Biology subjects

Stewart, I.

Publications and source records attributed to Stewart, I..

9 recordsLinked to original sources

bioRxiv2026

Multi-omics reveals a monocyte-macrophage-fibroblast axis in post-COVID-19 fibroinflammatory lung remodelling

Post-COVID-19 residual lung abnormalities (RLA) are associated with persistent respiratory symptoms and radiological changes, yet the underlying mechanisms remain unclear. We performed integrated multi-omic profiling of paired bronchoalveolar lavage and blood samples from patients with post-COVID-19 RLA and healthy controls, combining single-cell RNA sequencing, CITE-seq, single-cell T cell receptor sequencing, bronchoalveolar lavage fluid proteomics and functional fibroblast assays. In post-COVID-19 RLA lungs, we identified an increased abundance of profibrotic SPP1hi monocyte-derived alveolar macrophages, arising from an expanded circulating HLA-DRlowCD163+PDE4Dhi classical monocyte progenitor population, supporting a blood-lung myeloid axis. Cell-cell communication modelling positioned macrophages as central hubs of immune-stromal crosstalk, promoting monocyte recruitment with profibrotic priming, and fibroblast activation. Proteomic analysis of bronchoalveolar lavage fluid from post-COVID-19 RLA and idiopathic pulmonary fibrosis, compared with healthy controls, revealed shared and distinct signatures. These alveolar proteins in post-COVID-19 RLA were predominantly attributed to myeloid cells and predicted to engage fibroblast receptors. Bronchoalveolar lavage fluid induced fibroblast proliferation, differentiation and collagen deposition in vitro, with proliferation attenuated by the antifibrotic drug nintedanib. We also identified compartment-specific lymphoid dysregulation, including depletion of mucosal-associated invariant T (MAIT) cells in both the lung and blood, decreased natural killer (NK) cells with oligoclonal T cell expansion in the lung, and expansion of regulatory and cytotoxic T cells in the blood. These findings support a persistent monocyte-macrophage-fibroblast axis linking immune dysregulation to fibroproliferative remodelling after COVID-19 and highlights candidate therapeutic targets for post-viral lung fibrosis. We provide a publicly available atlas (on publication).

systems biology↗

bioRxiv2024

Multiscale single-cell assessment of the fibrotic niche in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease characterised by excessive extracellular matrix deposition within the lung. Recent advances in single-cell RNA sequencing have identified distinct fibrotic populations, yet their origins and spatial relationships remain incompletely understood. MethodsUsing spatial transcriptomics and Hyperion imaging mass cytometry we compared the cellular composition in formalin fixed paraffin embedded fibrotic lesions (n=9 patients) with control lung (n=9), and cellular interactions were inferred using CellChat V2 ligand-receptor analysis. Monolayers of airway epithelial cells were used to identify changes in keratin (KRT) expression following cell detachment and cyclical mechanical stretch. ResultsSpatial multiomics profiling of human lung cells confirmed the in situ localisation of previously described IPF-enriched populations, and identified a previously unrecognized KRT5low/KRT17 epithelial population derived from airway basal cells that progressively acquiring molecular features of aberrant basaloid cells, forming a unique fibrotic niche enriched with the Secreted Phosphoprotein 1 (SPP1) positive macrophages. Functional studies demonstrated that epithelial detachment and cyclical mechanical stretch drive KRT5 reduction, providing a mechanism for the emergence of this transitional state. In addition, we also identified distinct immune-stromal niches enriched in lymphocytes and alveolar fibroblasts. ConclusionThese findings delineate distinct fibrotic epithelial niches in IPF and support a model in which epithelial loss induces aberrant basaloid differentiation and fibroblast activation, with subsequent airway traction and epithelial detachment generating a secondary niche enriched in basal-derived KRT5low/KRT17 cells and SPP1 macrophages.

molecular biology↗

bioRxiv2024

Genetic burden of dysregulated cytoskeletal organisation in the pathogenesis of pulmonary fibrosis

BackgroundRare genetic variants contribute to pulmonary fibrosis (PF) risk and outcome, with known variants highlighting the importance of impaired telomere maintenance and surfactant biology. However, much of the disrupted genetic architecture of PF remains unexplained. This study aimed to identify genes with rare pathogenic coding variants that represented a burden at the exon level associated with the pathogenesis of PF. MethodsPF case cohorts included the PROFILE study of incident idiopathic pulmonary fibrosis (IPF) and PF-classified participants from the Genomics England 100K (GE100KGP) study. Whole genome sequencing data were used to test the burden of rare protein altering variants (PAVs; CADD score >20) defined by minor allele frequency <0.1% in the summary level gnomAD reference database (v3.1.2). A summary exon-level pathogenicity score was derived by standardising PAV predictions from functional annotation tools (AlphaMissense, REVEL, ClinPred, CADD, PolyPhen-2, SIFT) averaged across exons. SKAT-O based kernel regression associated exon-level pathogenicity features with disease progression and survival. Single cell lung transcriptomic datasets were harmonised to evaluate expression patterns and gene ontology characterised enriched molecular functions. ResultsAcross 507 PROFILE cases and 451 GE100KGP cases, each compared against 76,156 reference participants, 77 genes showed overlapping significant rare PAV burden, comprising 206 concordant PAVs. Of these, 15 genes had exons with pathogenicity scores that were associated with worse clinical outcomes in IPF, including FAT4 exon-10 and DNAH7 exon-43. Dysregulated gene expression of COL6A3 and FAT4 was observed in IPF lung fibroblasts, while DNAH7, DNAH12 and PCM1 showed high expression in IPF epithelial cells. The top enriched molecular functions were related to cytoskeletal motor activity. ConclusionsRare PAV burden highlighted that pathogenicity within specific exons was associated with PF risk and worse clinical outcomes, identifying genes with dysregulated expression in disease lung cells. The genetic architecture implicates disrupted cytoskeletal organisation in the pathogenesis of PF.

genetics↗

bioRxiv2024

Association between mosaic loss of chromosome Y and pulmonary fibrosis susceptibility and severity

Background Pulmonary fibrosis (PF) is a rare lung disease with diverse pathogenesis and multiple interconnected underlying biological mechanisms. Mosaic loss of chromosome Y (mLOY) is one of the most common forms of acquired chromosome abnormality in men, which has been reported to be associated with increased risk of various chronic progressive diseases including fibrotic diseases. However, the exact role of mLOY in the development of PF remains elusive and to be elucidated. MethodsWe adopted three complementary approaches to explore the role of mLOY in the pathogenesis of PF. We used copy number on chromosome Y to estimate mLOY comparing patients in PROFILE and gnomAD cohorts and between cases and control patients from the GE100KGP cohort. Correlation of mLOY with demographic and clinical variables was tested using patients from PROFILE cohort. Lung single-cell transcriptomic data were analysed to assess the cell types implicated in mLOY. We performed Mendelian randomisation to examine the causal relationship between mLOY, IPF, and telomere length. ResultsThe genetic analysis suggests that mLOY is found in PF from both case cohorts but when compared with an age matched population the effect is minimal (P = 0.0032). mLOY is related to age (P = 0.00021) and shorter telomere length (P = 0.0081) rather than PF severity or progression. Single-cell analysis indicates that mLOY appears to be found primarily in immune cells and appears to be related to presence and severity of fibrosis. Mendelian randomisation demonstrates that mLOY is not on the causal pathway for IPF, but partial evidence supports that telomere shortening is on the causal pathway for mLOY. ConclusionOur study confirms the existence of mLOY in PF patients and suggests that mLOY is not a major driver of IPF. The combined evidence suggests a triangulation model where telomere shortening leads to both IPF and mLOY.

genomics↗

bioRxiv2023

Pharmacokinetic considerations for optimizing inhaled spray-dried pyrazinoic acid formulations

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of anti-TB regimens, but its prolonged use has the potential to drive development of PZA resistant Mtb. PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA, and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung, hence direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA-mutant PZA-resistant Mtb. The objectives of the current study were to i) develop novel dry powder POA formulations ii) assess their feasibility for pulmonary delivery using physicochemical characterization, iii) evaluate their pharmacokinetics (PK) in the guinea pig model and iv) develop a mechanism based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3); (ii) the highest concentration in ELF (CmacELF: 171 nM) within 15.5 minutes, correlating with a fast transfer into ELF after pulmonary administration (kPM: 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product. Table of Contents (TOC)/Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/534965v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@c548fforg.highwire.dtl.DTLVardef@3b8efborg.highwire.dtl.DTLVardef@281d94org.highwire.dtl.DTLVardef@1ae07a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Stable nebulization and muco-trapping properties of Regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19

The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries around the world, can effectively trap SARS-CoV-2 virus-like-particles in fresh human airway mucus. IN-006, a reformulation of Regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.

bioengineering↗

bioRxiv2021

The neural cell adhesion molecule NrCAM regulates development of hypothalamic tanycytes

Hypothalamic tanycytes are neural stem and progenitor cells, but little is known of how they are regulated. Here we provide evidence that the cell adhesion molecule, NrCAM, regulates tanycytes in the adult niche. NrCAM is strongly expressed in adult mouse tanycytes. Immunohistochemical and in situ hybridization analysis revealed that NrCAM loss of function leads to both a reduced number of tanycytes and reduced expression of tanycyte-specific cell markers, along with a small reduction in tyrosine hydroxylase-positive arcuate neurons. Similar analyses of NrCAM mutants at E16 identify few changes in gene expression or cell composition, indicating that NrCAM regulates tanycytes, rather than early embryonic hypothalamic development. Neurosphere and organotypic assays support the idea that NrCAM governs cellular homeostasis. Single-cell RNA sequencing (scRNA-Seq) shows that tanycyte-specific genes, including a number that are implicated in thyroid hormone metabolism, show reduced expression in the mutant mouse. However, the mild tanycyte depletion and loss of markers observed in NrCAM-deficient mice were associated with only a subtle metabolic phenotype.

neuroscience↗

bioRxiv2018

Patient reported distress can aid clinical decision making in idiopathic pulmonary fibrosis: analysis of the PROFILE cohort

Idiopathic pulmonary fibrosis is a progressive and fatal interstitial lung disease. We aimed to determine if patient response to a palliative assessment survey could predict disease progression or death. We undertook a cross-sectional study in a UK clinical cohort of incident cases. Rasch-based methodology provided a disease distress value from an abridged 11 item model of the original 45 item survey. Distress values were compared with measures of lung function. Disease progression or mortality alone was predicted at twelve months from survey completion, with risk of death assessed at three, six and twelve months. Disease distress values were negatively correlated with lung function (r=-0.275 percent predicted DLCO). Expected survey scores computed from distress values could distinguish disease progression, 8.8 (p=0.004), and people who died, 10.2 (p=0.002), from those who did not progress, 6.9. Actual survey scores predicted disease progression and mortality with an area under the curve of 0.60 and 0.64, respectively. Each point increment in actual score increased risk of twelve-month mortality by 10%, almost 43% of people scoring above 18 did not survive beyond 105 days. We define a short questionnaire that can score disease distress and predict prognosis, assisting clinical decision making in progressive fibrosis.

clinical trials↗

bioRxiv2018

Characterization of missing values in untargeted MS-based metabolomics data and evaluation of missing data handling strategies

BACKGROUNDUntargeted mass spectrometry (MS)-based metabolomics data often contain missing values that reduce statistical power and can introduce bias in epidemiological studies. However, a systematic assessment of the various sources of missing values and strategies to handle these data has received little attention. Missing data can occur systematically, e.g. from run day-dependent effects due to limits of detection (LOD); or it can be random as, for instance, a consequence of sample preparation.\n\nMETHODSWe investigated patterns of missing data in an MS-based metabolomics experiment of serum samples from the German KORA F4 cohort (n = 1750). We then evaluated 31 imputation methods in a simulation framework and biologically validated the results by applying all imputation approaches to real metabolomics data. We examined the ability of each method to reconstruct biochemical pathways from data-driven correlation networks, and the ability of the method to increase statistical power while preserving the strength of established genetically metabolic quantitative trait loci.\n\nRESULTSRun day-dependent LOD-based missing data accounts for most missing values in the metabolomics dataset. Although multiple imputation by chained equations (MICE) performed well in many scenarios, it is computationally and statistically challenging. K-nearest neighbors (KNN) imputation on observations with variable pre-selection showed robust performance across all evaluation schemes and is computationally more tractable.\n\nCONCLUSIONMissing data in untargeted MS-based metabolomics data occur for various reasons. Based on our results, we recommend that KNN-based imputation is performed on observations with variable pre-selection since it showed robust results in all evaluation schemes.\n\nKey messagesO_LIUntargeted MS-based metabolomics data show missing values due to both batch-specific LOD-based and non-LOD-based effects.\nC_LIO_LIStatistical evaluation of multiple imputation methods was conducted on both simulated and real datasets.\nC_LIO_LIBiological evaluation on real data assessed the ability of imputation methods to preserve statistical inference of biochemical pathways and correctly estimate effects of genetic variants on metabolite levels.\nC_LIO_LIKNN-based imputation on observations with variable pre-selection and K = 10 showed robust performance for all data scenarios across all evaluation schemes.\nC_LI

systems biology↗