EBioMedicine

Background[RP1.1] In low-burden countries such as France, whole-genome sequencing (WGS) is increasingly integrated into routine tuberculosis (TB) surveillance to improve case management and transmission monitoring. However, applying WGS to all TB cases generates large volumes of data, requiring automated tools for timely interpretation and outbreak response. Methods Since November 2016, all clinical M. tuberculosis isolates diagnosed in eight hospitals from three cities of Auvergne-Rhone-Alpes in France have undergone WGS. In July 2023, an automated pipeline for anti-TB drug resistance prediction and unbiased detection of transmission clusters based on SNP distances was implemented. Epidemiological, microbiological and clinical data were collected, with contact duration classified as household, frequent, or occasional. Index cases were stratified by their level of extra-household transmission (EHT), and statistical analyses were performed to identify associated factors. Findings Among 1,152 TB patients diagnosed between 2016 and 2025, 75 clusters involving 247 patients (21.4%) were identified. WGS reliably detected resistance to first-line anti-TB drugs, leveraging the WHO mutation catalogue. Routine WGS enabled real-time alerts for TB control centres, leading to expanded field investigations, including community spillover, nosocomial transmissions, and school outbreak. Classical indicators of contagiousness (smear results, cavitary disease) were not associated with EHT level. Instead, lower TB severity indices and longer duration of symptoms were linked to higher EHT level. Interpretation Systematic WGS supports timely identification of drug resistance and transmission events and provides new insights into contagiousness factors. The automated pipeline enables direct interpretation by clinical microbiologists, facilitating real-time public health action. In this study, we demonstrate how, with the appropriate pipeline, WGS offered a time- and cost-effective solution for routine TB management. Funding This work was supported by SHAPE-Med@Lyon, a French government grant managed by the French National Research Agency under the France 2030 program (reference ANR-22-EXES-0012).

Rationale: Preclinical familial pulmonary fibrosis (FPF) represents an early stage of fibrotic lung disease, yet the compartment- and cell-specific molecular programs preceding fibrosis remain poorly understood. Objective: To define spatially organized molecular signatures associated with preclinical FPF and identify tissue-informed circulating biomarkers linked to early fibrotic remodeling. Methods: We performed integrated multi-omic profiling of histologically preserved and remodeled lung regions from subjects with preclinical FPF, Idiopathic Pulmonary Fibrosis (IPF), and controls using spatial transcriptomics, single-nucleus RNA sequencing (snRNAseq), and blood proteomics. Differential expression and pathway enrichment analyses were performed across spatial compartments and epithelial cell states. Results: Histologically preserved lung regions in preclinical FPF demonstrated transcriptional abnormalities including stress-response, ciliary, and extracellular matrix-associated programs despite minimal architectural distortion. Spatial analyses identified alterations in alveolar niche molecular programs accompanied by increasing profibrotic signaling across preserved and tissue remodeled lung compartments. Compared with advanced IPF, preclinical FPF retained epithelial repair and surfactant-associated signatures. Integration with snRNAseq demonstrated enrichment of alveolar and airway epithelial cell dysregulated states associated with transitional phenotypes previously implicated in IPF. Compartment- and epithelial-associated transcriptional signatures identified in lung tissue were partially represented in the peripheral blood. Conclusion: Preclinical FPF is characterized by compartment- and cell-specific molecular programs that precede established fibrosis. We identified distinct alveolar, airway, and vascular molecular signatures and epithelial remodeling states represented in the peripheral blood. These findings provide an initial framework for molecular classification of early stages of pulmonary fibrosis and support future studies evaluating minimally invasive approaches for disease stratification and precision therapeutics.

Lung adenocarcinoma (LUAD) is classified internationally into six histological subtypes that predict clinical outcomes. Mutation analyses identify targets but provide less prognostic information than histological appearances. Immunotherapy in LUAD is constrained by the unpredictable immune environment within tumours. We therefore characterised relationships between WHO histological classification, common mutations, and underlying transcriptomic and immune profiles in 89 LUAD cases. Mutation profiles poorly correlated with histology or survival. Global gene expression was structured into 12 modules, identifying different tumour cells and pathways within WHO subtypes. Tumour classes also held distinctive immune cell profiles. Transcripts within high-risk solid tumours indicated enrichment of CD8+ and activated CD4+ T-cells, suggesting responsivity to immunotherapy. Independently from histologic classification, 31 transcripts were strongly associated with survival and were enriched in macrophage and fibroblast derived networks. The results suggest histological subtype stratification and typing for survival-associated markers have the potential to inform clinical trials of LUAD.

BackgroundA bidirectional relationship has been observed between COVID-19 and respiratory disorders, where respiratory comorbidities increase severity and COVID-19 induces respiratory sequelae. The underlying biological and genetic mechanisms remain unclear. While previous studies have identified overlapping genetic loci, few have systematically disentangled the genetic factors shared between these conditions versus those specific to COVID-19, particularly at a multi-omics level. MethodsWe developed and applied a unified analytical framework to compare three COVID-19 phenotypes with eight respiratory disorders (including asthma, COPD, IPF, and pneumonia). Utilizing the cofdr method for shared genetic signal analysis and DDx/mtCOJO for differentiation, we integrated genome-wide association statistics with multi-omics data (transcriptome, splicing, and proteome). This approach allowed for the simultaneous identification of shared genetic signals (concordant or discordant) and disease-specific variants across expression (TWAS), alternative splicing (spTWAS), and protein abundance (PWAS). ResultsWe delineated a comprehensive atlas of 214 differential and numerous shared loci across 24 pairwise comparisons. The shared genetic architecture was characterized by pleiotropic effects in genes such as ATP11A (exhibiting opposing effects in COVID-19 vs. IPF) and GSDMB (shared with COPD). Crucially, differentiation analysis revealed that severe COVID-19 is genetically distinct from other respiratory infections (e.g., pneumonia and influenza) through dysregulated Type I/III interferon signaling and specific defects in alveolar epithelial and macrophage function, as well as GM-CSF/surfactant metabolism pathways. These findings provide direct genetic evidence supporting the use of GM-CSF modulators and interferon-lambda for COVID-19 treatment, therapies that have already entered clinical trials. Furthermore, multi-trait conditional analysis prioritized FYCO1 and HCN3 as potential COVID-19-specific risk genes. Splicing analysis underscored the critical role of alternative splicing in both shared and differential architectures, highlighting IFNAR2 isoform regulation as a key discriminator between COVID-19 and other respiratory traits. ConclusionThis study provides the first genome-wide, multi-omics map revealing the shared and differential genetic landscapes of COVID-19 and other respiratory phenotypes. By uncovering specific molecular mechanisms that distinguish COVID-19 pathology, specifically involving surfactant homeostasis and interferon pathways, our findings offer novel insights for targeted drug repurposing and precision risk stratification.

Background: Alpha-1 antitrypsin deficiency (AATD) caused by the PI*ZZ mutation (Glu342Lys) results in hepatic accumulation of misfolded AAT-Z protein and reduced circulating AAT levels, leading to progressive liver disease and emphysema. Gene correction therapy represents a potentially curative approach by directly correcting the underlying genetic defect. We report the first case of successful hepatic gene correction with early histological and functional assessment. Methods/Case presentation: We report the case of a 66-year-old male patient with PI*ZZ AATD who underwent gene correction therapy within the YOLT-202 phase I/Ia clinical trial (clinical trial.gov ID NCT07193615). Ten weeks post treatment a liver biopsy was performed to re-evaluate pre-existing F2 liver fibrosis as measured by elastography before entering the study. Serum samples allowed functional assessment of the AAT-mediated elastase inhibition. Results: Liver biopsy did not show signs of hepatic inflammation and demonstrated 54% (Sanger) and 57% (Illumina) gene correction rate of the PI*ZZ variant on the DNA level with no bystander edits or off-target effects. Following a transient elevation of transaminases during the early post-treatment period, liver enzymes normalized. Monthly serum AAT measurements demonstrated biologically active and stable therapeutic levels throughout follow-up. Conclusions: This case demonstrates efficient and precise hepatic gene correction without concerning histological alterations and with substantial improvement of functional parameters, supporting the feasibility and safety of gene editing approaches for AATD.

BackgroundInterleukin-6 (IL-6) is a central driver of pulmonary vascular remodeling in idiopathic, heritable, and connective tissue disease-associated pulmonary arterial hypertension (PAH). Elevated IL-6 correlates with right ventricular (RV) dysfunction and poor survival. However, the specific downstream mechanisms by which IL-6 drives pathogenesis remain poorly defined. We investigated the therapeutic impact of direct IL-6 neutralization and its regulation of a novel epigenetic signaling axis in PAH. Materials and MethodsWe evaluated species-specific IL-6-neutralizing antibodies in murine Sugen/hypoxia and rat monocrotaline models of PAH. RV structure and function were assessed using cardiac MRI and invasive hemodynamics. Lung transcriptomic profiling was performed by RNA sequencing mouse lung tissue. Key findings were validated in explanted human PAH lungs, serum, and peripheral blood mononuclear cells (PBMCs), and further interrogated through mechanistic in vitro studies in human pulmonary artery endothelial cells (PAECs). ResultsIL-6 neutralization significantly improved RV function, reduced pulmonary arterial pressures, and attenuated pulmonary vascular remodeling in both experimental models. Transcriptomic analysis identified a dysregulated FOXP3 signaling axis. Mechanistically, IL-6 induced cooperative binding of phosphorylated STAT3 and STAT4 to the FOXP3 promoter, facilitating DNMT1-mediated DNA methylation and stable gene silencing. IL-6 blockade restored downstream FOXP3 expression, rescued downstream BMPR2 signaling, and re-established endothelial homeostasis. In clinical PAH cohorts, FOXP3 expression was markedly reduced and inversely correlated with circulating IL-6 levels and indices of disease severity. ConclusionIL-6 drives pulmonary hypertension through STAT3/STAT4- and DNMT1-dependent epigenetic repression of FOXP3, linking chronic inflammation to BMPR2 dysfunction and pulmonary vascular remodeling. IL-6 neutralization reverses this pathogenic program in experimental PAH. FOXP3 emerges as a mechanistic biomarker of disease severity and a potential tool for precision stratification of patients likely to benefit from IL-6-targeted therapies. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISynergistic Gene Repression: The study demonstrates that phosphorylated STAT3 functional transcriptional complex that binds directly to the FOXP3 promoter. Acting as a molecular scaffold, this complex actively represses FOXP3 expression within the pulmonary endothelium. C_LIO_LIEpigenetic Silencing Mechanism: IL-6 does not merely induce a transient suppression of FOXP3. Instead, IL-6/STAT3 signaling recruits DNMT1 to the FOXP3 promoter, leading to site-specific DNA methylation and long-term epigenetic silencing. This mechanism provides a direct molecular link between chronic systemic inflammation and sustained pulmonary vascular injury. C_LIO_LIThe FOXP3-BMPR2 Connection: The findings identify FOXP3 as a critical positive regulator of BMPR2 expression. Neutralization of IL-6 disrupts this pathological inhibitory loop, permitting restoration of BMPR2 signaling, the central gatekeeper of pulmonary vascular integrity and homeostasis. C_LIO_LITranslational Biomarkers: Importantly, the molecular changes within the lung are reflected systemically. Suppressed FOXP3 expression is readily detectable in PBMCs and serum, offering a non-invasive biomarker that correlates with pulmonary hemodynamic status and vascular health. C_LI Clinical ImplicationsO_LIPrecision Patient Stratification: FOXP3 expression in PBMCs and serum functions as a surrogate marker of pulmonary vascular epigenetic health. This could identify a specific inflammatory endotype of PAH patients most likely to respond to IL-6-targeted therapies. C_LIO_LIPrognostic Biomarker: Because FOXP3 levels integrate both systemic inflammatory burden and dysfunction of the RV-pulmonary vascular axis, they may provide a more sensitive tool for monitoring therapeutic response and predicting clinical worsening than current standard-of-care markers. C_LI

I.BackgroundPulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-{beta} (TGF-{beta}) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-{beta} exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-{beta} axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-{beta} axis in the PAH pulmonary vasculature. MethodsCD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248-/-) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-{beta} signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. ResultsLC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-{beta} receptor I (T{beta}RI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-{beta}-induced gene expression (FN1, Col11, -SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of T{beta}RI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted T{beta}RI instability and attenuated the production of FN1, Col11, and -SMA in primary PAH PASMCs. ConclusionsThis work identifies CD248 as a previously unrecognized co-activator of T{beta}RI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-{beta} inhibition.

Adjunctive therapies that enhance the efficacy of existing antitubercular drugs are needed for drug-resistant tuberculosis. We evaluated the efficacy of intranasally administered recombinant D29 LysB, a mycobacteriophage-derived mycolylarabinogalactan esterase, in murine and guinea pig models of pulmonary tuberculosis. BALB/c mice and guinea pigs were aerosol-infected with Mycobacterium tuberculosis H37Rv and treated for 4 weeks with LysB alone or with standard antitubercular therapy (ATT: rifampicin, isoniazid, pyrazinamide). Outcomes included pulmonary and extrapulmonary bacterial burden (CFU), lung and spleen histopathology, cytokine profiling, and humoral immune responses. LysB monotherapy produced modest pulmonary CFU reductions. When given adjunctively with ATT, LysB produced an additional 0.6-0.7 log10 reduction in lung CFU compared with ATT alone and decreased splenic dissemination in both species. Combination therapy improved tissue pathology, reducing granulomatous involvement and preserving pulmonary architecture. LysB treatment increased TNF- with a moderate rise in IL-10, a profile consistent with enhanced antibacterial immunity without excessive inflammatory damage. Repeated intranasal administration was well tolerated; no IgE-mediated hypersensitivity was detected. LysB-specific IgG developed but did not diminish therapeutic efficacy. These results show that intranasal D29 LysB augments the bactericidal and histopathological effects of standard ATT in vivo and support further development of inhaled phage-derived lysins as adjunctive therapies for drug-resistant tuberculosis. ImportanceTuberculosis remains a major cause of infectious mortality worldwide, and the increasing burden of multidrug-resistant and extensively drug-resistant disease continues to challenge effective treatment. New therapeutic approaches that complement conventional antibiotics are urgently needed. In this study, intranasally delivered recombinant mycobacteriophage-derived LysB was well tolerated and enhanced treatment efficacy in experimental pulmonary tuberculosis. Adjunctive LysB improved bacterial clearance, reduced tissue pathology, and modulated host immune responses in both murine and guinea pig models. These findings highlight phage-derived endolysins as promising inhalable adjunctive therapeutics for drug-resistant tuberculosis.

Novel blood-based biomarkers for tuberculosis (TB) are needed to develop rapid, point-of-care diagnostics. We sought to use combined plasma metabolomics and high-density cytokine profiling to identify a biomarker signature that can differentiate pulmonary TB (PTB) from patients hospitalized with other respiratory diseases and ambulatory household contacts with TB symptoms. We analyzed plasma concentrations of 28 cytokines and 118 metabolites from 391 adults ([≥]18 years) presenting with respiratory symptoms suggestive of TB, of which 187 had PTB confirmed by Xpert MTB and/or M. tuberculosis sputum culture and 204 were controls in whom PTB was excluded. Our study identified a 5-marker signature (IFN.gamma, IL.22, IL.10, methionine and oxoproline) with an AUC of 0.97 (95% CI: 0.95-1.00) in the test set. The signature had 98% and 84% sensitivity at 70% and 98% specificity respectively, which meet WHO target product profiles for both non-sputum triage and diagnostic TB tests.

Alveolar capillary endothelial cells are positioned adjacent to the alveolar epithelium and contribute to lung homeostasis and injury responses. Single-cell studies have identified aerocyte capillary endothelial cells (aCap), which are specialized for gas exchange, and general capillary endothelial cells (gCap), which contribute to endothelial maintenance and inflammatory signaling. Apelin and its receptor are differentially enriched across these endothelial compartments, but their roles in emphysema development remain incompletely understood. Using an elastase-induced emphysema model in male C57BL/6J mice, we combined bulk RNA sequencing, CIBERSORTx-based cell-type deconvolution, histology, inflammatory assays, pulmonary function testing, and pharmacologic activation of the apelin receptor with [Pyr1]-Apelin-13. At 24 hours after elastase exposure, the inferred fraction of gCap was reduced, and lung expression of apelin and the apelin receptor was decreased. Early [Pyr1]-Apelin-13 administration reduced lung inflammatory mediator expression, Ly6G-positive neutrophil accumulation, bronchoalveolar lavage neutrophil counts, and matrix metalloproteinase-9 activity. Early treatment also attenuated subsequent airspace enlargement, whereas treatment initiated after emphysema was established did not improve physiological or histological outcomes. In a chronic {beta}ENaC-transgenic mouse model, the inferred gCap fraction was maintained, the aCap fraction was reduced, and apelin receptor activation did not improve disease phenotypes. These findings suggest that early activation of the apelin receptor modifies acute inflammatory and endothelium-associated responses following elastase injury and limits emphysematous remodeling in mice. Together, these results support a time-sensitive role for apelin-APJ signaling during the early phase of emphysema development.

Genome-wide association studies of idiopathic pulmonary fibrosis (IPF) have identified 35 common genetic risk loci associated with IPF susceptibility. In this study, we evaluated the effects of the reported variants in clinically curated non-European individuals. Despite limited sample sizes, we observed partial replication, limited transferability of some variants and evidence of ancestry-specific effects. The MUC5B promoter variant rs35705950 emerged as the dominant and most consistent signal across ancestries. Our findings highlight the need for larger, well-characterised studies in understudied populations to support robust discovery and translation.

Background: Host genetics alone explains limited susceptibility to tuberculosis (TB), particularly in people with HIV (PWH). Protein quantitative trait loci (pQTLs), genetic variants that regulate plasma protein levels, may bridge genetic and immunological mechanisms underlying TB progression. Methods: We conducted cis-pQTL mapping in 60 PWH who progressed to active TB and 194 matched controls from the Swiss HIV Cohort Study. Plasma proteomes were quantified via high-resolution mass spectrometry (dia-PASEF), and genotype-protein associations were analyzed separately in TB and control groups. Results: TB progressors harbored 26 cis-pQTLs linked to 12 proteins uniquely enriched in immune pathways (antigen presentation, complement activation, phagocytosis, and T-cell regulation). Controls showed 107 cis-pQTLs linked to 14 targets. Gene Ontology enrichment revealed 46 immune biological processes in TB versus only 1 in controls, with HLA-C, C4B, and CHIT1 as key TB-specific proteins. Conclusions: Integrating proteomics with genomics suggests differential regulation of immune proteins associated with TB progression in PWH. hese genetically anchored protein candidates support follow-up studies and future biomarker evaluation for TB risk prediction.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

BackgroundAfter the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. MethodsA global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. FindingsThe global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. InterpretationOur work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease in which the earliest cellular events driving fibrosis remain poorly defined. Here, we analyzed lung samples from three independent and unique cohorts of patients with early disease and preserved lung function (Florence, NIH, Forli), applying an integrated multi-modal approach combining single-nucleus RNA sequencing, bulk transcriptomics, immunostaining, and spatial transcriptomics. Single nuclear RNA sequencing of samples obtained by diagnostic bronchoscopic cryobiopsy (Florence, n= 22) revealed that early IPF is characterized by a marked shift in alveolar epithelial composition, with loss of AT1 and AT2 cells and the emergence of aberrant basaloid cells and alveolar epithelial intermediate cells. These populations exhibited transcriptional programs associated with epithelial plasticity and profibrotic signaling and closely resembled those observed in end-stage IPF. Higher proportions of aberrant basaloid and alveolar epithelial intermediate cells were associated with subsequent disease progression, whereas AT2 cell abundance correlated with preserved lung function. Fibrotic CTHRC1+ fibroblasts are largely restricted to advanced disease, while endothelial remodeling and inflammatory fibroblast states are already evident in early IPF. Spatial transcriptomic analyses confirmed early disruption of the alveolar niche, with replacement of normal epithelial-capillary interactions by aberrant epithelial and venous endothelial cells (Forli, n= 24); the findings were replicated through single cell RNA sequencing of samples obtained by video assisted thoracoscopy two decades earlier (NIH n=9). Together, these findings identify that alveolar niche remodeling with loss of its normal components, and emergence of aberrant basaloid cells are features of early IPF, highlighting epithelial dysfunction as a key potential target for therapeutic interventions in early disease.

Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

BackgroundIn extensively drug-resistant and pre-extensively drug-resistant TB, bacteriology-based monitoring often fails to capture structural lung recovery and patient-reported functional health. We aimed to characterize multidomain treatment response and examine host inflammatory and transcriptional features associated with incomplete recovery. MethodsWe conducted an ancillary analysis of a prospective, open-label, pilot study evaluating adjunctive ibuprofen in XDR-TB (NCT02781909). Participants were assessed at baseline and during treatment using TBS, chest radiography, sputum culture, SGRQ, blood cell indices, plasma cytokines, and whole-blood transcriptomic profiling. Clinical and laboratory measures were compared across outcome groups, and blood transcriptional profiles were analyzed in relation to treatment outcomes. ResultsHere we show that microbiological and symptomatic improvement occurred earlier than radiological and functional recovery. Higher baseline systemic inflammation, including elevated NLR, SII, and IL-6, as well as increased expression of interferon-related genes such as CD274 and GBP5, were associated with poorer radiological and SGRQ outcomes at 6 months. In contrast, transient elevations of IL-8 and IL-4 were associated with early bacteriological clearance. IL-8 was the only plasma biomarker consistently correlated with symptom severity, radiological findings, and functional health. ConclusionsTreatment response in drug-resistant TB is asynchronous across biological domains. Integrated host profiling identifies inflammatory and transcriptional features associated with incomplete structural and functional recovery, supporting the use of multidimensional endpoints to better capture long-term outcomes and inform individualized patient management. Plain Language SummaryPeople with highly drug-resistant tuberculosis can clear the infection but still experience lung damage and reduced quality of life after treatment. In this study, we examined recovery using several measures, including symptoms, chest X-rays, blood markers of inflammation, and gene activity, in addition to tests for tuberculosis bacteria. We analyzed data and stored samples from a small clinical trial to see how these measures changed over time. We found that lung structure and quality of life improved more slowly than bacterial clearance. People with higher levels of inflammation before treatment were more likely to have ongoing lung changes and poorer quality of life later. These results suggest that tuberculosis care should look beyond bacterial clearance and include monitoring inflammation to better support long-term recovery.

Background: Tuberculosis recurrence accounts for a substantial proportion of incident tuberculosis in many settings. Distinguishing between its mechanisms can inform public health interventions for prevention. Methods: We conducted a retrospective study of individuals with multiple culture-confirmed TB episodes and available sequential isolates from 2012 to 2023 in Dourados and Campo Grande, Mato Grosso do Sul state, Brazil. Patients were classified as having recurrent TB after treatment completion or retreatment following non-curative outcomes. Whole-genome sequencing was used to assess pairwise genetic distances between isolates, classifying relapse or persistent infection ([≤]12 single-nucleotide polymorphisms [SNPs]) versus reinfection or retreatment with reinfection (>12 SNPs). Results: Among 9,293 individuals with TB, 772 recurrent or retreatment episodes were identified. Paired isolates from 82 individuals were available for comparisons. Among individuals who completed treatment, reinfection accounted for 74.1% (40/54) of recurrent episodes, while 25.9% (14/54) were classified as relapse. Among individuals with non-curative outcomes, persistent infection (53.6%, 15/28) and retreatment with reinfection (46.4%, 13/28) occurred at similar frequencies. Persistent infection and relapse occurred earlier after the initial episode, whereas reinfection and retreatment with reinfection predominated after two years. Incarceration history was strongly associated with reinfection after treatment completion (92.5%, p=0.012) and after non-curative outcomes (76.9%, p=0.016). Conclusions: In this high-burden setting, reinfection drives TB recurrence among individuals who complete treatment, particularly at longer intervals after initial disease, reflecting sustained exposure risk. Relapse and persistent infection remain clinically important, especially following non-curative outcomes. These findings underscore the need for integrated strategies combining adherence support to prevent treatment-related recurrence with interventions to reduce transmission, particularly in high-risk settings.

BackgroundRheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by a heterogeneous clinical course with periods of remission and flare. Although biologic DMARDs (bDMARDs) have revolutionized RA treatment by enabling sustained disease control, their long-term use is associated with adverse effects and high costs, making dose tapering an attractive but clinically challenging strategy. The lack of reliable biomarkers to predict flare risk limits safe implementation of treatment de-escalation. This study aimed to identify novel circulating protein biomarkers associated with flare risk in RA patients undergoing bDMARDs tapering, useful to enable biomarker-guided treatment optimization strategies. MethodsA discovery proteomic analysis using mass spectrometry was performed on baseline serum samples from a subset of the OPTIBIO clinical trial (n=44), followed by validation in the full cohort (n=194) using ELISA. Functional pathway analysis explored biological processes associated with candidate biomarkers. In parallel, anti-cytokine autoantibodies were profiled using multiplex immunoassays. Logistic and Cox regression models were used to assess associations with flare risk. Predictive models integrating biomarkers and clinical variables were evaluated using receiver operating characteristic (ROC) analysis, sensitivity and specificity metrics, and decision curve analysis to assess clinical utility. ResultsMass spectrometry identified 806 proteins, of which 87 were differentially expressed at baseline between patients who flared and those who maintained remission during follow-up within the intervention (tapering) arm. Functional enrichment analysis highlighted immune-regulatory and innate immune pathways. Among the candidates, V-set immunoglobulin-domain-containing 4 (VSIG4) was validated as a biomarker associated with increased flare risk. Anti-interferon-{gamma} (anti-IFN{gamma}) autoantibodies were also associated with flare. A combined model including VSIG4, anti-IFN{gamma}, and the clinical variable DAS28-CRP improved predictive performance compared with clinical variables alone (AUC 0.76 vs 0.66), achieving significantly higher sensitivity. Decision curve analysis demonstrated higher net benefit of the combined model, indicating improved clinical decision-making. In a secondary analysis focused on patients with prolonged remission, representing the most suitable candidates for safe treatment tapering, the model performance further improved (AUC 0.84). ConclusionIntegration of novel serum proteomic and autoantibody biomarkers with clinical parameters improves prediction of flare during biologic tapering in RA and provides clinically relevant benefit for patient stratification. These findings support further development of biomarker-driven approaches for personalized treatment optimization strategies.