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Interpreting transcriptome data is an important yet challenging aspect of bioinformatic analysis. While gene set enrichment analysis is a standard tool for interpreting regulatory changes, we utilize deep learning techniques, specifically autoencoder architectures, to learn latent variables that drive transcriptome signals. We investigate whether simple, variational autoencoder (VAE), and beta-weighted VAE are capable of learning reduced representations of transcriptomes that retain critical biological information. We propose a novel VAE which utilizes priors from biological data to direct the network to learn a representation of the transcriptome that is based on understandable biological concepts.After training five different autoencoder architectures on 22310 transcriptomes, we benchmarked their performance on organ and disease classification tasks on separate selection of 5577 test samples. Every tested architecture succeeded in reducing the transcriptomes to 50 latent dimensions, which captured enough variation for accurate reconstruction. The simple, fully connected autoencoder, performs best across the benchmarks, but lacks the characteristic of having directly interpretable latent dimensions. The beta-weighted, prior-informed VAE implementation is able to solve the benchmarking tasks, and provide semantically accurate latent features equating to biological pathways.This study opens a new direction for differential pathway analysis in transcriptomics with increased transparency and interpretability.Author summaryThe ability to measure the human transcriptome has been a critical tool to studying health and disease. However, transcriptomes data sets are too large and complex for direct human interpretation. Deep learning techniques such as autoencoders are capable of distilling high-level features from complex data. However, even if deep learning models find patterns, these patterns are not necessarily represented in a way that humans can easily understand. By bringing in the prior knowledge of biological pathways, we have trained the model to “speak the language” of the biologist, and represent complex transcrtomes, in simpler concepts that are already familiar to biologists. We can then apply the tool to compare for example samples from lung cancer cells to healthy cells, and show which biological processes are perturbed.

Idiopathic pulmonary fibrosis (IPF) is an irreversible, age-related diffuse parenchymal lung disease of poorly defined etiology. Many patients with IPF demonstrate distinctive lymphocytic interstitial infiltrations within remodeled lung tissue with uncertain pathogenetic relevance. Histopathological examination of explant lung tissue of patients with IPF revealed accentuated lymphoplasmacellular accumulations in close vicinity to, or even infiltrating, remodeled lung tissue. Similarly, we found significant accumulations of B cells interfused with T cells within remodeled lung tissue in two murine models of adenoviral TGF-β1 or bleomycin (BLM)-induced lung fibrosis. Such B cell accumulations coincided with significantly increased lung collagen deposition, lung histopathology, and worsened lung function in wild-type (WT) mice. Surprisingly, B cell–deficient µMT knockout mice exhibited similar lung tissue remodeling and worsened lung function upon either AdTGF-β1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-β1– and BLM-exposed WT mice identified a large set of commonly regulated genes, but with significant enrichment observed for Gene Ontology terms apparently not related to lung fibrogenesis. Collectively, although we observed B cell accumulations in lungs of IPF patients as well as two experimental models of lung fibrosis, comparative profiling of characteristic features of lung fibrosis between WT and B cell–deficient mice did not support a major involvement of B cells in lung fibrogenesis in mice.

PDF file, 210K, Forty-five distinct peptides were predicted to bind with IC50< 1000 nM, with 60 peptide-allele combinations.

Purpose: Characterization of an approach to identify leukemia neoantigens arising in the context of drug resistance.Experimental Design: We assessed whether leukemia neoantigens could be generated from drug-resistant mutations in BCR-ABL after imatinib relapse in patients with chronic myelogenous leukemia (CML).Results: We computationally predicted that approximately 70 peptides derived from 26 BCR-ABL mutations would bind eight common alleles of MHC class I (IC50 < 1,000 nmol/L). Seven of nine imatinib-resistant CML patients were predicted to generate at least 1 peptide that binds autologous HLA alleles. We predicted and confirmed that an E255K mutation-derived peptide would bind HLA-A3 with high affinity (IC50 = 28 nmol/L), and showed that this peptide is endogenously processed and presented. Polyfunctional E255K-specific CD8+ T cells were detected in two imatinib-resistant HLA-A3+ CML patients concurrent with an effective anti-CML response to further therapy.Conclusions: Our in vitro studies support the hypothesis that leukemia-driven genetic alterations are targeted by the immune system in association with a clinical response, and suggest the possibility of immunizing relapsed patients with CML against newly acquired tumor neoantigens. Clin Cancer Res; 18(20); 5761–72. ©2012 AACR.

PDF file, 135K, Analysis of potential peptide binders for common mutations related to dasatinib and nilotinib.

Nowadays laparoscopic interventions enable the collection of resident macrophage populations out of the human cavities. We employed this technique to isolate pleural monocytes/macrophages from healthy young adults who underwent a correction of pectus excavatum. High quality CD14

The SERPINA1 gene encodes alpha1-antitrypsin (AAT), an acute phase glycoprotein and serine protease inhibitor that is mainly (80-90%) produced in the liver. Point mutations in the SERPINA1 gene can lead to the misfolding, intracellular accumulation, and deficiency of circulating AAT protein, increasing the risk of developing chronic liver diseases or chronic obstructive pulmonary disease. Currently, siRNA technology can knock down the SERPINA1 gene and limit defective AAT production. How this latter affects other liver genes is unknown. Livers were taken from age- and sex-matched C57BL/6 wild-type (WT) and Serpina1 knockout mice (KO) aged from 8 to 14 weeks, all lacking the five serpin A1a-e paralogues. Total RNA was isolated and RNA sequencing, and transcriptome analysis was performed. The knockout of the Serpina1 gene in mice changed inflammatory, lipid metabolism, and cholesterol metabolism-related gene expression in the liver. Independent single-cell sequencing data of WT mice verified the involvement of Serpina1 in cholesterol metabolism. Our results from mice livers suggested that designing therapeutic strategies for the knockout of the SERPINA1 gene in humans must account for potential perturbations of key metabolic pathways and consequent mitigation of side effects. RNA sequencing was supported by the grant ISCIII-AESI PI20CIII/00015. Sí

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To explore the relationship between cancer cell SREBF1 expression, lipid droplets (LDs) formation, and the sensitivity to chemotherapies, we cultured lung adenocarcinoma cells H1299 (with LD) and H1563 (without LD) in a serum-free basal medium (BM) or neutrophil degranulation products containing medium (NDM), and tested cell responses to cisplatin and etoposide. By using the DESeq2 Bioconductor package, we detected 674 differentially expressed genes (DEGs) associated with NDM/BM differences between two cell lines, many of these genes were associated with the regulation of sterol and cholesterol biosynthesis processes. Specifically

The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN. The transfection of TNBC cell lines HCC38, HCC1937 and HCC1395 with microRNA-449a mimics led to induced apoptosis, reduced cell proliferation, and reduced expression of genes in homology directed repair (HDR) in microarray analyses. EME1 was identified as a new target gene by immunoprecipitation and luciferase assays. The reduced expression of EME1 led to an increased frequency of ultrafine bridges, 53BP1 foci, and micronuclei. The induced expression of microRNA-449a elevated CIN beyond tolerable levels and induced apoptosis in TNBC cell lines by two different mechanisms: (I) promoting chromatid mis-segregation by targeting endonuclease EME1 and (II) inhibiting HDR by downregulating key players of the HDR network such as E2F3, BIRC5, BRCA2 and RAD51. The ectopic expression of microRNA-449a enhanced the toxic effect of PARPi in cells with pathogenic germline BRCA1 variants. The newly identified role makes microRNA-449a an interesting therapeutic target for TNBC.

RationaleIn adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children.ObjectivesTo define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages.MethodsIn the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28.Measurements and main resultsBased on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: “atopy-only”, “eosinophils-only”, “T2-high” (eosinophilia + atopy) and “T2-low” (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (pConclusionsUsing easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Background Despite the significant contribution of transcriptomics to the fields of biological and biomedical research, interpreting long lists of significantly differentially expressed genes remains a challenging step in the analysis process. Gene set enrichment analysis is a standard approach for summarizing differentially expressed genes into pathways or other gene groupings. Here, we explore an alternative approach to utilizing gene sets from curated databases. We examine the method of deriving custom gene sets which may be relevant to a given experiment using reference data sets from previous transcriptomics studies. We call these data-derived gene sets, “gene signatures” for the biological process tested in the previous study. We focus on the feasibility of this approach in analyzing immune-related processes, which are complicated in their nature but play an important role in the medical research. Results We evaluate several statistical approaches to detecting the activity of a gene signature in a target data set. We compare the performance of the data-derived gene signature approach with comparable GO term gene sets across all of the statistical tests. A total of 61 differential expression comparisons generated from 26 transcriptome experiments were included in the analysis. These experiments covered eight immunological processes in eight types of leukocytes. The data-derived signatures were used to detect the presence of immunological processes in the test data with modest accuracy (AUC = 0.67). The performance for GO and literature based gene sets was worse (AUC = 0.59). Both approaches were plagued by poor specificity. Conclusions When investigators seek to test specific hypotheses, the data-derived signature approach can perform as well, if not better than standard gene-set based approaches for immunological signatures. Furthermore, the data-derived signatures can be generated in the cases that well-defined gene sets are lacking from pathway databases and also offer the opportunity for defining signatures in a cell-type specific manner. However, neither the data-derived signatures nor standard gene-sets can be demonstrated to reliably provide negative predictions for negative cases. We conclude that the data-derived signature approach is a useful and sometimes necessary tool, but analysts should be weary of false positives.

Purpose There is an ongoing demand for easily accessible biomarkers that reflect the physiological and pathophysiological mechanisms of COPD. To test if an exercise challenge could help to identify clinically relevant metabolic biomarkers in COPD. Patients and methods We performed two constant-load exercise challenges separated by 4 weeks including smokers with COPD (n=23/19) and sex- and age-matched healthy smokers (n=23/20). Two hours after a standardized meal venous blood samples were obtained before, 5 mins after the start, at the end of submaximal exercise, and following a recovery of 20 mins. Data analysis was performed using mixed- effects model, with the metabolite level as a function of disease, time point and interaction terms and using each individual's resting level as reference. Results Exercise duration was longer in healthy smokers but lactate levels were comparable between groups at all four time points. Glucose levels were increased in COPD. Glutamine was lower, while glutamate and arginine were higher in COPD. Branched-chain amino acids showed a stronger decline during exercise in healthy smokers. Carnitine and the acyl-carnitines C16 and C18:1 were increased in COPD. These metabolite levels and changes were reproducible in the second challenge. Conclusion Higher serum glucose, evidence for impaired utilization of amino acids during exercise and a shift of energy metabolism to enhanced consumption of lipids could be early signs for a developing metabolic syndrome in COPD. In COPD patients, deviations of energy and nitrogen metabolism are amplified by an exercise challenge.

Background There is an association between persistent preschool wheezing phenotypes and school-age asthma. These wheezing/asthma phenotypes likely represent clinical entities having specific genetic risk factors. The SERPINA1 gene encodes α 1 -antitrypsin (AAT), and mutations in the gene are important in the pathophysiology of pulmonary diseases. We hypothesized that there might be an association between SERPINA1 gene polymorphisms and the risk of developing wheezing/school age asthma. Objective To examine 10 single nucleotide polymorphisms (SNPs) of SERPINA1 (rs6647, rs11832, rs17580, rs709932, rs1243160, rs2854254, rs8004738, rs17751769, rs28929470, and rs28929474) and relate them to childhood wheezing phenotypes and doctor-diagnosed asthma in the population-based Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Methods Wheeze data, reports of physician-diagnosed asthma and data on the SERPINA1 gene SNPs, were available for 7964 children. Binary logistic regression was used to assess the associations between allele prevalence and wheezing and asthma phenotypes. P values were adjusted to account for multiple hypotheses using the Benjamini-Hochberg false discovery rate. Results Only within a subgroup of children with asthma who had no prior diagnosis of preschool wheeze was there a trend for association between rs28929474 (Glu342Lys, Pi*Z causing AAT deficiency; P = .0058, adjusted P = .058). No SNP was associated with wheezing and asthma in those with preschool wheeze. Conclusion Analyzed SNPs in SERPINA1 are not associated with wheezing/asthma phenotypes. Only rs28929474, the most common pathologic SNP (Pi*Z) in the SERPINA1 gene, might be associated with a risk of developing school-age asthma without exhibiting preschool wheeze.

RationaleDendritic cells (DC) accumulate in the lungs of patients with idiopathic lung fibrosis, but their pathogenetic relevance is poorly defined.ObjectivesTo assess the role of the FMS-like tyrosine kinase-3 ligand (Flt3L)-lung dendritic cell axis in lung fibrosis.Measurements and main resultsWe demonstrate in a model of adenoviral gene transfer of active TGF-β1 that established lung fibrosis was accompanied by elevated serum Flt3L levels and subsequent accumulation of CD11bpos DC in the lungs of mice. Patients with idiopathic pulmonary fibrosis also demonstrated increased levels of Flt3L protein in serum and lung tissue and accumulation of lung DC in explant subpleural lung tissue specimen. Mice lacking Flt3L showed significantly reduced lung DC along with worsened lung fibrosis and reduced lung function relative to wild-type (WT) mice, which could be inhibited by administration of recombinant Flt3L. Moreover, therapeutic Flt3L increased numbers of CD11bpos DC and improved lung fibrosis in WT mice exposed to AdTGF-β1. In this line, RNA-sequencing analysis of CD11bpos DC revealed significantly enriched differentially expressed genes within extracellular matrix degrading enzyme and matrix metalloprotease gene clusters. In contrast, the CD103pos DC subset did not appear to be involved in pulmonary fibrogenesis.ConclusionsWe show that Flt3L protein and numbers of lung DC are upregulated in mice and humans during pulmonary fibrogenesis, and increased mobilisation of lung CD11bpos DC limits the severity of lung fibrosis in mice. The current study helps to inform the development of DC-based immunotherapy as a novel intervention against lung fibrosis in humans.

Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host’s misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation., eLife digest Proteins can lose their structure and form polymers because of mutations or changes in their immediate environment which can lead to cell damage and disease. Interestingly, polymers formed by a variety of proteins can reduce the levels of CX3C chemokine receptor 1 (CX3CR1 for short) that controls the behaviour of immune cells and is implicated in a range of illnesses. Inherited ZZ alpha-1 antitrypsin deficiency is a rare genetic condition that highly increases the risk of liver and lung diseases. This disorder is characterised by mutant alpha-1 antitrypsin proteins (AAT for short) reacting together to form polymers; yet it remains unclear how the polymers affect different cells or organs, and lead to diseases. To investigate this question, Tumpara et al. examined whether polymers of mutant AAT influence the level of the CX3CR1 protein in specific classes of immune cells. Experiments revealed that in people with AAT deficiency, certain blood immune cells express lower levels of CX3CR1. Regardless of age, clinical diagnosis, or treatment regimen, all individuals with ZZ alpha-1 antitrypsin deficiency had AAT polymers circulating in their blood: the higher the levels of polymers measured, the lower the expression of CX3CR1 recorded in the specific immune cells. When Tumpara et al. added polymers of mutant AAT to the immune cells of healthy donors, the expression of CX3CR1 dropped in a manner dependent on the polymer concentration. According to microscopy data, AAT polymers occurred inside cells alongside the CX3CR1 protein, suggesting that the two molecular actors interact. In the future, new drugs that remove these polymers, either from inside cells or as they circulate in the body, could help patients suffering from conditions associated with this abnormal protein aggregation.

The CX3CR1 (chemokine (C-X3-C motif) receptor 1) expression levels on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host‘s misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, we present a novel example that polymers of human ZZ alpha1-antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 expression in human PBMCs. We also show that extracellular polymers of Z-AAT are internalized by PBMCs, which parallels with increased intracellular levels of CX3CR1 protein. Our findings support the role of extracellular misfolded proteins in CX3CR1 regulation and encourage conducting further studies on this issue.

Bronchoscopy is an established procedure routinely used by pediatric pulmonologists. Despite its frequent application, data on complications and specific risk factors are scarce and sometimes conflicting. AIM The aim of this study was to evaluate frequency and severity of clearly defined complications of bronchoscopy in children that occur both during and after the procedure, and to identify potential risk factors. METHOD A retrospective single-center analysis of 670 elective bronchoscopies in 522 children aged 0-17 years during the time period of 2008-2012 was performed. Procedures in intensive care unit patients and children after lung transplantation were excluded. RESULTS Mean patient age was 5.58 years, 61.5% had underlying chronic diseases. Intraprocedural complications occurred in 7.2% of all procedures; of these, hypoxemia was the most common, occuring in 4.8% of cases. Postprocedural adverse events were documented in 25.8%, the most frequent of which were fever in 14.2% and transient oxygen dependency in 13.4% of cases. No bronchoscopy related deaths occurred. Multivariate logistic regression was used to identify risk factors for (1) any complication, or (2) severe complications. Age below two years (OR 1.837 [1.224-2.757], P = 0.003) and primary ciliary dyskinesia (OR 4.821 [2.018-11.552], P

Background: Increased Interleukin 17 (IL-17) levels are observed in inflammatory diseases such as allergic asthma, psoriasis, and rheumatoid arthritis. Nevertheless, with the exception of its role in attracting neutrophils to inflammatory sites, how IL-17 influences other leukocytes is yet to be fully elucidated. Objective: Our aim in this study is to understanding how IL-17 influences transcriptomic patterns of conventional pulmonary dendritic cells both in steady and antigen exposed conditions Methods: We made a comprehensive comparative transcriptomic analysis of lungs and lung draining lymph nodes derived conventional dendritic cells from wild type as well as IL-17A/F double knockout mice in an experimental model of allergic asthma. Results: We used previously described gene sets in order to carry out a comprehensive comparative analysis between IL-17 sufficient versus IL-17 deficient cDCs transcriptomes. We hereby show that, absence of IL-17 affects various transcriptional programs associated with the ability of DCs to migrate, present antigen as well as co-stimulation of effector cells. We have used bioinformatics tools in order to illustrate affected migration, antigen presentation and co-stimulation pathways. Conclusions: Our transcriptomic analysis shows that IL-17 enhances migratory as well as antigen presenting potential of cDCs. We thus conclude that functional potential of DC could be impaired through targeting IL-17 in order to control inflammation.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease with different courses. Median survival without treatment remains poor at 3 years, with acute exacerbation (AE) being a leading cause of mortality. On a histopathological level acute exacerbations shares many features of ARDS, but only very few studies investigated underlying molecular mechanisms. Objective: in order to identify key pathomechanisms of acute exacerbation of IPF we tested the transcriptome of BAL cells. Methods: The transcriptome of BAL cells from 9 IPF patients with AE and 59 patients without exacerbation was analysed using microarrays on the Agilent platform. Statistical analyses were performed on the R platform using different bioinformatics tools. We identified differentially expressed genes (DEGs) and underlying pathways in a comparative analysis. We also tested for enrichment of the identified DEGs in the BAL transcriptome signature predictive of mortality in IPF which we recently published. Results: We found 527 genes differentially expressed (DEGs, FDR Conclusions: Our data indicate that AE of IPF is not a completly different disease state rather most of the inflammatory and developmental pathways upregulated at AE are already present at baseline IPF.

Purpose There is an ongoing demand for easily accessible biomarkers that reflect the physiological and pathophysiological mechanisms of COPD. To test if an exercise challenge could help to identify clinically relevant metabolic biomarkers in COPD. Patients and Methods We performed two constant-load exercise challenges separated by 4 weeks including smokers with COPD (n=23/19) and sex- and age-matched healthy smokers (n=23/20). Two hours after a standardized meal venous blood samples were obtained before, 5 mins after the start, at the end of submaximal exercise, and following a recovery of 20 mins. Data analysis was performed using mixed- effects model, with the metabolite level as a function of disease, time point and interaction terms and using each individual's resting level as reference. Results Exercise duration was longer in healthy smokers but lactate levels were comparable between groups at all four time points. Glucose levels were increased in COPD. Glutamine was lower, while glutamate and arginine were higher in COPD. Branched-chain amino acids showed a stronger decline during exercise in healthy smokers. Carnitine and the acyl-carnitines C16 and C18:1 were increased in COPD. These metabolite levels and changes were reproducible in the second challenge. Conclusion Higher serum glucose, evidence for impaired utilization of amino acids during exercise and a shift of energy metabolism to enhanced consumption of lipids could be early signs for a developing metabolic syndrome in COPD. In COPD patients, deviations of energy and nitrogen metabolism are amplified by an exercise challenge.

Low plasma level of alpha1-antitrypsin (AAT) is an established risk factor for early-onset chronic obstructive lung disease (COPD). However, less attention is given to the levels of AAT in the general population.This is a part of a multicentre, population-based study conducted at 11 sites throughout Spain. Plasma levels of AAT were available for 837 persons with a mean (SD) age of 58.05 (11.3) years: 328-smokers, 272-ex-smokers and 237 non-smokers. Out of 837, 303 (36.2%) had a diagnosis of COPD, 222 (26.5%) had respiratory symptoms but no COPD, and 312 (37.3%) were healthy controls.In the whole cohort, the mean level of plasma AAT was 1.51 (0.47)g/L. Levels were higher in COPD patients [1.55 (0.45)g/L] and individuals with respiratory symptoms [1.57 (0.47)g/L] than in controls [1.43 (0.47)g/L], p0.001, a finding which persisted after correction for age and CRP. Plasma AAT levels were negatively associated with FEV1/FVC ratio, after adjustment for age, sex, smoking status, CRP, TNFα, fibrinogen and albumin. The risk for COPD was significantly associated with higher AAT levels in univariate and multivariate models, with odds ratios of 1.8 and 1.5, respectively. In the univariate and multivariate models smoking status, gender, and CRP levels were also associated with COPD probability, demonstrating that they act independently.Increased circulating levels of AAT, similarly to CRP and other markers of systemic inflammation, is an important feature of COPD. Our results highlight a complex interrelationship between levels of AAT and health of respiratory system.

The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community. This work was supported by the National Institutes of Health (HHSN261200800001E (Leidos Prime contract with NCI); 10XS170 (NDRI), 10XS171 (Roswell Park Cancer Institute), 10X172 (Science Care Inc.), 12ST1039 (IDOX); 10ST1035 (Van Andel Institute); HHSN268201000029C (Broad Institute); and R01 DA006227-17 (U Miami Brain Bank).

Introduction: Idiopathic pulmonary fibrosis is characterized by a vast remodeling of the alveolar compartment. Recent data indicate a role for airway basal cells in IPF. Methods: Bronchial epithelial cells of 30 patients with IPF and 18 age-matched healthy volunteers were harvested by bronchial brushes during routine diagnostic bronchoscopy. Airway basal cells were isolated in accordance to recently published protocols. Gene expression was analyzed by RNAseq using HiSeq/ Illumina platform. Cell function was tested by in vitro and in vivo assays. Results: RNAseq data revealed an abundant reprogramming of airway basal cells in IPF compared to healthy cells. Similarities between IPF and cancer as well as organ development in regards to underlying mechanisms/ pathways became evident by Ingenuity analyses. Involved upstream regulators and signaling pathways were identified. The pivotal role of these pathways in airway basal cell function was then tested in a 3D organoid model and a humanized mouse model. Airway basal cell proliferation and bronchosphere generation were inhibited by siRNA and small molecules blocking the involved pathways. Moreover, in a humanized mouse model using human IPF airway basal cells the blockade of these pathways resulted in less airway basal cell engraftment and reduced pulmonary fibrosis. Conclusion: airway basal cells derived from distal airways are reprogrammed in IPF which results in malfunction and mislead repair processes.

The transcription factor SOX2 is an essential regulator of pluripotent stem cells and promotes development and maintenance of squamous epithelia. We previously reported that SOX2 is an oncogene and subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs). Here, we have further characterized the function of SOX2 in SCC. Using ChIP-seq analysis, we compared SOX2-regulated gene profiles in multiple SCC cell lines to ES cell profiles and determined that SOX2 binds to distinct genomic loci in SCCs. In SCCs, SOX2 preferentially interacts with the transcription factor p63, as opposed to the transcription factor OCT4, which is the preferred SOX2 binding partner in ES cells. SOX2 and p63 exhibited overlapping genomic occupancy at a large number of loci in SCCs; however, coordinate binding of SOX2 and p63 was absent in ES cells. We further demonstrated that SOX2 and p63 jointly regulate gene expression, including the oncogene ETV4, which was essential for SOX2-amplified SCC cell survival. Together, these findings demonstrate that the action of SOX2 in SCC differs substantially from its role in pluripotency. The identification of the SCC-associated interaction between SOX2 and p63 will enable deeper characterization the downstream targets of this interaction in SCC and normal squamous epithelial physiology.

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch-signaling—mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively. SF3B1 mutation appears to be a mechanism by which changes in diverse cancer-related pathways are generated.

Purpose: Characterization of an approach to identify leukemia neoantigens arising in the context of drug resistance. Experimental Design: We assessed whether leukemia neoantigens could be generated from drug-resistant mutations in BCR-ABL after imatinib relapse in patients with chronic myelogenous leukemia (CML). Results: We computationally predicted that approximately 70 peptides derived from 26 BCR-ABL mutations would bind eight common alleles of MHC class I (IC50 < 1,000 nmol/L). Seven of nine imatinib-resistant CML patients were predicted to generate at least 1 peptide that binds autologous HLA alleles. We predicted and confirmed that an E255K mutation-derived peptide would bind HLA-A3 with high affinity (IC50 = 28 nmol/L), and showed that this peptide is endogenously processed and presented. Polyfunctional E255K-specific CD8+ T cells were detected in two imatinib-resistant HLA-A3+ CML patients concurrent with an effective anti-CML response to further therapy. Conclusions: Our in vitro studies support the hypothesis that leukemia-driven genetic alterations are targeted by the immune system in association with a clinical response, and suggest the possibility of immunizing relapsed patients with CML against newly acquired tumor neoantigens. Clin Cancer Res; 18(20); 5761–72. ©2012 AACR.

MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes. NetMHCpan and NetMHCIIpan are used as prediction engines. The 13 HLA Class I supertypes are A1, A2, A3, A24, B7, B8, B27, B44, B58, B62, C1, and C4. The 13 HLA Class II DR supertypes are DR1, DR3, DR4, DR6, DR7, DR8, DR9, DR11, DR12, DR13, DR14, DR15, and DR16. In total, MULTIPRED2 enables prediction of peptide binding to 1077 variants representing 26 HLA supertypes. MULTIPRED2 has visualization modules for mapping promiscuous T-cell epitopes as well as those regions of high target concentration – referred to as T-cell epitope hotspots. Novel graphic representations are employed to display the predicted binding peptides and immunological hotspots in an intuitive manner and also to provide a global view of results as heat maps. Another function of MULTIPRED2, which has direct relevance to vaccine design, is the calculation of population coverage. Currently it calculates population coverage in five major groups in North America. MULTIPRED2 is an important tool to complement wet-lab experimental methods for identification of T-cell epitopes. It is available at http://cvc.dfci.harvard.edu/multipred2/.

We describe a novel approach to genetic association analyses with proteins sub-divided into biologically relevant smaller sequence features (SFs), and their variant types (VTs). SFVT analyses are particularly informative for study of highly polymorphic proteins such as the human leukocyte antigen (HLA), given the nature of its genetic variation: the high level of polymorphism, the pattern of amino acid variability, and that most HLA variation occurs at functionally important sites, as well as its known role in organ transplant rejection, autoimmune disease development and response to infection. Further, combinations of variable amino acid sites shared by several HLA alleles (shared epitopes) are most likely better descriptors of the actual causative genetic variants. In a cohort of systemic sclerosis patients/controls, SFVT analysis shows that a combination of SFs implicating specific amino acid residues in peptide binding pockets 4 and 7 of HLA-DRB1 explains much of the molecular determinant of risk.

There have been several attempts over the years to identify positions in the peptide-binding region (PBR) of human leukocyte antigens (HLA) that influence the specificity of bound amino acids (AAs) at each position in the peptide. Originally, six pockets (A-F) were defined by calculating the surface area of the PBR on the crystal structure of HLA-A2 molecules. More recent crystallographic analyses of a variety of HLA alleles have led to broader pocket definitions. In this study, we examined the peptide-binding specificity of HLA-B*41 alleles and compared our results with the available pocket definitions. By generating recombinant HLA-B molecules and studying the eluted peptides by mass spectrometry and pool sequencing, we detected two different POmega peptide motifs within the B*41 group: Leu vs Val/Pro. Specificity was dependent on the presence of Leu (B*4102, B*4103, and B*4104) vs Trp (B*4101, B*4105, and B*4106) at AA position 95 in the HLA molecule, whose impact on POmega has been a subject of controversy in current pocket definitions. In contrast, the Arg97Ser mutation did not affect pocket F binding specificity in B*41 subtypes although residue 97 was previously identified as a modulator of peptide binding for several HLA class I alleles. According to most pocket definitions, this study shows that the Asn80Lys substitution in B*4105 impels the peptide's POmega anchor toward more promiscuity. Our sequencing results of peptides eluted from HLA-B*41 variants demonstrate the limitations of current pocket definitions and underline the need for an extended peptide motif database for improved understanding of peptide-major histocompatibility complex interactions.

Expression, genetic variation, and tissues Human genomes show extensive genetic variation across individuals, but we have only just started documenting the effects of this variation on the regulation of gene expression. Furthermore, only a few tissues have been examined per genetic variant. In order to examine how genetic expression varies among tissues within individuals, the Genotype-Tissue Expression (GTEx) Consortium collected 1641 postmortem samples covering 54 body sites from 175 individuals. They identified quantitative genetic traits that affect gene expression and determined which of these exhibit tissue-specific expression patterns. Melé et al. measured how transcription varies among tissues, and Rivas et al. looked at how truncated protein variants affect expression across tissues. Science , this issue p. 648 , p. 660 , p. 666 ; see also p. 640