortuitum (7.2%), and M. abscessus (6.6%). The most common mycobacteria among definitive cases of NTM lung disease were M. avium (42/351, 12.0%), M. intracellulare (19/111, 17.1%), M. abscessus (11/50, 22.0%), M. massiliense (4/13, 30.8%), and M. fortuitum (4/54, 7.4%). Clinically significant cases of NTM lung disease increased from 4 patients in 2007 to 32 in 2011. The mean patient age was 64 yr (range: 35-88 yr), and 58 (64%) patients were women. Patients suffered from cough, productive sputum, and hemoptysis. In summary, the most common mycobacteria causing NTM lung disease were M. avium and M. intracellulare; however, cases of M. massiliense and M. abscessus infection are on the rise in Korea.
Adult ; Aged ; Aged, 80 and over ; Bronchoalveolar Lavage Fluid/microbiology ; DNA, Bacterial/analysis ; Female ; Hospitals, General/standards/*trends ; Humans ; Lung Diseases/diagnosis/*microbiology ; Male ; Middle Aged ; Mycobacterium Infections, Nontuberculous/diagnosis/*microbiology ; Nontuberculous Mycobacteria/genetics/*physiology ; Nucleic Acid Hybridization ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Republic of Korea ; Sputum/microbiology

As the first line of immune defense for Mycobacterium tuberculosis (Mtb), macrophages also provide a major habitat for Mtb to reside in the host for years. The battles between Mtb and macrophages have been constant since ancient times. Triggered upon Mtb infection, multiple cellular pathways in macrophages are activated to initiate a tailored immune response toward the invading pathogen and regulate the cellular fates of the host as well. Toll-like receptors (TLRs) expressed on macrophages can recognize pathogen-associated-molecular patterns (PAMPs) on Mtb and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). In addition, Vitamin D receptor (VDR) and Vitamin D-1-hydroxylase are up-regulated in Mtb-infected macrophages, by which Vitamin D participates in innate immune responses. The signaling pathways that involve TNF, type I IFNs and Vitamin D are inter-connected, which play critical roles in the regulation of necroptosis, apoptosis, and autophagy of the infected macrophages. This review article summarizes current knowledge about the interactions between Mtb and macrophages, focusing on cellular fates of the Mtb-infected macrophages and the regulatory molecules and cellular pathways involved in those processes.
Animals ; Apoptosis ; Autophagy ; Humans ; Interferon Type I ; metabolism ; Macrophages ; immunology ; metabolism ; Mycobacterium tuberculosis ; physiology ; Receptors, Calcitriol ; metabolism ; Steroid Hydroxylases ; metabolism ; Toll-Like Receptors ; metabolism ; Tuberculosis ; immunology ; metabolism ; pathology ; Tumor Necrosis Factors ; metabolism

BACKGROUNDChronic hepatic inflammation is characterized by the accumulation of lymphocytes as a consequence of increased recruitment from the blood and retention within the tissue at sites of infection. CXC chemokine ligand 16 (CXCL16) mRNA has been detected in both inflamed and normal liver tissues and is strongly upregulated in the injured liver tissues in a murine model. The aim of this study was to investigate the effect of cefodizime on CXCL16 mRNA of liver tissues in mice with immunological hepatic injury.

METHODSThe murine model of immunological hepatic injury was induced by Bacillus Calmette Guerin and Lipoposaccharide. The mice with immunological hepatic injury were randomly assigned to the model group, the cefodizime group and the ceftriaxone group. The three groups were continuously given agents for seven days and CXCL16 mRNA of liver tissue was determined and contrasted with the control group treated by normal saline. Reverse transcription-polymerase chain reaction was used to assay CXCL16 mRNA levels in liver tissues.

RESULTSThe expressions of CXCL16 mRNA were significantly higher in the model group and the ceftriaxone group than in the control group and the cefodizime group (P < 0.05), indicating the mice in the model group and the ceftriaxone group were immunodeficient. There was no statistical difference in the expressions of CXCL16 mRNA between the control group and the cefodizime group. Similarly, no statistical difference in the expressions of CXCL16 mRNA between the model group and the ceftriaxone group was detected (P > 0.05).

CONCLUSIONCefodizime effectively reduces the infiltration of lymphocytes into liver tissues and alleviates the liver damage by decreasing CXCL16 mRNA in liver tissues in mice with immunological hepatic injury.

Animals ; Cefotaxime ; analogs & derivatives ; therapeutic use ; Chemokine CXCL16 ; Chemokine CXCL6 ; genetics ; Chemokines ; Lipopolysaccharides ; toxicity ; Liver ; drug effects ; metabolism ; microbiology ; Mice ; Mycobacterium bovis ; physiology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of early intervention on CD4+CD25+ regulatory T cells and TLR4 expression with Mycobacterium phlei F.U.36 in asthmatic mice.

METHODSThirty female BALB/c mice were randomly divided into three groups: control, asthma model and Mycobacterium phlei F.U.36 treated asthma groups. Asthma was induced by sensitization and challenges with ovalbumin (OVA) in the later two groups. Mycobacterium phlei F.U.36 was intraperitoneally injected 2 weeks before the first sensitization (0.57 μg/time, once every other day for three times) in the intervention group. After 24 hrs of the last challenge, the mice were sacrificed and the left lung tissues were obtained for the observation of lung pathological changes. Splenic mononuclear cells were isolated. The percentage of CD4+CD25+ regulatory T cells in CD4+ T cells and the mean fluorescence intensity of TLR4 on CD4+ CD25+ T cells were detected by flow cytometry.

RESULTSThe percentage of CD4+CD25+ regulatory T cells in the asthma model group was significantly lower than that in the control group (P<0.01), but the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells was not significantly different from the control group. The percentage of CD4+CD25+ regulatory T cells and the mean fluorescence intensity of TLR4 on CD4+CD25+ regulatory T cells increased significantly in asthmatic mice receiving Mycobacterium phlei F.U.36 treatment compared with the asthma group (P<0.01).

CONCLUSIONSEarly intervention with Mycobacterium phlei F.U.36 can increase TLR4 expression on CD4+CD25+ cells and the number of CD4+CD25+ regulatory T cells, and thus provides therapeutic effects in asthmatic mice.

Animals ; Asthma ; immunology ; pathology ; Female ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Mycobacterium phlei ; T-Lymphocytes, Regulatory ; immunology ; Toll-Like Receptor 4 ; physiology