Mycobacterium Tuberculosis Infection Research Articles

Investigating the treatment shortening potential of a combination of bedaquiline, delamanid and moxifloxacin with and without sutezolid, in a murine tuberculosis model with confirmed drug exposures.

New and shorter regimens against multi-drug resistant tuberculosis (TB) remain urgently needed. To inform treatment duration in clinical trials, this study aimed to identify human pharmacokinetic equivalent doses, antimycobacterial and sterilizing activity of a novel regimen, containing bedaquiline, delamanid, moxifloxacin and sutezolid (BDMU), in the standard mouse model (BALB/c) of Mycobacterium tuberculosis (Mtb) infection. Treatment of mice with B25D0.6M200U200, B25D0.6M200, B25D0.6M200(U2003) or H10R10Z150E100 (isoniazid, rifampicin, pyrazinamide, ethambutol, HRZE), started 3 weeks after Mtb infection. Bactericidal activity was assessed after 1, 2, 3 and 4 months of treatment and relapse rates were assessed 3 months after completing treatment durations of 2, 3 and 4 months. B25D0.6M200U200 generated human equivalent exposures in uninfected BALB/c mice. After 1 month of treatment, a higher bactericidal activity was observed for the B25D0.6M200U200 and the B25D0.6M200 regimen compared to the standard H10R10Z150E100 regimen. Furthermore, 3 months of therapy with both BDM-based regimens resulted in negative lung cultures, whereas all H10R10Z150E100 treated mice were still culture positive. After 3 months of therapy 7% and 13% of mice relapsed receiving B25D0.6M200U200 and B25D0.6M200, respectively, compared to 40% for H10R10Z150E100 treatment showing an increased sterilizing activity of both BDM-based regimens. BDM-based regimens, with and without sutezolid, have a higher efficacy than the HRZE regimen in the BALB/c model of TB, with some improvement by adding sutezolid. By translating these results to TB patients, this novel BDMU regimen should be able to reduce treatment duration by 25% compared to HRZE therapy.

USP25 Promotes the Antimycobacterial Response of Macrophages Through Stabilizing B-Raf and C-Raf.

Ubiquitin-specific peptidase 25 (USP25) is one of the best-characterized deubiquitinating enzymes and plays a vital regulatory role in various biological processes, especially in cancer development and immune regulation. However, the exact role of USP25 and its underlying mechanisms in macrophage activation and immunogenicity during Mycobacterium tuberculosis infection remain unclear. In this study, we found that M tuberculosis infection induced USP25 expression in human and mouse macrophages. In particular, USP25 expression is elevated in multiple cell types, especially monocytes, in patients with tuberculosis. Additionally, USP25 deficiency in macrophages and mice resulted in compromised immunity against M tuberculosis infection, accompanied by reduced expressions of various proinflammatory cytokines and chemokines. Mechanistically, USP25 in macrophages promoted the activation of the ERK signaling pathway through deubiquitination and stabilization of B-Raf and C-Raf. These findings collectively suggest the critical roles of USP25 in M tuberculosis infection and its potential as a therapeutic target.

Tuberculosis susceptibility in genetically diverse mice reveals functional diversity of neutrophils.

Tuberculosis (TB) is a heterogenous disease in humans with individuals exhibiting a wide range of susceptibility. This heterogeneity is not captured by standard laboratory mouse lines. We used a new collection of 19 wild-derived inbred mouse lines collected from diverse geographic sites to identify novel phenotypes during Mycobacterium tuberculosis ( Mtb ) infection. Wild derived mice have heterogenous immune responses to infection that result in differential ability to control disease at early timepoints. Correlation analysis with multiple parameters including sex, weight, and cellular immune responses in the lungs revealed that enhanced control of infection is associated with increased numbers of CD4 T cells, CD8 T cells and B cells. Surprisingly, we did not observe strong correlations between IFN-γ production and control of infection. Although in most lines high neutrophils were associated with susceptibility, we identified a mouse line that harbors high neutrophils numbers yet controls infection. Using single-cell RNA sequencing, we identified a novel neutrophil signature associated with failure to control infection.

Assay for interferon gamma release as a novel marker in pediatric patients with systemic lupus erythematosus

BackgroundThe interferon-gamma (IFN-γ) release assay (IGRA) is an important laboratory diagnosis for latent Mycobacterium tuberculosis (TB) infection. The TB-IGRA measures the release of IFN-γ from peripheral blood cells, who are exposed to TB antigen (Ag), mitogen (MT), or negative/nil control (NL) in vitro. While, an exceptional higher TB Ag-NL level will reflect an elevation of peripheral lymphocytes released IFN-γ in a same condition. Therefore, we found that the elevated levels of TB Ag-NL could become a new biomarker for the diagnosis and treatment of pediatric systemic lupus erythematosus (SLE) patients.MethodsWe have analyzed the clinical data of 776 children who are underwent TB-IGRA testing in the Department of Allergy and Rheumatology of Guangzhou Women and Children’s Medical Center from 2018 to 2020. To investigate the association between TB Ag-NL and SLE, we have analyzed the clinical data of 47 SLE patients and TB Ag-NL testing results, and then evaluated the association between TB Ag-NL and SLE disease activity.ResultsThe TB Ag-NL levels were significantly higher in patients with active SLE than those in inactive SLE (p = 0.0002). The TB Ag-NL levels were positively correlated with the SLE disease activity index (SLEDAI) and laboratory diagnosis parameters. The mean value of TB Ag-NL in SLE patients (0.04191 ± 0.07955, IU/mL) were significantly higher than those in patients with juvenile dermatomyositis (JDM) (0.0158 ± 0.0337, IU/mL, p = 0.036), juvenile idiopathic arthritis (JIA) (0.0162 ± 0.0388, IU/mL, p = 0.001), and healthy controls (HC) (0.0001 ± 0.0027, IU/mL, p = 0.0003). Therefore, the elevated TB Ag-NL levels could serve as a potential diagnostic biomarker of SLE, especially for the active SLE.ConclusionThe detection of IFN-γ release levels by the TB-IGRA may be useful to assess SLE disease activity in pediatric patients with active SLE.

Specific Cytokines Analysis Incorporating Latency-Associated Antigens Differentiates Mycobacterium tuberculosis Infection Status: An Exploratory Study.

Current immunologic methods cannot distinguish Mycobacterium tuberculosis (Mtb) infection statuses, especially to discriminate active tuberculosis (ATB) from latent tuberculosis infection (LTBI). This study explored the potential of latency-associated antigens (Rv1733cSLP and Rv2028c) and multifactorial cytokine detection to distinguish tuberculosis infection states. ATB patients (20), LTBI healthcare workers (25), fever patients (11), and healthy controls (10) were enrolled. Cytokine levels (IFN-γ, TNF-α, IL-2, IL-6, IP-10, IL-1Ra, CXCL-1, and MCP-1) were measured using Luminex with/without MTB-specific virulence factor and latency-associated antigens stimulation. Without antigen stimulation, IL-6, IP-10, MCP-1, and IL-1Ra were higher in the ATB group than in the LTBI group (p<0.05), but no significant differences between the ATB group and the fever group. Stimulated with the four antigens, respectively, the cytokines, including IP-10Esat-6, IP-10CFP-10, IFN-γRv1733cSLP, IFN-γRv2028c, IL-6Esat-6, IL-6Rv1733cSLP, IL-6Rv2028c, IL-2Rv1733cSLP, IL-2 Rv2028c, IL-1RaEsat-6, IL-1RaCFP-10, IL-1RaRv2028c, CXCL-1Esat-6, CXCL-1CFP-10, CXCL-1Rv1733cSLP, CXCL-1Rv2028c, MCP-1Esat-6 and MCP-1CFP-10, demonstrated accurate discrimination between ATB and LTBI (p<0.05). Additive concentrations demonstrated significant secretion differences of IFN-γ, IP-10 and IL-2, primarily by virulence factors in ATB and latency-associated antigens in LTBI. Latency-associated antigens synergized with virulence factors, enhancing TH1-type cytokine diagnostic efficacy for discriminating ATB from LTBI, the AUC for TNF-α increased from 0.696 to 0.820 (p=0.038), IFN-γ increased from 0.806 to 0.962 (p=0.025), and IL-2 increased from 0.565 to 0.868 (p=0.007). Model selected by forward likelihood method indicated combined detection of IFN-γCFP-10, IFN-γRv1733cSLP, IP-10Rv1733cSLP, and CXCL-1Rv1733cSLP achieved ATB diagnosis (AUC=0.996) and ATB-LTBI differentiation (AUC=0.992). Combined detection of IFN-γCFP-10 and IFN-γRv1733cSLP achieved tuberculosis infection diagnosis (AUC=0.943). Latency-associated antigens enhance multiple cytokine discriminatory ability, particularly TH1-type cytokines, for differentiating Mtb infection statuses.

The International Consensus for Early TB framework (ICE-TB): Implications from a low-incidence setting.

<sec><title>BACKGROUND</title>In recent years, there has been increasing recognition of the public health significance of the spectrum of TB disease presentation, and the existing classification systems of asymptomatic infection and symptomatic TB have been limited in terms of explanatory power. Accordingly, in 2022-2023, a new International Consensus framework for Early TB (ICE-TB) was developed, categorising the spectrum of TB infection and disease into five states based on the presence or absence of macroscopic pathology, host infectiousness, and symptoms and signs.</sec><sec><title>METHODS</title>We used the ICE-TB framework to re-analyse existing notification data for 2022 within a low-incidence setting to explore the potential utility and future challenges for its public health application.</sec><sec><title>RESULTS</title>Existing notification data were sufficient to allow substantial reclassification of currently recognised active disease states, but did not systematically capture Mycobacterium tuberculosis infection or subclinical TB. Fifty percent of existing TB notifications would be classified as 'Clinical, infectious', with the potential need to consider further subclassification.</sec><sec><title>CONCLUSION</title>Our exploration highlighted limitations in existing classification systems and diagnostic approaches and should encourage researchers and programmatic implementers to emphasise person-centred and programmatic needs in the development of new tools for TB management.</sec>.

Effectiveness of preventive treatment among different age groups and Mycobacterium tuberculosis infection status: a systematic review and individual-participant data meta-analysis of contact tracing studies.

Tuberculosis is a preventable disease. However, there is debate regarding which individuals would benefit most from tuberculosis preventive treatment and whether these benefits vary in settings with a high burden and low burden of tuberculosis. We aimed to compare the effectiveness of tuberculosis preventive treatment in exposed individuals of differing ages and Mycobacterium tuberculosis infection status while considering tuberculosis burden of the settings. In this systematic review and individual-participant meta-analysis, we investigated the development of incident tuberculosis in people closely exposed to individuals with tuberculosis. We searched for studies published between Jan 1, 1998, and April 6, 2018, in MEDLINE, Web of Science, BIOSIS, and Embase. We restricted our search to cohort studies; case-control studies and outbreak reports were excluded. Two reviewers evaluated titles, abstracts, and full text articles for eligibility. At each stage, two reviewers discussed discrepancies and re-evaluated articles until a consensus was reached. Individual-participant data and a pre-specified list of variables, including characteristics of the exposed contact, the index patient, and environmental characteristics, were requested from authors of all eligible studies; contacts exposed to a drug-resistant tuberculosis index patient were excluded. The primary study outcome was incident tuberculosis. We estimated adjusted hazard ratios (aHRs) for incident tuberculosis with mixed-effects Cox regression models with a study-level random effect. We estimated the number-needed-to-treat (NNT) to prevent one person developing tuberculosis. Propensity score matching procedures were used in all analyses. This study is registered with PROSPERO (CRD42018087022). After screening 25 358 records for eligibility, 439 644 participants from 32 cohort studies were included in the individual-participant data meta-analysis. Participants were followed for 1 396 413 person-years (median of 2·7 years [IQR 1·3-4.4]), during which 2496 people were diagnosed with incident tuberculosis. Overall, effectiveness of preventive treatment was 49% (aHR 0·51 [95% CI 0·44-0·60]). Participants with a positive tuberculin-skin-test (TST) or IFNγ release assay (IGRA) result at baseline benefitted from greater protection, regardless of age (0·09 [0·05-0·17] in children younger than 5 years, 0·20 [0·15-0·28] in individuals aged 5-17 years, and 0·17 [0·13-0·22] in adults aged 18 years and older). The effectiveness of preventive treatment was greater in high-burden (0·31 [0·23-0·40]) versus low-burden (0·58 [0·47-0·72]) settings. The NNT ranged from 9 to 34 depending on age among participants with a positive TST or IGRA in both high-burden and low-burden settings; among all contacts (regardless of TST or IGRA test result), the NNT ranged from 29 to 43 in high-burden settings and 213 to 455 in low-burden settings. Our findings suggest that a risk-targeted strategy prioritising contacts with evidence of M tuberculosis infection might be indicated in low-burden settings, and a broad approach including all contacts should be considered in high-burden settings. Preventive treatment was similarly effective among contacts of all ages. None.

A multistage protein subunit vaccine as BCG-booster confers protection against Mycobacterium tuberculosis infection in murine models

The eradication of tuberculosis remains a global challenge. Despite being the only licensed vaccine, Bacillus Calmette-Guérin (BCG) confers limited protective efficacy in adults and individuals with latent tuberculosis infections (LTBI). There is an urgent need to develop novel vaccines that can enhance the protective effect of BCG. Protein subunit vaccines have garnered significant research interest due to their safety and plasticity. Based on previous studies, we selected three antigens associated with LTBI (Rv2028c, Rv2029c, Rv3126c) and fused them with an immunodominant antigen Ag85A, resulting in the construction of a multistage protein subunit vaccine named A986. We evaluated the protective effect of recombinant protein A986 adjuvanted with MPL/QS21 as a booster vaccine for BCG against Mycobacterium tuberculosis (Mtb) infection in mice. The A986 + MPL/QS21 induced the secretion of antigen-specific Th1 (IL-2+, IFN-γ+ and TNF-α+) and Th17 (IL-17A+) cytokines in CD4+ and CD8+ T cells within the lung and spleen of mice, while also increased the frequency of central memory and effector memory T cells. Additionally, it also induced the enhanced production of IgG antibodies. Compared to BCG alone, A986 + MPL/QS21 boosting significantly augmented the proliferation of antigen-specific multifunctional T cells and effectively reduced bacterial load in infected mice. Taken together, A986 + MPL/QS21 formulation induced robust antigen-specific immune responses and provided enhanced protection against Mtb infection as a booster of BCG vaccine.

Can We Exploit Inflammasomes for Host-Directed Therapy in the Fight against Mycobacterium tuberculosis Infection?

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a major global health issue, with around 10 million new cases annually. Advances in TB immunology have improved our understanding of host signaling pathways, leading to innovative therapeutic strategies. Inflammasomes, protein complexes organized by cytosolic pattern recognition receptors (PRRs), play a crucial role in the immune response to M. tb by activating caspase 1, which matures proinflammatory cytokines IL1β and IL18. While inflammation is necessary to fight infection, excessive or dysregulated inflammation can cause tissue damage, highlighting the need for precise inflammasome regulation. Drug-resistant TB strains have spurred research into adjunctive host-directed therapies (HDTs) that target inflammasome pathways to control inflammation. Canonical and non-canonical inflammasome pathways can trigger excessive inflammation, leading to immune system exhaustion and M. tb spread. Novel HDT interventions can leverage precision medicine by tailoring treatments to individual inflammasome responses. Studies show that medicinal plant derivatives like silybin, andrographolide, and micheliolide and small molecules such as OLT1177, INF39, CY-09, JJ002, Ac-YVAD-cmk, TAK-242, and MCC950 can modulate inflammasome activation. Molecular tools like gene silencing and knockouts may also be used for severe TB cases. This review explores these strategies as potential adjunctive HDTs in fighting TB.

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

Necrosis drives susceptibility to Mycobacterium tuberculosis in POLG mtDNA mutator mice.

The genetic and molecular determinants that underlie the heterogeneity of Mycobacterium tuberculosis (Mtb) infection outcomes in humans are poorly understood. Multiple lines of evidence demonstrate that mitochondrial dysfunction can exacerbate mycobacterial disease severity and mutations in some mitochondrial genes confer susceptibility to mycobacterial infection in humans. Here, we report that mutations in mitochondria DNA (mtDNA) polymerase gamma (POLG) potentiate susceptibility to Mtb infection in mice. POLG mutator mtDNA mice fail to mount a protective innate immune response at an early infection timepoint, evidenced by high bacterial burdens, reduced M1 macrophages, and excessive neutrophil infiltration in the lungs. Immunohistochemistry reveals signs of enhanced necrosis in the lungs of Mtb-infected POLG mice and POLG mutator macrophages are hyper-susceptible to extrinsic triggers of necroptosis ex vivo. By assigning a role for mtDNA mutations in driving necrosis during Mtb infection, this work further highlights the requirement for mitochondrial homeostasis in mounting balanced immune responses to Mtb.

Evaluation of GeneNAT Real-Time Polymerase Chain Reaction Analyzer and Pre-loaded Chip-Based Mycobacterium tuberculosis and Multidrug-Resistant Tuberculosis Detection in the Diagnosis of Pulmonary Tuberculosis.

Tuberculosis (TB) is still the second causative agent of death worldwide after COVID-19. It is caused by Mycobacterium tuberculosis (MTB) infection. The aim of the current study was to compare the performance of GeneNAT real-time polymerase chain reaction analyzer and pre-loaded chip-based MTB screening and multidrug-resistant tuberculosis (MDR-TB) detection kit (Smart SureTM MTB & MDR-TB, Genetix Biotech Asia Pvt. Ltd., New Delhi, India) against the established WHO-approved GeneXpert Ultra (MTB/rifampicin (RIF)), line probe assay (LPA), and mycobacteriagrowthindicatortube(MGIT) culture at point of care (POC) level. A total of 450 pulmonary TB (PTB) suspect patients were enrolled from October 2023 to March 2024 at the Intermediate Reference Laboratory, Department of Medicine, All India Instituteof Medical Sciences, New Delhi, India. GeneXpert and GeneNAT tests were done directly from sputum specimens. However, processed sputum specimens were used for both LPA (GenoType MTBDRplus) and liquid culture and drug susceptibility testing (MGIT culture and drug susceptibility testing (DST)). On comparing with GeneXpert, for the detection of MTB and rifampicin (RIF), Smart SureTM showed a sensitivity of 98.18% and 97.5% with a specificity of 100% and 98.92%, respectively. While comparing mutations in therpoB gene with LPA, the Smart SureTM MDR-TB kit exhibited sensitivity and specificity of 96.77% and 99.12%, respectively. For katG and inhA genes, sensitivity and specificity were 97.6% & 85.71% and 98.66% & 98.01%, respectively, for both genes. Smart SureTM MDR-TB showed comparable results with MGIT-DST with sensitivity and specificity of 96.88% & 96.15% and 98.99%& 99.02%, respectively, for both RIF and isoniazid (INH) drugs. The GeneNAT system test may provide the status of RIF and INH resistance in PTB cases in a short time with the use of minimal specimens. It required very little infrastructurewith less skilled laboratory staffin comparison with other WHO-approved diagnostics used in resource-limited countries with TB and drug-resistant TB burdens.

Type I IFN signaling in the absence of IRGM1 promotes M. tuberculosis replication in immune cells by suppressing T cell responses

Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm, Irgm1, is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infection in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1−/− mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact TH1 immune responses.

Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis infection

Compounds targeting host control of infectious diseases provide an attractive alternative to antimicrobials. A phenotypic screen of a kinase library identified compounds targeting glycogen synthase kinase 3 as potent inhibitors of Mycobacterium tuberculosis (Mtb) intracellular growth in the human THP-1 cell line and primary human monocytes-derived macrophages (hMDM). CRISPR knockouts and siRNA silencing showed that GSK3 isoforms are needed for the growth of Mtb and that a selected compound, P-4423632 targets GSK3β. GSK3 inhibition was associated with macrophage apoptosis governed by the Mtb secreted protein tyrosine phosphatase A (PtpA). Phospho-proteome analysis of macrophages response to infection revealed a wide array of host signaling and apoptosis pathways controlled by GSK3 and targeted by P-4423632. P-4423632 was additionally found to be active against other intracellular pathogens. Our findings strengthen the notion that targeting host signaling to promote the infected cell's innate antimicrobial capacity is a feasible and attractive host-directed therapy approach.

Plasma Immune Biomarkers Predictive of Progression to Active Tuberculosis in Household Contacts of TB Patients.

The progression from Mycobacterium tuberculosis infection to active tuberculosis (TB) disease varies among individuals, and identifying biomarkers to predict progression is crucial for guiding interventions. In this study, we aimed to determine plasma immune biomarker profiles in healthy household contacts of index pulmonary TB (PTB) patients who either progressed to TB or remained as non-progressors. A cohort of household contacts of adults with PTB was enrolled, consisting of 15 contacts who progressed to TB disease and 15 non-progressors. Plasma samples were collected at baseline, 4 months, and 12 months to identify predictive TB progression markers. Our findings revealed that individuals in the progressor group exhibited significantly decreased levels of IFNγ, IL-2, TNFα, IL1α, IL1β, IL-17A, and IL-1Ra at baseline, months 4 and 12. In contrast, the progressor group displayed significantly elevated levels of IFNα, IFNβ, IL-6, IL-12, GM-CSF, IL-10, IL-33, CCL2, CCL11, CXCL8, CXCL10, CX3CL1, VEGF, Granzyme-B and PDL-1 compared to the non-progressor group at baseline, months 4 and 12. ROC analysis identified IFNγ, GM-CSF, IL-1Ra, CCL2 and CXCL10 as the most promising predictive markers, with an AUC of ≥90. Furthermore, combinatorial analysis demonstrated that GM-CSF, CXCL10 and IL-1Ra, when used in combination, exhibited high accuracy in predicting progression to active TB disease. Our study suggests that a specific set of plasma biomarkers GM-CSF, CXCL10 and IL-1Ra, can effectively identify household contacts at significant risk of developing TB disease. These findings have important implications for early intervention and preventive strategies in TB-endemic regions.

Study on the role and molecular mechanism of METTL3-mediated miR-29a-3p in the inflammatory response of spinal tuberculosis

BackgroundSpinal Tuberculosis (STB) is a common cause of spinal malformation caused by extrapulmonary tuberculosis in developing countries, which seriously affects the quality of life of patients. It was found that the expression of miRNAs in macrophages was stable, specific and readily available after Mycobacterium tuberculosis (MTB) infection. Our research group determined the differential expression of miR-29a-3p in the vertebra of spinal tuberculosis and intervertebral disc lesions through RNAs chip screening and bioinformatics analysis. However, the specific molecular mechanism of miR-29a-3p in the inflammatory response of spinal tuberculosis remains unclear. ObjectiveIn this study, we mainly discussed the expression of miR-29a-3p in the focal tissue of spinal tuberculosis and the role and molecular mechanism of miR-29a-3p mediated by METTL3 in the inflammatory response of spinal tuberculosis. MethodsThe tissues of 15 cases of lumbar degenerative diseases and vertebral lesions of spinal tuberculosis were collected, and the THP-1 macrophage model infected by Mycobacterium tuberculosis was constructed, and verified by qRT-PCR, Western blot, fluorescence in situ hybridization, immunohistochemistry, Cell fluorescence and ELISA. Results and conclusionWe found that the expression of miR-29a-3p was significantly down-regulated in the vertebral body and disc lesion tissues of patients with spinal tuberculosis. Overexpression of miR-29a-3p inhibited the levels of inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and inhibition of miR-29a-3p expression promoted the release of the levels of TNF-α, IL-1β and IL-6 inflammatory factors. Our study also shows that knockout of methyltransferase 3 (METTL3) can significantly reduce the expression of miR-29a-3p and promote the release of pro-inflammatory cytokines in macrophages.

5-Fluoroindole Reduces the Bacterial Burden in a Murine Model of Mycobacterium tuberculosis Infection.

Tuberculosis is a disease caused by a single pathogen that leads to a death toll estimated to be more than a million per year. Mycobacterium tuberculosis (Mtb), which affects mainly the lungs, spreads by airborne transmission when infectious respiratory particles from an infected human enter the respiratory tract of another person. Despite diagnosis and treatment being well established, the rise of cases of patients infected with Mtb strains with multidrug resistance to the antibiotics used in the regimen against the disease is alarming. Indole used as a core molecule has been described as a promising structure to treat several diseases. 5-Fluoroindole (5-FI) compound, evaluated in the free base and in the hydrochloride (5-FI.HCl) forms, inhibited the growth of pan-sensitive Mtb H37Rv strain in the same range (4.7-29.1 μM) of clinical isolates that have resistance to at least two first-line drugs. Although 5-FI showed no cytotoxicity in Vero and HepG2 cells, high permeability (2.4.10-6 cm/s) in the PAMPA assay, and high metabolic stability (Clint 9.0 mL/min/kg) in rat liver microsomes, limited solubility at plasmatic and intestinal pH values prompted formation and employment of its salt form (5-FI.HCl). Although the 5-FI.HCl compound showed increased solubility at pH values of 7.4 and 9.1 and increased stability in aqueous solutions, data for intrinsic clearance (Clint = 48 mL/min/kg) and a half-life (t 1/2 = 12 min) showed decreased metabolic stability. As 5-FI.HCl showed both good absorption and ability to reach the systemic circulation of animals without the need to use vehicles containing cosolvents or surfactants, it was chosen to evaluate its effectiveness in the model of tuberculosis in mice. The in vivo results showed the concentration of the compound in plasma increasing within 30 min in the systemic circulation and the capacity of reducing the Mtb burden in the lungs at the concentration of 200 μmol/kg after 21 days of infection, with no toxicity in mice.

Lung-resident CD3-NK1.1+CD69+CD103+ Cells Play an Important Role in Bacillus Calmette-Guérin Vaccine-Induced Protective Immunity against Mycobacterium tuberculosis Infection.

Tissue-resident immune cells play important roles in local tissue homeostasis and infection control. There is no information on the functional role of lung-resident CD3-NK1.1+CD69+CD103+ cells in intranasal Bacillus Calmette-Guérin (BCG)-vaccinated and/or Mycobacterium tuberculosis (Mtb)-infected mice. Therefore, we phenotypically and functionally characterized these cells in mice vaccinated intranasally with BCG. We found that intranasal BCG vaccination increased CD3-NK1.1+ cells with a tissue-resident phenotype (CD69+CD103+) in the lungs during the first 7 d after BCG vaccination. Three months post-BCG vaccination, Mtb infection induced the expansion of CD3-NK1.1+CD69+CD103+ (lung-resident) cells in the lung. Adoptive transfer of lung-resident CD3-NK1.1+CD69+CD103+ cells from the lungs of BCG-vaccinated mice to Mtb-infected naive mice resulted in a lower bacterial burden and reduced inflammation in the lungs. Our findings demonstrated that intranasal BCG vaccination induces the expansion of CD3-NK1.1+CD69+CD103+ (lung-resident) cells to provide protection against Mtb infection.

Boosting Immunogenicity of a Recombinant Mycobacterium smegmatis Strain via Zinc-Dependent Ribosomal Proteins.

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that a live vaccine strain of Mycobacterium smegmatis, previously designated as IKEPLUS, conferred a higher survival benefit than the Bacillus Calmette-Guerin (BCG) in a murine model of intravenous Mycobacterium tuberculosis (Mtb) infection. We have shown that there was a significant increase in the expression of the Rv0282 gene, which is encoded in the esx-3 locus, which played an important role in iron uptake when IKEPLUS was grown in both low zinc and iron-containing Sauton medium. We then confirmed using in vitro assays of biofilm formation that zinc plays a vital role in the growth and formation of M. smegmatis biofilms. IKEPLUS grown in low zinc media led to the better protection of mice after intravenous challenge with a very high dosage of Mtb. We also showed that various variants of IKEPLUS induced apoptotic cell-death of infected macrophages at a higher rate than wild-type M. smegmatis. We next attempted to determine if zinc containing ribosomal proteins such as rpmb2 could contribute to protective efficacy against Mtb infection. Since BCG has an established role in anti-mycobacterial efficacy, we boosted BCG vaccinated mice with rmpb2, but this did not lead to an increment in the protection mediated by BCG.

Characteristics of Pulmonary Tuberculosis patients with HIV in Regional General Hospital Dr Harjono Ponorogo period January 2018 – December 2022

Tuberculosis (TB) is the most common opportunistic infection in people living with HIV/AIDS in Indonesia, and HIV infection promotes Mycobacterium tuberculosis infection. In ODHIV, the probability of contracting TB is 10% per year. According to the Indonesian Ministry of Health, East Java is in second place with 71,909 cases and has the second highest number of AIDS patients and 824,000 TB cases in Indonesia. This study aims to determine the characteristics of pulmonary tuberculosis patients with HIV at DR Harjono Ponorogo Hospital for the period January 2018 - December 2022. This study applied descriptive research methodology with quantitative techniques with secondary data from medical records of pulmonary TB patients with HIV at Dr. Harjono Ponorogo Hospital for the period January 2018 - December 2022. The results showed a total sample of 130 medical records, there were several characteristics of TB patients with HIV, namely the highest age group of 30-39 years by 29.2%, more in male gender 73%. More patients had last high school education 38.5%, more treatment duration in patients who did for 9 months 61.5% and the results showed more patients with poor prognosis 56%. In conclusion, the characteristics of TB patients with HIV with the highest group at the age of 3-39 years, having male gender, more high school education, with more treatment duration of 9 months, and the results showed a poor prognosis.