Tuberculosis

Phage therapy offers promise to combat antimicrobial resistance, including drug-resistant tuberculosis (TB). Understanding phage activity against Mycobacterium tuberculosis (Mtb) adapted to physiologic microenvironments, such as hypoxia and acidity in granulomas, is essential since these conditions induce non-replicating states. We evaluated a phage combination against Mtb under hypoxic, acidic (pH 5.5), and stationary-phase conditions in vitro. In planktonic Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in Mtb H37Rv load, which was maintained over 31 days. Phage addition prevented regrowth was observed with rifampicin and isoniazid alone. Individual phage stability was differentially affected by acidic media conditions, resulting in variability of antimycobacterial activity. In hypoxic conditions and stationary growth experiments, phage titers remained stable over time with no change in mycobacterial load compared to controls. Model-based predictions were able to adequately capture phage-mycobacterial interactions with and without rifampicin. The lack of antimycobacterial activity in assays with non-replicating mycobacteria suggest that phages need actively replicating mycobacteria to exert lytic activity. Stable phage concentrations in assays with non-replicating mycobacteria suggests low grade phage replication in these conditions. Established models can support future study design through simulations of different experimental scenarios.

Background: Tuberculosis (TB) remains the leading infectious cause of death worldwide, with India accounting for nearly one-fourth of global TB cases. Ni-kshay, the countrys digital case-based TB notification platform is rich in data pertaining to the continuum of care of TB patients. This study aims to develop a standardized analytical approach to programmatic data to identify predictors of unfavourable treatment outcomes and mortality among adult drug-sensitive TB patients at the state level for Maharashtra during 2021 and 2022. Methods: Two separate analyses were undertaken comparing treatment success with: (1) unfavourable outcomes (death, treatment failure, loss to follow-up, regimen change, or not evaluated); and (2) mortality. Multivariate logistic regression was used to compute adjusted odds ratios (aOR) for key risk factors, adjusting for age, gender, and weight. Results: The final cohort included 323,124 cases for unfavourable outcome analysis and 315,579 cases for mortality analysis. Increasing age, male gender, lower body weight, known HIV and diabetes comorbidities, tobacco and alcohol consumption, and "unknown" status for behavioural risks and comorbidity status were significantly associated with increased odds of both unfavourable outcomes and mortality. Conclusions: This study highlights the utility of programmatic data in identifying high-risk TB patients and offers a reproducible analytic framework.

Background: Without tuberculosis preventive therapy (TPT), approximately 5% of individuals infected with M. tuberculosis progress to active tuberculosis (TB) disease. Recent studies have identified body mass index (BMI) < 25 kg/m2 as a predictor of TB progression, but additional markers are needed to better identify persons at increased risk. Methods: Close contacts of patients with culture-confirmed pulmonary TB were enrolled in the Regional Prospective Observational Research in Tuberculosis (RePORT)-Brazil cohort from 2015 to 2019 and followed for up to 24 months. Analyses were restricted to interferon-{gamma} release assay (IGRA)-positive contacts who did not receive TPT or received <30 days of isoniazid. Prediction models to identify close contacts at increased TB risk were constructed using two complementary approaches: incremental models used BMI as the base predictor and evaluated whether baseline whole-blood transcriptomic signatures, human genetic polymorphism risk scores derived from low-pass whole-genome sequencing, and BMI-related plasma biomarkers improved model discrimination. Agnostic models did not impose BMI in the model and used penalized regression for predictor selection. Results: Among 285 close contacts, 15 (5%) progressed to TB. The model with BMI as unique predictor had a C-index of 0.66 (95% confidence interval [CI] 0.55; 0.77). Adding Rajan5 or Duffy9 transcriptomic signature scores to BMI improved discrimination compared with BMI alone, with C-indices of 0.78 (95% CI 0.62; 0.99) and 0.75 (95% CI 0.61; 0.89), respectively, but did not further improve discrimination after accounting for adiponectin. Adding adiponectin to BMI increased the C-index to 0.80 (95% CI 0.68; 0.91), while adiponectin alone captured most of the discriminatory performance in agnostic models (C-index, 0.80, 95% CI 0.69; 0.91). Genetic risk scores, leptin, and the adiponectin:leptin ratio did not improve model discrimination compared with the BMI-only model. In exploratory post hoc analyses, higher adiponectin was associated with increased risk of progression to TB, with each two-fold increase associated with a higher hazard of TB (HR 2.91, 95% CI 1.73; 4.91, p < 0.001). Conclusions: Baseline adiponectin strongly predicted progression to TB among close contacts and captured most of the discriminatory information contained in epidemiological and transcriptomic variables. Its consistent selection across modelling approaches supports adiponectin as a promising biomarker for TB risk stratification.

Pulmonary vascular remodelling is common in patients with chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factors (VEGFs) are key mediators in angiogenesis and vascular remodelling and exist in different isoforms. VEGF-A is the most potent angiogenic member binding to VEGF receptor 2 (VEGFR2). There are, however, few studies on other isoforms, as VEGF-C, and its receptor VEGFR3 in COPD and subsequent impact of cAMP therapies on VEGF isoforms. Our aim was to evaluate the VEGF isoform synthesis in primary distal lung fibroblasts from control subjects (non-smokers (n=6) and ex-smokers (n=4), and COPD subjects with GOLD stage II (n=4) or GOLD stage IV (n=6), and the expression of VEGFR2 and VEGFR3 in human lung tissue. Primary lung fibroblasts were exposed to the cAMP generating therapies formoterol, iloprost, or roflumilast, the adenylyl cyclase activator forskolin or to transforming growth factor (TGF)-b1. VEGF isoforms were evaluated with ELISA. VEGF-C release was not significantly altered by TGF-{beta}1, in contrast to the increased levels of VEGF-A, in all fibroblasts. VEGF-C was significantly decreased by iloprost, forskolin and formoterol, whereas VEGF-A was significantly increased by iloprost and forskolin, with differences in release pattern between and within fibroblasts from control and COPD subjects. Exposure to VEGF-C specifically towards VEGFR3 decreased proliferative rate in human lung fibroblasts and bronchial epithelial cells. VEGFR2 and VEGFR3 were both present in parenchymal lung tissue and VEGFR2 in pulmonary blood vessels. in both healthy and COPD, whereas there was elevated expression of VEGFR3 in bronchial epithelium. In conclusion, TGF-{beta}1 and cAMP generating compounds have significant effects on VEGF-C and VEGF-A synthesis, which appear dysregulated in lung fibroblasts from ex-smokers and patients with COPD. Increased VEGFR3 expression in the bronchial epithelium in lung tissue, and studies into their functional impact, warrants further investigations.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become a global concern over the last two decades. Bovine TB primarily affects cattle, but other domestic livestock are also affected and it is more common in less developed and developing countries. The significant loss of livestock leads to trade restrictions and economic crises. Zoonotic potential of bovine TB raises health concerns for the public. Currently, no effective treatment is available and animal slaughtering is usually undertaken to reduce the burden of it in the environment. Antibiotic therapy can be used on animals living in captivity, but it is not reliable for herd or free-grazing animals. The BCG vaccine is another option available for treating the disease, but it shows limited efficacy in cattle. The prevention of bovine TB is a long-term goal that can only be accomplished by developing a more effective vaccine than BCG and designing new drugs. In this research, we propose therapeutic drug targets and vaccine for treating bovine TB. The conceptual framework for vaccine developed in this study uses a number of bioinformatics approaches to identify potential vaccine candidates and construct an in-silico epitope-based vaccine. Our holistic framework identified potential therapeutic candidates by directly analysing the proteome of TB bacterial strains. Specifically, we performed a comparative proteomic analysis of 11 Mycobacterium bovis strains to cover the diversity and identify conserved proteins among those strains for developing the bovine TB vaccine. An extensive reverse vaccinology and immunoinformatics analysis provided 26 highly immunogenic, non-toxic and non-allergenic epitopes (CTL epitopes-8, HTL epitopes-2 and B-cell epitopes-16) for Mycobacterium bovis required for three-dimensional structure construction of TB vaccine. The constructed epitope-based vaccine showed a potent interaction inside the host, thus generating efficient cell-mediated and humoral immune responses. Next, a framework based on a novel subtractive proteomic approach was developed for identifying bovine TB drug targets. We performed this approach on the 11 Mycobacterium bovis strains and identified nine drug targets that are conserved, essential, antigenic and have unique metabolic pathways in Mycobacterium bovis. These drug targets could further help investigate therapeutic drugs for the treatment of bovine TB. Several bioinformatics prediction tools were used together to ensure checks and balances, aiming to reduce the chance of errors and provide accurate results. The vaccine and drug targets developed in this study can be tested experimentally with confidence for further validation as therapeutics with the potential to eradicate bovine TB globally. The strategies implemented in the study are generic and can be used for other zoonotic infectious diseases. This study would be a game changer in the field of bovine tuberculosis treatment.

Background: Diabetes mellitus (DM) worsens pulmonary tuberculosis (TB) and drives systemic hyper-inflammation, but the underlying mechanisms remain unknown. Neutrophils have key roles in TB immunopathology and lung cavitation. Here, we determine the role of neutrophils in DMTB patients and in driving TB immunopathology. Methods: Sputum and plasma from 30 TB and 30 DMTB patients were analysed for proteases and cytokines using Luminex bead array. Whole blood transcriptomics identified transcriptional differences. Single-cell RNA sequencing characterised neutrophil subsets and dysregulated pathways. Neutrophil function of poorly-controlled DM patients (HbA1c>8%) and healthy controls (HC) were examined following Mycobacterium tuberculosis stimulation, including reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and phagocytosis. Pathways were interrogated using chemical inhibitors, protein array and western blot. Results: Compared to non-diabetic TB patients, poorly-controlled DMTB patients showed up-regulated sputum MMP-8 and MMP-9, associated with increased collagen-destruction and lung cavity formation. Circulating neutrophil count and neutrophil-derived plasma MMP-8 were up-regulated, alongside transcriptional enrichment of extracellular matrix degradation and inflammatory pathways including TNF and RAGE. Single-cell profiling identified reduced cycling neutrophil subset and myelocytes in DMTB, with overall reduced antibacterial and cell-killing signatures. Ex vivo mycobacterial stimulation of DM neutrophils increased ROS and MMP-9 with impaired NETs and delayed phagocytosis. TNFR1, TNFR2, and RAGE were up-regulated. RAGE inhibition with rosiglitazone mitigated Mtb-induced ROS and MMP-8 release. Conclusion: DM worsens neutrophil-driven tissue destruction and inflammation in TB via dysregulated TNF and RAGE-signalling, priming neutrophils towards immunopathology. Targeting RAGE alongside tight glycaemic control may dampen neutrophil hyper-inflammatory responses to limit tissue destruction.

Summary- In a cross-sectional study, we calculated direct and indirect costs incurred by people prior to starting tuberculosis (TB) treatment in primary healthcare facilities in KwaZulu-Natal, South Africa. We related the total costs to patient income to explore the economic impact of TB care-seeking and contribute to the literature by exploring differences between those with and without TB symptoms. Background- Patient costs during tuberculosis (TB) treatment in South Africa are high. There are fewer data about the costs incurred prior to starting treatment. We measured pre-TB treatment costs for people in rural KwaZulu-Natal, South Africa. Design/methods- In the context of a TB case-contact study, we interviewed people starting TB treatment at primary healthcare facilities in rural South Africa. We estimated total direct and indirect costs incurred by respondents and their households in the three months prior to starting TB treatment. We estimated other coping costs, such as selling productive assets, as well as the value of any loans taken. Results- Among 98 participants (52 female, median age 36 years), 86/98 (88%) reported one or more symptoms from the WHO 4-symptom TB screening tool prior to starting treatment. The median total pre-treatment cost for TB affected households was USD 10.78 (IQR: [4.13 -- 20.23]). Total, pre-treatment costs for those with TB symptoms were USD 10.78 (IQR: [4.83 -- 20.23]) compared to USD 8.91 (IQR: [1.27 -- 22.19]) for those without TB symptoms. Conclusions- Whilst TB testing and care is free in South African public health facilities, patients still face costs that are burdensome. Our results indicate people affected by TB, including patients and their families, also face an economic burden. Our study highlights the need for further consideration of social protection policies to reduce the economic effects of asymptomatic TB.

Tuberculosis (TB) continues to pose a significant global public health challenge with substantial patient morbidity and mortality. Current TB patient biomarkers lack sufficient resolution to inform treatment response and patient stratification. This necessitates the development of sensitive and reliable host biomarkers. We previously demonstrated the efficacy of TruCulture whole blood stimulation for differentiating asymptomatic TB from active pulmonary TB disease patients in endemic regions. Our systems immunology study expands upon this previous work by evaluating the potential of TruCulture to monitor longitudinal responses to TB treatment in patients from the Predict-TB trial before, during, and after 6 months of antibiotic therapy. We stimulated whole blood from TB patients (n=40) using TruCulture under four conditions (Null, Mycobacterium tuberculosis-antigen, LPS, and IL-1{beta}) at baseline (week 0), during treatment (weeks 16 and 24), and one-year follow-up post- treatment (week 72). 20/25 measured cytokines exhibited significant changes throughout treatment, with several continuing to evolve during post-therapy follow-up. Machine learning based analysis identified Mtb-Ag-induced IL-1RA (AUC = 0.90, 0.92, 0.95 at weeks 16, 24, 72) and LPS-induced NLRP3 (AUC = 0.94 at week 16) as the best protein and transcriptional biomarkers for distinguishing treated from untreated patients, strongly implicating the inflammasome response. Combining these results with the extent of lung disease assessed by FDG PET/CT scans, we showed direct disease relevance for these blood-based biomarkers. The identified biomarker profiles hold promise for improving TB patient care through early prediction of treatment responses, real-time therapy monitoring, and informed development of host-directed therapeutic strategies for clinical decision-making. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/723467v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@14a32eforg.highwire.dtl.DTLVardef@55f3d4org.highwire.dtl.DTLVardef@fb0137org.highwire.dtl.DTLVardef@10cf39e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Predict-TB clinical study overview and summary of TB-specific biomarkers identified from TruCulture whole blood stimulation system. C_FIG

Macrophages are crucial for host defense against the pathogen. However, pathogens such as Mycobacterium tuberculosis (Mtb) have evolved mechanisms to alter macrophage physiology and exploit these cells as their primary niche. Mtb-infected macrophages upregulate several metabolic pathways including glutamine metabolism. We previously showed that inhibiting glutamine metabolism with the pleiotropic glutamine metabolism antagonist prodrug JHU083 has dual antibacterial and immunomodulatory effects in a mouse model of tuberculosis. In the present study, using single-cell RNA sequencing and LS-MS/MS metabolomics, we showed that JHU083-mediated glutamine metabolism inhibition increased the population of interstitial macrophages in Mtb-infected lungs. JHU083 treatment also increased inflammatory signatures while lowering immunosuppressive markers on these macrophages. Metabolically, these macrophages exhibited marked depletion of complex lipids, accumulation of free fatty acids, and increased expression of transcripts associated with the {beta}-oxidation pathway. Additionally, JHU083-treatment also improved phagocytic activity of macrophages, as measured by using fluorescent E. coli as a bait. In conclusion, JHU083-mediated glutamine metabolism inhibition metabolically reprograms macrophages, increasing both their lipid utilization as well as phagocytic activity, potentially driving their antimycobacterial activity that we had observed earlier.

Tuberculosis (TB) remains a formidable global health challenge, exacerbated by the emergence of drug-resistant Mycobacterium tuberculosis strains that threaten to render existing drug therapies and vaccine ineffective. Despite the availability of the Bacillus Calmette-Guerin (BCG) vaccine, its limited efficacy--primarily in infants and young children--falls short of reducing TB prevalence or offering adequate protection to adults. Therefore, developing a new TB vaccine with enhanced efficacy and the capability to generate a robust reservoir of memory cells is essential. Addressing the challenge of drug-resistant tuberculosis requires a deep understanding of bacterial evolution and developing robust countermeasures. This study aims to design a next-generation TB vaccine that provides broad-spectrum protection against various Mycobacterium tuberculosis strains, including drug-resistant ones. By conducting an in-depth investigation into pathogen-human interactions, the research proposes a holistic framework that leverages computational vaccinology to tackle challenges posed by pathogen polymorphism and overcome the limitations of conventional vaccines. By targeting conserved proteins across diverse TB strains and enhancing both humoral and cell-mediated immunity, this study proposes a new strategy for an epitope-based vaccine that provides long-lasting, universal coverage. An extensive proteomic, reverse vaccinology and immunoinformatics analysis of 159 TB strains yielded 27 highly conserved, immunogenic, non-toxic, and non-allergenic epitopes. These epitopes, consisting of 14-cytotoxic T-lymphocytes (CTL), 5-helper T-lymphocytes (HTL), and 8-B-cell epitopes, were used to construct a three-dimensional, multi-epitope TB vaccine designed based on a new concept introduced in this research for maximising vaccine efficacy. Molecular docking and immune simulation studies demonstrated a significant affinity between the vaccine constructs and toll-like receptors, indicating a strong potential for effective immune system engagement. The crucial features of the epitope-based TB vaccine constructed in this research include sequence conservancy, robust antigenicity, exclusion of self-peptides and potential for diverse allelic interactions. The proposed epitope-based vaccine is poised to be highly effective, safe, and capable of providing universal coverage, potentially paving the way for global TB eradication. Validation in laboratory and clinical settings will be essential to confirm its efficacy and real-world applicability.

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Background: As tuberculosis (TB) incidence declines, transmission increasingly concentrates into vulnerable populations. There is an urgent need for affordable surveillance strategies to monitor trends, identify high-risk groups and target interventions. Mycobacterium tuberculosis (Mtb) immunoreactivity surveys indirectly detect transmission and therefore undiagnosed infectious disease. Methods: We conducted a cross-sectional community-based interferon-gamma release assay (IGRA) survey in children aged 1-4 years in Blantyre, Malawi. Community-representative participants were recruited using novel convenience sampling in health facilities alongside random household sampling, and tested for Mtb immunoreactivity using QFT-Plus IGRA. We constructed hierarchical Bayesian logistic regression models for IGRA positivity, with neighbourhood-level random effects. Findings: Of 1,545 participants, 102 (6.6%) had a positive IGRA: an annual risk of Mtb infection (ARTI) of 2.7% (95% CrI 2.2-3.2%). Immunoreactivity was higher in the poorest third of households (8.7% vs 4.9%; adjusted odds ratio: 1.88, 95% CrI 1.08-3.01) compared to the richest, but was not associated with HIV exposure, malnutrition or reported household TB exposure. There was substantial between-neighbourhood heterogeneity (ARTI range 1.1-4.1%). There was no association between neighbourhood-level TB case notifications and ARTI. Interpretation: An innovative convenience sampling approach identified a high burden and substantial spatial variation of recent TB transmission, which did not correspond to case notification rates. This strategy could support identification of high-risk populations, monitoring of trends and targeted public health interventions.

RationaleFibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and fibroproliferative remodeling in acute respiratory distress syndrome (ARDS), are characterized by increased extracellular matrix (ECM) deposition. However, measuring collagen accumulation alone does not capture differences in ECM organization or biochemical maturation that may distinguish persistent fibrosis from potentially reversible remodeling. ObjectivesTo examine collagen organization characteristics and mature (pyridinoline) collagen crosslinking amount in established end stage fibrotic lung disease (IPF) and fibroproliferation following an acutely damaged lung (non-resolving (NR) ARDS) and to investigate any relationships in these parameters and temporal tissue remodeling. MethodsHuman lung tissue samples from control subjects, patients with IPF, and NR-ARDS were analyzed. Collagen amount and fiber organization were digitally quantified using picrosirius red staining. Mature collagen crosslinking was assessed by quantification of pyridinoline crosslinks. Measurements and Main ResultsLung tissue from both IPF and NR-ARDS lungs had higher collagen content compared with controls. Collagen fiber organization differed between groups. IPF lungs exhibited collagen architectures consistent with established fibrosis, whereas NR-ARDS lungs showed altered but less stabilized collagen organization despite similarly elevated collagen levels. Mature collagen crosslinks were significantly higher in IPF lungs but not in NR-ARDS lungs compared to controls. Integrated analyses identified distinct disease-associated ECM phenotypes, indicating that higher collagen abundance in NR-ARDS, unlike IPF, is not accompanied by more mature and persistent collagen crosslinking. ConclusionsDespite shared increases in collagen content, IPF and NR-ARDS lungs differ fundamentally in collagen organization and crosslinking maturity, suggesting differences in the reversibility of these conditions.

Objectives Tuberculosis (TB) in the United States disproportionately affects non-U.S.-born individuals. While testing this population for TB infection is recommended, little is known about individuals' willingness to take treatment for latent TB infection (LTBI). To address this gap, we conducted a pilot preference survey among individuals from countries with high TB incidence. Design Cross-sectional survey supported by language concordant community health workers. Setting Federally qualified health center, serving a primarily Asian immigrant community, in San Francisco. Participants Adults eligible for risk-based LTBI testing based on place of birth seeking primary care. Outcome measures Perspectives on TB disease, risk of reinfection, and willingness to accept treatment if diagnosed with LTBI conditional on different factors, such as side effects, costs, and other treatment burden. Results Among 60 participants, the median age was 48 years (interquartile range 35-63 years), 52% were women, and 100% spoke Chinese. Infecting others (n=35, 58%), risk of death (n=30, 50%), and potential isolation (n=25, 42%) were the most worrisome consequences of TB disease. Reinfection risk, risk of liver damage, cost, TB progression risk, clinic visits, and blood draws were most often considered moderately or very important when deciding whether to take LTBI treatment (n=53 to 57, 88-95%). While most participants (n=56, 93%) were willing to take treatment if diagnosed with LTBI even at a 10-year TB progression risk below 1% and willing to accept a risk of liver damage (n=41, 68%), less than half would accept LTBI treatment if there were any associated cost (n=28, 47%). Finally, many participants had concerns about their reinfection risk after completing LTBI treatment (n=34, 57%). Conclusions Amongst surveyed participants, TB disease and its consequences such as hospitalization, death and infecting others were worrisome, and participants had a high level of willingness to take treatment if diagnosed with LTBI. Future assessments of how people weigh tradeoffs regarding LTBI diagnosis and treatment could inform interventions to increase LTBI treatment acceptance and completion.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

In vitro methods to characterize drug combinations typically involve phenotypic screenings using checkerboard assays (CBA) or, more recently, DiaMOND. Such approaches rely on the Fractional Inhibitory Concentration Index (FICI), a fixed-time measurement of growth inhibition that, nonetheless, necessitates secondary validation by time-kill assays (TKA). Longitudinal time-kinetics of bacterial killing are considered the gold standard in vitro proxy for antimicrobial activity, but they required increased assay complexity, particularly against the slow growing Mycobacterium tuberculosis. Here, we developed a new methodology named OPTIKA (Optimized Time Kill Assays) that enhances the capacity of traditional TKA by over 1000-fold. This allows for easy and dynamic examination of n-way drug interactions by simultaneously monitoring bactericidal and sterilizing capacities in a longitudinal manner. We then replicated previous DiaMOND studies and performed comparisons using CBA and OPTIKA methodologies. We demonstrate that selection of the efficacy parameters (either routed on bacteriostatic, bactericidal or sterilizing properties) affects the interpretation of in vitro drug interactions and, consequently, its potential translational value. The increased assay throughput provided by OPTIKA offers a novel framework for developing tuberculosis treatment regimens. TeaserOPTIKA is a new methodology that increases time-kill assay performance against Mycobacterium tuberculosis by over 1,000-fold

BackgroundCalcific aortic valve disease (CAVD) is the most common valvular heart disease in developed countries, yet no pharmacological therapy is available to slow or halt its progression. CAVD is driven by progressive calcification of aortic valve leaflets, in which myeloid cells play a central role. While macrophages have been implicated in CAVD pathogenesis, the contribution of their precursors, monocytes, remains poorly understood. We hypothesized that circulating monocytes acquire a pro-calcific and pro-inflammatory phenotype contributing to valve remodelling and CAVD progression. MethodsWe profiled circulating CD14+ monocytes from healthy volunteers (Vol), patients with CAVD, and without CAVD (NCAVD). Peripheral blood mononuclear cells (PBMCs) were isolated, and monocyte subpopulations were phenotyped by flow cytometry. Transcriptome profiling by RNA sequencing identified disease-associated gene signatures, which were validated by RT-qPCR. The CD14+ monocyte secretome was analysed using multiplex assays. Functional ability of CAVD-derived CD14+ monocytes to induce myofibroblastic transdifferentiation (MT) and osteoblastic differentiation (OD) of human valvular interstitial cells (VICS) was evaluated by immunocytochemistry and quantitative o-cresolphthalein complexone assays. ResultsIn PBMCs, CAVD monocytes displayed a subpopulation shift, with an increased proportion of CD14CD16- classical monocytes and a reduced CD14CD16 non-classical monocyte levels. In CD14+ monocytes, transcriptomic analysis revealed upregulation of inflammation-related (PDK4) and calcification-related (ATP2B1) genes, alongside downregulation of immunomodulatory genes (DDR1, IKBKE). Secretome analysis showed reduced production of immunomodulatory and anti-osteoblastogenic cytokines (IL-4, CCL3) while promoting gene expression of factors promoting MT and OD in VICS. These alterations were associated with a marked monocyte-induced increase in SMA and OPN expression in VICS and a two-fold increase in calcification. ConclusionWe demonstrate for the first time that circulating monocytes from patients with CAVD exhibit enhanced pro-inflammatory and pro-calcific properties that may contribute to CAVD progression. Additionally, we identify dysregulated gene sets within these monocytes that represent potential novel therapeutic targets for CAVD.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health challenge. Currently treatment of drug-sensitive TB, involves a six-month regimen consisting of a combination of four anti-TB drugs, with drug-resistant TB requiring over two years of treatment and additional drugs. As toxicity of anti-TB drugs often leads to poor compliance, disease relapse and the emergence of drug-resistant strains, new strategies to reduce drug toxicity and shorten treatment duration are critical. We report nanocarrier-based drug delivery systems targeting macrophages, which primarily support replication and survival of Mtb. We have developed mannose-functionalized nanoparticles that bind to mannose receptors on macrophages and feature a pH-sensitive core which releases an encapsulated drug in the acidic lysosomal environment of macrophages. Rifampicin (RIF), a main anti-TB drug currently in use clinically, was encapsulated within the nanoparticles. We demonstrate that antibiotic-containing nanocarriers efficiently accumulated in macrophages without causing toxicity. Encapsulated RIF showed enhanced efficacy against both BCG and Mtb in primary macrophages. Biodistribution studies in mice revealed that the nanoparticles have extended circulation time and do not induce toxicity. In addition, the encapsulated RIF showed better targeting of mycobacteria when compared to free RIF in a murine model of mycobacterial infection. Such an enhanced bacterial killing using mannose-functionalised nanocarriers loaded with the key anti-TB drug rifampicin offers excellent potential for TB therapy.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

BackgroundSystematic screening for tuberculosis (TB) is recommended in people with diabetes; however, data on the accuracy of screening tools in this population are lacking. We assessed the accuracy of symptom and chest X-ray screening among people with diabetes. MethodsWe consecutively enrolled adults with diabetes attending routine care in South Africa. All participants underwent symptom screening and chest X-ray. A single sputum specimen was collected from all participants and tested by Xpert Ultra. A positive Xpert Ultra result was used as the reference standard. ResultsWe enrolled 673 participants. The median age was 54 years (interquartile range 47-60 years), and 63.8% were female. HIV prevalence was 17.2%. Prevalent TB was diagnosed in nine participants (1.33%). Any cough had a sensitivity of 22.2% (95% confidence interval [CI] 2.1-60.0%) and a specificity of 97.5% (95%CI 96.0-98.6%). Expanding the symptom definition to include any of cough, fever, weight loss, or night sweats did not improve sensitivity (22.2 %, 95 % CI 2.1-60.0) and slightly reduced specificity to 96.0 % (95 % CI 94.2-97.0). Chest X-ray abnormalities suggestive of TB demonstrated a sensitivity of 55.6% (95%CI 21.2-86.3%) and a specificity of 95.4% (95%CI 93.4-97.0%). The specificity of chest X-ray was significantly lower in participants with prior TB (87.6%, 95% CI: 79.8-90.6%), compared to 97.2% (95% CI: 95.2-98.5%) in those without (p < 0.01). ConclusionSymptom-based TB screening has poor sensitivity in people with diabetes. Although chest X-ray improved the sensitivity, it remained suboptimal, with a reduced specificity in people with previous TB.