Consensus clustering with missing labels (ccml): a consensus clustering tool for multi-omics integrative prediction in cohorts with unequal sample coverage.

Multi-omics data integration is a complex and challenging task in biomedical research. Consensus clustering, also known as meta-clustering or cluster ensembles, has become an increasingly popular downstream tool for phenotyping and endotyping using multiple omics and clinical data. However, current consensus clustering methods typically rely on ensembling clustering outputs with similar sample coverages (mathematical replicates), which may not reflect real-world data with varying sample coverages (biological replicates). To address this issue, we propose a new consensus clustering with missing labels (ccml) strategy termed ccml, an R protocol for two-step consensus clustering that can handle unequal missing labels (i.e. multiple predictive labels with different sample coverages). Initially, the regular consensus weights are adjusted (normalized) by sample coverage, then a regular consensus clustering is performed to predict the optimal final cluster. We applied the ccml method to predict molecularly distinct groups based on 9-omics integration in the Karolinska COSMIC cohort, which investigates chronic obstructive pulmonary disease, and 24-omics handprint integrative subgrouping of adult asthma patients of the U-BIOPRED cohort. We propose ccml as a downstream toolkit for multi-omics integration analysis algorithms such as Similarity Network Fusion and robust clustering of clinical data to overcome the limitations posed by missing data, which is inevitable in human cohorts consisting of multiple data modalities. The ccml tool is available in the R language (https://CRAN.R-project.org/package=ccml, https://github.com/pulmonomics-lab/ccml, or https://github.com/ZhoulabCPH/ccml).

Radiomultiomics: quantitative CT clusters of severe asthma associated with multi-omics.

RATIONALE: Lung quantitative computed tomographic (qCT) severe asthma clusters have been reported, but their replication and underlying disease mechanisms are unknown. We identified and replicated qCT clusters of severe asthma in two independent asthma cohorts and determined their association with molecular pathways. METHODS: We used consensus clustering on qCT measurements of airway and lung CT scans, performed in 105 severe asthmatic adults from the U-BIOPRED cohort. The same qCT measurements were used to replicate qCT clusters in a subsample of the ATLANTIS asthma cohort (n=97). We performed integrated enrichment analysis using blood, sputum, bronchial biopsies, bronchial brushings and nasal brushings transcriptomics and blood and sputum proteomics to characterize radiomultiomic-associated clusters (RACs). RESULTS: qCT clusters and clinical features in U-BIOPRED were replicated in the matched ATLANTIS cohort. In the U-BIOPRED cohort, RAC1 (n=30) was predominantly female with elevated BMI, mild airflow limitation, normal qCT parameters and upregulation of the complement pathway. RAC2 (n=34) subjects had a lower degree of airflow limitation, airway wall thickness and dilatation, with upregulation of proliferative pathways, including neurotrophic receptor tyrosine kinase 2/tyrosine kinase receptor B (NTRK2/TRKB), and down-regulation of semaphorin pathways. RAC3 (n=41) showed increased lung attenuation area and air trapping, severe airflow limitation, hyperinflation, and upregulation of cytokine signaling and signaling by interleukin pathways, and matrix metallopeptidase 1, 2 and 9. CONCLUSIONS: U-BIOPRED severe asthma qCT clusters were replicated in a matched independent asthmatic cohort and associated with specific molecular pathways. Radiomultiomics might represent anovel strategy to identify new molecular pathways in asthma pathobiology.

Multiomics integration-based molecular characterizations of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly became a global health challenge, leading to unprecedented social and economic consequences. The mechanisms behind the pathogenesis of SARS-CoV-2 are both unique and complex. Omics-scale studies are emerging rapidly and offer a tremendous potential to unravel the puzzle of SARS-CoV-2 pathobiology, as well as moving forward with diagnostics, potential drug targets, risk stratification, therapeutic responses, vaccine development and therapeutic innovation. This review summarizes various aspects of understanding multiomics integration-based molecular characterizations of COVID-19, which to date include the integration of transcriptomics, proteomics, genomics, lipidomics, immunomics and metabolomics to explore virus targets and developing suitable therapeutic solutions through systems biology tools. Furthermore, this review also covers an abridgment of omics investigations related to disease pathogenesis and virulence, the role of host genetic variation and a broad array of immune and inflammatory phenotypes contributing to understanding COVID-19 traits. Insights into this review, which combines existing strategies and multiomics integration profiling, may help further advance our knowledge of COVID-19.

Large airway T cells in adults with former bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary Dysplasia (BPD) in infants born prematurely is a risk factor for chronic airway obstruction later in life. The distribution of T cell subtypes in the large airways is largely unknown. OBJECTIVE: To characterize cellular and T cell profiles in the large airways of young adults with a history of BPD. METHODS: Forty-three young adults born prematurely (preterm (n = 20), BPD (n = 23)) and 45 full-term-born (asthma (n = 23), healthy (n = 22)) underwent lung function measurements, and bronchoscopy with large airway bronchial wash (BW). T-cells subsets in BW were analyzed by immunocytochemistry. RESULTS: The proportions of both lymphocytes and CD8 + T cells in BW were significantly higher in BPD (median, 6.6%, and 78.0%) when compared with asthma (3.4% and 67.8%, p = 0.002 and p = 0.040) and healthy (3.8% and 40%, p < 0.001 and p < 0.001). In all adults born prematurely (preterm and BPD), lymphocyte proportion correlated negatively with forced vital capacity (r= -0.324, p = 0.036) and CD8 + T cells correlated with forced expiratory volume in one second, FEV1 (r=-0.448, p = 0.048). Correlation-based network analysis revealed that lung function cluster and BPD-birth cluster were associated with lymphocytes and/or CD4 + and CD8 + T cells. Multivariate regression analysis showed that lymphocyte proportions and BPD severity qualified as independent factors associated with FEV1. CONCLUSIONS: The increased cytotoxic T cells in the large airways in young adults with former BPD, suggest a similar T-cell subset pattern as in the small airways, resembling features of COPD. Our findings strengthen the hypothesis that mechanisms involving adaptive and innate immune responses are involved in the development of airway disease due to preterm birth.

Country-specific determinants for COVID-19 case fatality rate and response strategies from a global perspective: an interpretable machine learning framework.

BACKGROUND: There are significant geographic inequities in COVID-19 case fatality rates (CFRs), and comprehensive understanding its country-level determinants in a global perspective is necessary. This study aims to quantify the country-specific risk of COVID-19 CFR and propose tailored response strategies, including vaccination strategies, in 156 countries. METHODS: Cross-temporal and cross-country variations in COVID-19 CFR was identified using extreme gradient boosting (XGBoost) including 35 factors from seven dimensions in 156 countries from 28 January, 2020 to 31 January, 2022. SHapley Additive exPlanations (SHAP) was used to further clarify the clustering of countries by the key factors driving CFR and the effect of concurrent risk factors for each country. Increases in vaccination rates was simulated to illustrate the reduction of CFR in different classes of countries. FINDINGS: Overall COVID-19 CFRs varied across countries from 28 Jan 2020 to 31 Jan 31 2022, ranging from 68 to 6373 per 100,000 population. During the COVID-19 pandemic, the determinants of CFRs first changed from health conditions to universal health coverage, and then to a multifactorial mixed effect dominated by vaccination. In the Omicron period, countries were divided into five classes according to risk determinants. Low vaccination-driven class (70 countries) mainly distributed in sub-Saharan Africa and Latin America, and include the majority of low-income countries (95.7%) with many concurrent risk factors. Aging-driven class (26 countries) mainly distributed in high-income European countries. High disease burden-driven class (32 countries) mainly distributed in Asia and North America. Low GDP-driven class (14 countries) are scattered across continents. Simulating a 5% increase in vaccination rate resulted in CFR reductions of 31.2% and 15.0% for the low vaccination-driven class and the high disease burden-driven class, respectively, with greater CFR reductions for countries with high overall risk (SHAP value > 0.1), but only 3.1% for the ageing-driven class. CONCLUSIONS: Evidence from this study suggests that geographic inequities in COVID-19 CFR is jointly determined by key and concurrent risks, and achieving a decreasing COVID-19 CFR requires more than increasing vaccination coverage, but rather targeted intervention strategies based on country-specific risks.

Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms.

In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies.

Mucins 3A and 3B Are Expressed in the Epithelium of Human Large Airway.

Aberrant mucus secretion is a hallmark of chronic obstructive pulmonary disease (COPD). Expression of the membrane-tethered mucins 3A and 3B (MUC3A, MUC3B) in human lung is largely unknown. In this observational cross-sectional study, we recruited subjects 45-65 years old from the general population of Stockholm, Sweden, during the years 2007-2011. Bronchial mucosal biopsies, bronchial brushings, and bronchoalveolar lavage fluid (BALF) were retrieved from COPD patients (n = 38), healthy never-smokers (n = 40), and smokers with normal lung function (n = 40). Protein expression of MUC3A and MUC3B in bronchial mucosal biopsies was assessed by immunohistochemical staining. In a subgroup of subjects (n = 28), MUC3A and MUC3B mRNAs were quantified in bronchial brushings using microarray. Non-parametric tests were used to perform correlation and group comparison analyses. A value of p < 0.05 was considered statistically significant. MUC3A and MUC3B immunohistochemical expression was localized to ciliated cells. MUC3B was also expressed in basal cells. MUC3A and MUC3B immunohistochemical expression was equal in all study groups but subjects with emphysema had higher MUC3A expression, compared to those without emphysema. Smokers had higher mRNA levels of MUC3A and MUC3B than non-smokers. MUC3A and MUC3B mRNA were higher in male subjects and correlated negatively with expiratory air flows. MUC3B mRNA correlated positively with total cell concentration and macrophage percentage, and negatively with CD4/CD8 T cell ratio in BALF. We concluded that MUC3A and MUC3B in large airways may be a marker of disease or may play a role in the pathophysiology of airway obstruction.

Development of a Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry Platform for the Quantitative Metabolic Profiling of Octadecanoid Oxylipins.

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.

Increased cytotoxic T-cells in the airways of adults with former bronchopulmonary dysplasia.

RATIONALE: Bronchopulmonary dysplasia (BPD) in preterm-born infants is a risk factor for chronic airway obstruction in adulthood. Cytotoxic T-cells are implicated in COPD, but their involvement in BPD is not known. OBJECTIVES: To characterise the distribution of airway T-cell subsets in adults with a history of BPD. METHODS: Young adults with former BPD (n=22; median age 19.6 years), age-matched adults born preterm (n=22), patients with allergic asthma born at term (n=22) and healthy control subjects born at term (n=24) underwent bronchoalveolar lavage (BAL). T-cell subsets in BAL were analysed using flow cytometry. RESULTS: The total number of cells and the differential cell counts in BAL were similar among the study groups. The percentage of CD3+CD8+ T-cells was higher (p=0.005) and the proportion of CD3+CD4+ T-cells was reduced (p=0.01) in the BPD group, resulting in a lower CD4/CD8 ratio (p=0.007) compared to the healthy controls (median 2.2 versus 5.3). In BPD and preterm-born study subjects, both CD3+CD4+ T-cells (rs=0.38, p=0.03) and CD4/CD8 ratio (rs=0.44, p=0.01) correlated positively with forced expiratory volume in 1 s (FEV1). Furthermore, CD3+CD8+ T-cells were negatively correlated with both FEV1 and FEV1/forced vital capacity (rs= -0.44, p=0.09 and rs= -0.41, p=0.01, respectively). CONCLUSIONS: Young adults with former BPD have a T-cell subset pattern in the airways resembling features of COPD. Our findings are compatible with the hypothesis that CD3+CD8+ T-cells are involved in mechanisms behind chronic airway obstruction in these patients.

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

Urinary Leukotriene E4 and Prostaglandin D2 Metabolites Increase in Adult and Childhood Severe Asthma Characterized by Type 2 Inflammation. A Clinical Observational Study.

Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE2 pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE2 metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE4 and the PGD2 metabolite 2,3-dinor-11ß-PGF2α. High concentrations of LTE4 and PGD2 metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).

High-Precision Automated Workflow for Urinary Untargeted Metabolomic Epidemiology.

Urine is a noninvasive biofluid that is rich in polar metabolites and well suited for metabolomic epidemiology. However, because of individual variability in health and hydration status, the physiological concentration of urine can differ >15-fold, which can pose major challenges in untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. Although numerous urine normalization methods have been implemented (e.g., creatinine, specific gravity-SG), most are manual and, therefore, not practical for population-based studies. To address this issue, we developed a method to measure SG in 96-well-plates using a refractive index detector (RID), which exhibited accuracy within 85-115% and <3.4% precision. Bland-Altman statistics showed a mean deviation of -0.0001 SG units (limits of agreement: -0.0014 to 0.0011) relative to a hand-held refractometer. Using this RID-based SG normalization, we developed an automated LC-MS workflow for untargeted urinary metabolomics in a 96-well-plate format. The workflow uses positive and negative ionization HILIC chromatography and acquires mass spectra in data-independent acquisition (DIA) mode at three collision energies. Five technical internal standards (tISs) were used to monitor data quality in each method, all of which demonstrated raw coefficients of variation (CVs) < 10% in the quality controls (QCs) and < 20% in the samples for a small cohort (n = 87 urine samples, n = 22 QCs). Application in a large cohort (n = 842 urine samples, n = 248 QCs) demonstrated CVQC < 5% and CVsamples < 16% for 4/5 tISs after signal drift correction by cubic spline regression. The workflow identified >540 urinary metabolites including endogenous and exogenous compounds. This platform is suitable for performing urinary untargeted metabolomic epidemiology and will be useful for applications in population-based molecular phenotyping.

Distal Lung Microenvironment Triggers Release of Mediators Recognized as Potential Systemic Biomarkers for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model's applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.

Smoking-associated increase in mucins 1 and 4 in human airways.

RATIONALE: Smoking-related chronic obstructive pulmonary disease (COPD) is associated with dysregulated production of mucus. Mucins (MUC) are important both for mucus secretion and epithelial defense. We have examined the distribution of MUC1 and MUC4 in the airway epithelial cells of never-smokers and smokers with and without COPD. METHODS: Mucosal biopsies and bronchial wash samples were obtained by bronchoscopy from age- and sex-matched COPD-patients (n = 38; GOLD I-II/A-B), healthy never-smokers (n = 40) and current smokers with normal lung function (n = 40) from the Karolinska COSMIC cohort (NCT02627872). Cell-specific expressions of MUC1, MUC4 and regulating factors, i.e., epithelial growth factor receptor (EGFR) 1 and 2, were analyzed by immunohistochemistry. Soluble MUC1 was measured by quantitative immunodetection on slot blot. RESULTS: The levels of cell-bound MUC1 expression in basal cells and in soluble MUC1 in bronchial wash were increased in smokers, regardless of airway obstruction. Patients with chronic bronchitis had higher MUC1 expression. The expression of MUC4 in cells with goblet cell phenotype was increased in smokers. The expression of EGFR2, but not that of EGFR1, was higher in never-smokers than in smokers. CONCLUSIONS: Smoking history and the presence of chronic bronchitis, regardless of airway obstruction, affect both cellular and soluble MUC1 in human airways. Therefore, MUC1 may be a novel marker for smoking- associated airway disease.

Pulmonary outcomes in adults with a history of Bronchopulmonary Dysplasia differ from patients with asthma.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a risk factor for respiratory disease in adulthood. Despite the differences in underlying pathology, patients with a history of BPD are often treated as asthmatics. We hypothesized that pulmonary outcomes and health-related quality of life (HRQoL) were different in adults born preterm with and without a history of BPD compared to asthmatics and healthy individuals. METHODS: We evaluated 96 young adults from the LUNAPRE cohort ( clinicaltrials.gov/ct2/show/NCT02923648 ), including 26 individuals born preterm with a history of BPD (BPD), 23 born preterm without BPD (preterm), 23 asthmatics and 24 healthy controls. Extensive lung function testing and HRQoL were assessed. RESULTS: The BPD group had more severe airway obstruction compared to the preterm-, (FEV1- 0.94 vs. 0.28 z-scores; p ≤ 0.001); asthmatic- (0.14 z-scores, p ≤ 0.01) and healthy groups (0.78 z-scores, p ≤ 0.001). Further, they had increased ventilation inhomogeneity compared to the preterm- (LCI 6.97 vs. 6.73, p ≤ 0.05), asthmatic- (6.75, p = 0.05) and healthy groups (6.50 p ≤ 0.001). Both preterm groups had lower DLCO compared to healthy controls (p ≤ 0.001 for both). HRQoL showed less physical but more psychological symptoms in the BPD group compared to asthmatics. CONCLUSIONS: Lung function impairment and HRQoL in adults with a history of BPD differed from that in asthmatics highlighting the need for objective assessment of lung health.

An LC-MS/MS workflow to characterize 16 regio- and stereoisomeric trihydroxyoctadecenoic acids.

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived oxylipins with potential physiological relevance in inflammatory processes as well as in maintaining an intact skin barrier. Due to the high number of possible TriHOME isomers with only subtle differences in their physicochemical properties, the stereochemical analysis is challenging and usually involves a series of laborious analytical procedures. We herein report a straightforward analytical workflow that includes reversed-phase ultra-HPLC-MS/MS for rapid quantification of 9,10,13- and 9,12,13-TriHOME diastereomers and a chiral LC-MS method capable of resolving all sixteen 9,10,13-TriHOME and 9,12,13-TriHOME regio- and stereoisomers. We characterized the workflow (accuracy, 98-120%; precision, coefficient of variation ≤6.1%; limit of detection, 90-98 fg on column; linearity, R2 = 0.998) and used it for stereochemical profiling of TriHOMEs in bronchoalveolar lavage fluid (BALF) of individuals with chronic obstructive pulmonary disease (COPD). All TriHOME isomers were increased in the BALF of COPD patients relative to that of smokers (P ≤ 0.06). In both COPD patients and smokers with normal lung function, TriHOMEs with the 13(S) configuration were enantiomerically enriched relative to the corresponding 13(R) isomers, suggesting at least partial enzymatic control of TriHOME synthesis. This method will be useful for understanding the synthetic sources of these compounds and for elucidating disease mechanisms.

Integration of multi-omics datasets enables molecular classification of COPD.

Chronic obstructive pulmonary disease (COPD) is an umbrella diagnosis caused by a multitude of underlying mechanisms, and molecular sub-phenotyping is needed to develop molecular diagnostic/prognostic tools and efficacious treatments.The objective of these studies was to investigate whether multi-omics integration improves the accuracy of molecular classification of COPD in small cohorts.Nine omics data blocks (comprising mRNA, micro RNA, proteomes and metabolomes) collected from several anatomical locations from 52 female subjects were integrated by similarity network fusion (SNF). Multi-omics integration significantly improved the accuracy of group classification of COPD patients from healthy never-smokers and from smokers with normal spirometry, reducing required group sizes from n=30 to n=6 at 95% power. Seven different combinations of four to seven omics platforms achieved >95% accuracy.For the first time, a quantitative relationship between multi-omics data integration and accuracy of data-driven classification power has been demonstrated across nine omics data blocks. Integrating five to seven omics data blocks enabled 100% correct classification of COPD diagnosis with groups as small as n=6 individuals, despite strong confounding effects of current smoking. These results can serve as guidelines for the design of future systems-based multi-omics investigations, with indications that integrating five to six data blocks from several molecular levels and anatomical locations suffices to facilitate unsupervised molecular classification in small cohorts.

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study.

BACKGROUND: Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren's syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown. METHODS: Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16). RESULTS: A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio. CONCLUSIONS: These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease.

Long-term smoking alters abundance of over half of the proteome in bronchoalveolar lavage cell in smokers with normal spirometry, with effects on molecular pathways associated with COPD.

BACKGROUND: Smoking represents a significant risk factor for many chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). METHODS: To identify dysregulation of specific proteins and pathways in bronchoalveolar lavage (BAL) cells associated with smoking, isobaric tags for relative and absolute quantitation (iTRAQ)-based shotgun proteomics analyses were performed on BAL cells from healthy never-smokers and smokers with normal lung function from the Karolinska COSMIC cohort. Multivariate statistical modeling, multivariate correlations with clinical data, and pathway enrichment analysis were performed. RESULTS: Smoking exerted a significant impact on the BAL cell proteome, with more than 500 proteins representing 15 molecular pathways altered due to smoking. The majority of these alterations occurred in a gender-independent manner. The phagosomal- and leukocyte trans endothelial migration (LTM) pathways significantly correlated with FEV1/FVC as well as the percentage of CD8+ T-cells and CD8+CD69+ T-cells in smokers. The correlations to clinical parameters in healthy never-smokers were minor. CONCLUSION: The significant correlations of proteins in the phagosome- and LTM pathways with activated cytotoxic T-cells (CD69+) and the level of airway obstruction (FEV1/FVC) in smokers, both hallmarks of COPD, suggests that these two pathways may play a role in the molecular events preceding the development of COPD in susceptible smokers. Both pathways were found to be further dysregulated in COPD patients from the same cohort, thereby providing further support to this hypothesis. Given that not all smokers develop COPD in spite of decades of smoking, it is also plausible that some of the molecular pathways associated with response to smoking exert protective mechanisms to smoking-related pathologies in resilient individuals. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02627872 ; Retrospectively registered on December 9, 2015.

Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype.

BACKGROUND: Smoking is the main risk factor for chronic obstructive pulmonary disease (COPD). Women with COPD who smoke experienced a higher risk of hospitalization and worse decline of lung function. Yet the mechanisms of these gender-related differences in clinical presentations in COPD remain unknown. The aim of our study is to identify proteins and molecular pathways associated with COPD pathogenesis, with emphasis on elucidating molecular gender difference. METHOD: We employed shotgun isobaric tags for relative and absolute quantitation (iTRAQ) proteome analyses of bronchoalveolar lavage (BAL) cells from smokers with normal lung function (n = 25) and early stage COPD patients (n = 18). Multivariate modeling, pathway enrichment analysis, and correlation with clinical characteristics were performed to identify specific proteins and pathways of interest. RESULTS: More pronounced alterations both at the protein- and pathway- levels were observed in female COPD patients, involving dysregulation of the FcγR-mediated phagocytosis-lysosomal axis and increase in oxidative stress. Alterations in pathways of the phagocytosis-lysosomal axis associated with a female-dominated COPD phenotype correlated well with specific clinical features: FcγR-mediated phagocytosis correlated with FEV1/FVC, the lysosomal pathway correlated with CT < -950 Hounsfield Units (HU), and regulation of actin cytoskeleton correlated with FEV1 and FEV1/FVC in female COPD patients. Alterations observed in the corresponding male cohort were minor. CONCLUSION: The identified molecular pathways suggest dysregulation of several phagocytosis-related pathways in BAL cells in female COPD patients, with correlation to both the level of obstruction (FEV1/FVC) and disease severity (FEV1) as well as emphysema (CT < -950 HU) in women. TRIAL REGISTRATION: No.: NCT02627872 , retrospectively registered on December 9, 2015.