Human Bronchial Epithelial Cell Transcriptome Changes in Response to Serum from Patients with Different Status of Inflammation.

PURPOSE: To investigate the transcriptome of human bronchial epithelial cells (HBEC) in response to serum from patients with different degrees of inflammation. METHODS: Serum from 19 COVID-19 patients obtained from the Hannover Unified Biobank was used. At the time of sampling, 5 patients had a WHO Clinical Progression Scale (WHO-CPS) score of 9 (severe illness). The remaining 14 patients had a WHO-CPS of below 9 (range 1-7), and lower illness. Multiplex immunoassay was used to assess serum inflammatory markers. The culture medium of HBEC was supplemented with 2% of the patient's serum, and the cells were cultured at 37 °C, 5% CO2 for 18 h. Subsequently, cellular RNA was used for RNA-Seq. RESULTS: Patients with scores below 9 had significantly lower albumin and serum levels of E-selectin, IL-8, and MCP-1 than patients with scores of 9. Principal component analysis based on 500 "core genes" of RNA-seq segregated cells into two subsets: exposed to serum from 4 (I) and 15 (II) patients. Cells from a subset (I) treated with serum from 4 patients with a score of 9 showed 5566 differentially expressed genes of which 2793 were up- and 2773 downregulated in comparison with cells of subset II treated with serum from 14 patients with scores between 1 and 7 and one with score = 9. In subset I cells, a higher expression of TLR4 and CXCL8 but a lower CDH1, ACE2, and HMOX1, and greater effects on genes involved in metabolic regulation, cytoskeletal organization, and kinase activity pathways were observed. CONCLUSION: This simple model could be useful to characterize patient serum and epithelial cell properties.

An association between plasma levels of α2-macroglobulin and α1-antitrypsin in PiMM and PiZZ individuals differing in COPD presentation.

INTRODUCTION: Compared to normal PiMM, individuals with severe α1-antitrypsin (AAT) PiZZ (Glu342Lys) genotype deficiency are at higher risk of developing early-onset chronic obstructive pulmonary disease (COPD)/emphysema associated with Z-AAT polymers and neutrophilic inflammation. We aimed to investigate putative differences in plasma levels of acute phase proteins (APP) between PiMM and PiZZ subjects and to determine plasma Z-AAT polymer levels in PiZZ subjects. MATERIALS AND METHODS: Nephelometric analysis of seven plasma APPs was performed in 67 PiMM and 44 PiZZ subjects, of whom 43 and 42, respectively, had stable COPD. Of the PiZZ-COPD patients, 21 received and 23 did not receive intravenous therapy with human AAT preparations (IV-AAT). Plasma levels of Z-AAT polymers were determined by Western blotting using specific mouse monoclonal antibodies (2C1 and LG96). RESULTS: In addition to lower plasma AAT, PiZZ patients had higher α2-macroglobulin (A2MG) levels than PiMM patients. In contrast, PiZZ who received IV-AAT had higher AAT values but lower A2MG values than PiZZ without IV-AAT. Regardless of the AAT genotype, AAT levels were inversely correlated with A2MG, and the AAT/A2MG ratio was correlated with lung diffusion capacity (DCLO%). All PiZZ patients had circulating Z-AAT polymer levels that correlated directly with A2MG. In PiZZ without IV-AAT therapy polymer levels correlated inversely with the ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC). CONCLUSION: Combined measurement of plasma AAT and A2MG levels may be of clinical value in assessing the progression of COPD and requires further attention.

Pleural CD14+ monocytes/macrophages of healthy adolescents show a high expression of metallothionein family genes.

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+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized (MDM-IL4). Transcriptome analysis revealed 7166 and 7076 differentially expressed genes (DEGs) in plMo/Mφ relative to natural MDM-0 and polarized MDM-IL4, respectively. The gene set enrichment analysis, which was used to compare RNA-seq data from plMo/Mφ with single-cell (scRNA-seq) data online from human bronchial lavage macrophages, showed that plMo/Mφs are characterized by a high expression of genes belonging to the metallothionein (MT) family, and that the expression of these genes is significantly higher in plMo/Mφ than in MDM-0 or MDM-IL4. Our results provide additional insights on high MTs-expressing macrophage subsets, which seem to be present not only in bronchial lavage of healthy adults or in pleural exudates of lung cancer patients but also in pleural fluid of healthy young adults. Macrophage subsets expressing high MTs may have specific roles in lung defense, repair, and homeostasis, and require further investigations.

Lung Adenocarcinoma Cell Sensitivity to Chemotherapies: A Spotlight on Lipid Droplets and SREBF1 Gene.

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, SREBF1 markedly declined in both cell lines cultured in NDM or when treated with chemotherapeutics. Despite the latter, H1563 exhibited LD formation and resistance to etoposide, but not to cisplatin. Although H1299 cells preserved LDs, these cells were similarly sensitive to both drugs. In a cohort of 292 patients with non-small-cell lung cancer, a lower SREBF1 expression in tumors than in adjacent nontumor tissue correlated with overall better survival, specifically in patients with adenocarcinoma at stage I. Our findings imply that a direct correlation between SREBF1 and LD accumulation can be lost due to the changes in cancer cell environment and/or chemotherapy. The role of LDs in lung cancer development and response to therapies remains to be examined in more detail.

Loss of Serpina1 in Mice Leads to Altered Gene Expression in Inflammatory and Metabolic Pathways.

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.

Single cell versus single nucleus: transcriptome differences in the murine kidney after ischemia-reperfusion injury.

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.NEW & NOTEWORTHY Single-cell and single-nucleus RNA sequencing technologies provide powerful new tools to examine complex tissues such as the kidney. This research reference paper provides practical information on the differences between the two technologies when examining murine kidneys after ischemia-reperfusion injury. The results will serve those who are debating which protocols to use in their given study.

MicroRNA-449a Inhibits Triple Negative Breast Cancer by Disturbing DNA Repair and Chromatid Separation.

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.

B Cells Are Not Involved in the Regulation of Adenoviral TGF-ß1- or Bleomycin-Induced Lung Fibrosis in Mice.

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-ß1 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-ß1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-ß1- 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.

The Delivery of α1-Antitrypsin Therapy Through Transepidermal Route: Worthwhile to Explore.

Human α1-antitrypsin (AAT) is an abundant acute phase glycoprotein expressing anti-protease and immunomodulatory activities, and is used as a biopharmaceutical to treat patients with inherited AAT deficiency. The pleiotropic properties of AAT provide a rationale for using this therapy outside of inherited AAT deficiency. Therapy with AAT is administrated intravenously, yet the alternative routes are being considered. To examine the putative transepidermal application of AAT we used epiCS®, the 3D human epidermis equivalents reconstructed from human primary epidermal keratinocytes. We topically applied various concentrations of AAT protein with a constant volume of 50 µl, prepared in Hank's balance solution, HBSS, to epiCS cultured under bas\al condition or when culture medium supplemented with 100 µg/ml of a combined bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) mixture. AAT freely diffused across epidermis layers in a concentration and time-dependent manner. Within 18 h topically provided 0.2 mg AAT penetrated well the stratum corneum and localizes within the keratinocytes. The treatments with AAT did not induce obvious morphological changes and damages in keratinocyte layers. As expected, LPS/PGN triggered a strong pro-inflammatory activation of epiCS. AAT exhibited a limited capacity to neutralize the effect of LPS/PGN, but more importantly, it lowered expression of IL-18 and IL-8, and preserved levels of filaggrin, a key protein for maintaining the epidermal barrier integrity. Our findings suggest that the transepidermal route for delivering AAT is worthwhile to explore further. If successful, this approach may offer an easy-to-use therapy with AAT for skin inflammatory diseases.

IL-17 regulates DC migration to the peribronchial LNs and allergen presentation in experimental allergic asthma.

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.

Smokers with COPD Show a Shift in Energy and Nitrogen Metabolism at Rest and During Exercise.

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.

Serum Levels of Alpha1-antitrypsin and Their Relationship With COPD in the General Spanish Population / Niveles séricos de alfa-1 antitripsina y su relación con la EPOC en la población española

BACKGROUND: 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. METHODS: 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. RESULTS: 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], p < 0.001, 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. CONCLUSION: 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

INTRODUCCIÓN: Los niveles plasmáticos bajos de alfa-1 antitripsina (AAT) constituyen un factor de riesgo para el desarrollo temprano de la enfermedad pulmonar obstructiva crónica (EPOC). Sin embargo, se ha prestado una menor atención a los niveles de AAT en la población general. MÉTODOS: Este trabajo forma parte de un estudio poblacional multicéntrico llevado a cabo en 11 centros españoles. Se incluyeron niveles de AAT de 837 personas con una edad media (DE) de 58,05 (11,3) años: 328 fumadores, 272 exfumadores y 237 no fumadores. De los 837, a 303 (36,2%) se les diagnosticó EPOC, 222 (36,5%) presentaron síntomas respiratorios pero no EPOC y 312 (37,3%) eran controles sanos. RESULTADOS: En la cohorte total, los niveles plasmáticos medios de AAT fueron 1,51 (0,47) g/l. Los pacientes con EPOC y los individuos con síntomas respiratorios presentaron niveles más elevados (1.55 [0.45] g/l y 1.57 [0.47] g/l respectivamente) que los sujetos control (1.43 [0.47] g/l, p < 0.001). Este resultado se mantuvo tras la corrección por edad y niveles de proteína C reactiva (PCR). Los niveles plasmáticos de AAT se asociaron negativamente con la relación FEV1/FVC tras el ajuste por edad, sexo, hábito tabáquico, niveles de PCR, TNFα, fibrinógeno y albúmina. El riesgo de EPOC se asoció significativamente con niveles más elevados de AAT tanto en el modelo univariante como el multivariante, con odds ratios de 1,8 y 1.5 respectivamente. En los modelos univariantes y multivariantes, el hábito tabáquico, el sexo, y los niveles de PCR también se asociaron con la probabilidad de sufrir EPOC, lo cual demostró que se trataba de variables independientes. CONCLUSIÓN: Un aumento de los niveles circulantes de AAT, al igual que los niveles de PCR y otros marcadores de inflamación sistémica, son características importantes de la EPOC. Nuestros resultados evidencian la compleja interrelación existente entre los niveles de AAT y la salud del aparato respiratorio

A comparison of curated gene sets versus transcriptomics-derived gene signatures for detecting pathway activation in immune cells.

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.

Serum Levels of Alpha1-antitrypsin and Their Relationship With COPD in the General Spanish Population.

BACKGROUND: 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. METHODS: 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. RESULTS: 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], p<0.001, 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. CONCLUSION: 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.

Molecular profiling of rheumatoid arthritis patients reveals an association between innate and adaptive cell populations and response to anti-tumor necrosis factor.

BACKGROUND: The goal of this study is to use comprehensive molecular profiling to characterize clinical response to anti-TNF therapy in a real-world setting and identify reproducible markers differentiating good responders and non-responders in rheumatoid arthritis (RA). METHODS: Whole-blood mRNA, plasma proteins, and glycopeptides were measured in two cohorts of biologic-naïve RA patients (n = 40 and n = 36) from the Corrona CERTAIN (Comparative Effectiveness Registry to study Therapies for Arthritis and Inflammatory coNditions) registry at baseline and after 3 months of anti-TNF treatment. Response to treatment was categorized by EULAR criteria. A cell type-specific data analysis was conducted to evaluate the involvement of the most common immune cell sub-populations. Findings concordant between the two cohorts were further assessed for reproducibility using selected NCBI-GEO datasets and clinical laboratory measurements available in the CERTAIN database. RESULTS: A treatment-related signature suggesting a reduction in neutrophils, independent of the status of response, was indicated by a high level of correlation (ρ = 0.62; p < 0.01) between the two cohorts. A baseline, response signature of increased innate cell types in responders compared to increased adaptive cell types in non-responders was identified in both cohorts. This result was further assessed by applying the cell type-specific analysis to five other publicly available RA datasets. Evaluation of the neutrophil-to-lymphocyte ratio at baseline in the remaining patients (n = 1962) from the CERTAIN database confirmed the observation (odds ratio of good/moderate response = 1.20 [95% CI = 1.03-1.41, p = 0.02]). CONCLUSION: Differences in innate/adaptive immune cell type composition at baseline may be a major contributor to response to anti-TNF treatment within the first 3 months of therapy.

SERPINA1 gene polymorphisms in a population-based ALSPAC cohort.

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.

Complications and risk factors in pediatric bronchoscopy in a tertiary pediatric respiratory center.

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 < 0.001) significantly contributed to risk of any complication. Age below 2 years (OR 2.478 [1.072-5.728], P = 0.034) and underlying cardiovascular disease (OR 2.678 [1.013-7.077], P = 0.047) were independent risk factors for severe complications. CONCLUSION: Bronchoscopy in children is relatively safe. Nevertheless, adverse events can occur and knowledge of risk factors may help prevent complications.

Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia.

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 leads to diverse changes in CLL-related pathways.

Filtration and Normalization of Sequencing Read Data in Whole-Metagenome Shotgun Samples.

Ever-increasing affordability of next-generation sequencing makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches for the analysis of microbiome samples. The advantage of whole-metagenome sequencing is that it allows direct inference of the metabolic capacity and physiological features of the studied metagenome without reliance on the knowledge of genotypes and phenotypes of the members of the bacterial community. It also makes it possible to overcome problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sufficient sequence similarity of the "universal" 16S primers to some of the target 16S genes. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes. To overcome this difficulty, we present a model of GC-bias in sequencing metagenomic samples as well as filtration and normalization techniques necessary for accurate quantification of microbial organisms. While there has been substantial research in normalization and filtration of read-count data in such techniques as RNA-seq or Chip-seq, to our knowledge, this has not been the case for the field of whole-metagenome shotgun sequencing. The presented methods assume that complete genome references are available for most microorganisms of interest present in metagenomic samples. This is often a valid assumption in such fields as medical diagnostics of patient microbiota. Testing the model on two validation datasets showed four-fold reduction in root-mean-square error compared to non-normalized data in both cases. The presented methods can be applied to any pipeline for whole metagenome sequencing analysis relying on complete microbial genome references. We demonstrate that such pre-processing reduces the number of false positive hits and increases accuracy of abundance estimates.

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.

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.