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Relatives of patients with familial interstitial pneumonia are at increased risk for pulmonary fibrosis, and develop preclinical pulmonary fibrosis (PrePF).We defined the incidence and progression of new onset PrePF; its relationship to survival among first degree relatives of families with familial interstitial pneumonia.This is a cohort study of family members with familial interstitial pneumonia who were initially screened with a questionnaire and chest HRCT scan, and approximately 4 years later, the evaluation was repeated. 493 asymptomatic first-degree relatives of patients with familial interstitial pneumonia were evaluated at baseline, and 296 (60%) of the original subjects participated in subsequent evaluation.The median interval between HRCTs was 3.9 years (IQ range 3.5-4.4). 252 subjects who agreed to repeat evaluation were originally determined not to have PrePF at baseline; 16 developed PrePF. A conservative estimate of the annual incidence of PrePF is 1,023 per 100, 000 person years (95% CI 511-1831). Of 44 subjects with PrePF at baseline, 38.4% subjects had worsening dyspnea compared to 15.4% of those without PrePF (P=0.002). Usual interstitial pneumonia (UIP) by HRCT (P0.0002) and baseline quantitative fibrosis score (P0.001) are also associated with worsening dyspnea. PrePF at the initial screen is associated with decreased survival (P0.001).The incidence of PrePF in this at-risk population is at least 100-fold higher than that reported for sporadic idiopathic pulmonary fibrosis (IPF). While PrePF and IPF represent distinct entities, our study demonstrates that PrePF, like IPF, is progressive and associated with decreased survival.

Rationale: Idiopathic pulmonary fibrosis is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to non-genetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants genome-wide may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ≤0.01) within genes or regions. We also identified individual variants that are influential within genes and estimated the heritability of IPF based on rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP-heritability of IPF was estimated to be 32% (s.e. 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or development of therapies for IPF that focus on TERT, RTEL1, common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

A subset of patients with hypersensitivity pneumonitis (HP) develop lung fibrosis that is clinically similar to idiopathic pulmonary fibrosis (IPF). To address the aetiological determinants of fibrotic HP, we investigated whether the common IPF genetic risk variants were also relevant in study subjects with fibrotic HP. Our findings indicate that common genetic variants in TERC, DSP, MUC5B and IVD were significantly associated with fibrotic HP. These findings provide support for a shared etiology and pathogenesis between fibrotic HP and IPF.

Molecular patterns and pathways in idiopathic pulmonary fibrosis (IPF) have been extensively investigated but few studies have assimilated multi-omic platforms to provide an integrative understanding of molecular patterns that are relevant in IPF. Herein, we combine coding and non-coding transcriptome, DNA methylome, and proteome from IPF and healthy lung tissue to identify molecules and pathways associated with this disease. RNA sequencing, Illumina MethylationEPIC array, and liquid chromatography-mass spectrometry (LC-MS) proteomic data were collected on lung tissue from 24 IPF cases and 14 control subjects. Significant differential features were identified using linear models adjusting for age and sex, inflation and bias where appropriate. Data Integration Analysis for Biomarker discovery using a Latent component method for Omics studies (DIABLO) was used for integrative multi-omic analysis. We identified 4,643 differentially expressed transcripts aligning to 3,439 genes, 998 differentially abundant proteins, 2,500 differentially methylated regions (DMRs), and 1,269 differentially expressed lncRNAs that were significant after correcting for multiple tests (false discovery rate [FDR]0.8) with MMP7. Therefore, using a systems biology approach, we have identified novel molecular relationships in IPF.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF). Objectives: To determine the common and unique molecular features of CHP and IPF. Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons. Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing. Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, also known as Job's syndrome) is caused by mutations in STAT3. These patients present with immunodeficiency accompanied by severe nonimmunological features, including skeletal, connective tissue, and vascular abnormalities, poor postinfection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here, we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin-wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3-controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia-inducible factor 1α (HIF-1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF-1α expression and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling using cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF-1α-stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and suggests potential treatment options for this rare disease.

Background: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by progressive pulmonary fibrosis. The histopathological and clinical features of CHP are variable, however, a subset of CHP patients have usual interstitial pneumonia and behave clinically similar to patients with idiopathic pulmonary fibrosis (IPF). To determine the common and unique molecular features of CHP and IPF, we conducted a transcriptome analysis of lung samples from CHP (N=82), IPF (N=103), and unaffected controls (N=103). Methods: Gene expression in lung tissue was determined adjusting for sex, race, age, explant site, and smoking history, and using false discovery rate (FDR) to control for multiple comparisons. Findings: When compared to controls, we identified 413 upregulated and 317 downregulated genes in CHP, and 861 upregulated and 322 downregulated genes in IPF. Concordantly up/down-regulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene co-expression network analysis (WGCNA), we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing. Interpretation: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP. Funding Statement: NHLBI (R01-HL097163, P01-HL092870, UH3-HL123442, and DoD W81XWH17-1-0597). Declaration of Interests: None of the authors report conflicts of interest relevant to this study. D.A.S. is the founder and chief scientific officer of Eleven P15, a company focused on the early diagnosis and treatment of pulmonary fibrosis. D.A.S. has an awarded patent (US patent no: 8,673,565) for the treatment and diagnosis of fibrotic lung disease. Ethics Approval Statement: This study conformed to the Declaration of Helsinki and was approved by the Colorado Multiple Institution Board (COMIRB) 15-1147.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

BackgroundRelatives of patients with familial interstitial pneumonia (FIP) are at increased risk for pulmonary fibrosis. We assessed the prevalence and risk factors for preclinical pulmonary fibrosis (PrePF) in first-degree relatives of patients with FIP and determined the utility of deep learning in detecting PrePF on CT.MethodsFirst-degree relatives of patients with FIP over 40 years of age who believed themselves to be unaffected by pulmonary fibrosis underwent CT scans of the chest. Images were visually reviewed, and a deep learning algorithm was used to quantify lung fibrosis. Genotyping for common idiopathic pulmonary fibrosis risk variants in MUC5B and TERT was performed.FindingsIn 494 relatives of patients with FIP from 263 families of patients with FIP, the prevalence of PrePF on visual CT evaluation was 15.6% (95% CI 12.6 to 19.0). Compared with visual CT evaluation, deep learning quantitative CT analysis had 84% sensitivity (95% CI 0.72 to 0.89) and 86% sensitivity (95% CI 0.83 to 0.89) for discriminating subjects with visual PrePF diagnosis. Subjects with PrePF were older (65.9, SD 10.1 years) than subjects without fibrosis (55.8 SD 8.7 years), more likely to be male (49% vs 37%), more likely to have smoked (44% vs 27%) and more likely to have the MUC5B promoter variant rs35705950 (minor allele frequency 0.29 vs 0.21). MUC5B variant carriers had higher quantitative CT fibrosis scores (mean difference of 0.36%), a difference that remains significant when controlling for age and sex.InterpretationPrePF is common in relatives of patients with FIP. Its prevalence increases with age and the presence of a common MUC5B promoter variant. Quantitative CT analysis can detect these imaging abnormalities.

EgeUn###, Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 3 102295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence. Copyright © 2019 by the American Thoracic Society, Háskóli Íslands, HI University of Chicago University of Ulsan, UOU Monash University, MU University of Nottingham University of Edinburgh University of Colorado Denver Universitat de Barcelona University of Pittsburgh, Pitt Harvard School of Dental Medicine Massachusetts General Hospital, MGH University of Pennsylvania, Penn University College Cork, UCC City, University of London, City University of California, San Francisco, UCSF School of Medicine, Vanderbilt University Ege Üniversitesi University of Virginia, UV Aarhus Universitetshospital Central Manchester University Hospitals NHS Foundation Trust Columbia University Novartis Sunovion COPD Foundation Pfizer National Heart, Lung, and Blood Institute, NHLBI: R01-HL097163, UH3-HL123442, P01-HL092870 U.S. Department of Defense, DOD: W81XWH-17-1-0597, U01 HL089897, U01 HL089856 AstraZeneca Siemens Foundation North Carolina GlaxoSmithKline Foundation, 1National Jewish Health, Denver, Colorado; 2School of Public Health; 3Department of Medicine, and 54Department of Immunology, University of Colorado Denver, Denver, Colorado; 4Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; 5Brigham and Women’s Hospital, Harvard School of Medicine, Boston, Massachusetts; 6Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma;7Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 8MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; 9Respiratory Medicine Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 10Biomedical Research Centre, University of Nottingham, Nottingham, United Kingdom; 11Royal Brompton Hospital and Imperial College, London, United Kingdom; 12Simmons Center for Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania; 13Department of Medicine, University of California, San Francisco, San Francisco, California; 14Gilead Sciences, Foster City, California; 15Department of Medicine, University of Chicago, Chicago, Illinois; 16Department of Medicine, University of Virginia, Charlottesville, Virginia; 17Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; 18National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan; 19National Hospital Organization Tokyo National Hospital, Tokyo, Japan; 20Helmholtz Zentrum München, Neuherberg, Germany; 21University Hospital Munich, Munich, Germany; 22Department of Pulmonology, Ege University Hospital, Bornova, Izmir, Turkey; 23Royal Prince Alfred Hospital and University of Sydney, Sydney, Australia; 24Alfred Hospital and Monash University, Melbourne, Australia; 25Pulmonary Medicine, GB Morgagni Hospital, Forlì, Italy; 26Department of Diseases of the Thorax, Ospedale GB Morgagni, Forlì, Italy; 27Université Paris Diderot and Hôpital Bichat, Paris, France; 28Royal Papworth Hospital and Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom; 29Respiratory Department, University Hospital of Bellvitge, University of Barcelona, Barcelona, Spain; 30National University Hospital of Iceland, University of Iceland, Reykjavik, Iceland; 31Department of Medicine, Columbia University Irving Medical Center, New York, New York; 32Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, Massachusetts; 33Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark; 34Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas,” México City, México; 35Universidad Nacional Autónoma de México, México City, México; 36Cork University Hospital and University College Cork, Cork, Ireland; 37St. Vincent’s University Hospital, Dublin, Ireland; 38School of Medicine, University College Dublin, Dublin, Ireland; 39Department of Respiratory Medicine, First Faculty of Medicine Charles University and Thomayer Hospital, Prague, Czech Republic; 40University of British Columbia, Vancouver, Canada; 41Tokyo Medical and Dental University, Tokyo, Japan; 42Institute for Respiratory Health and; 43Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences, The University of Western Australia, Perth, Australia; 44Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island; 45Advanced Lung Disease and Transplant Program, Inova Fairfax Hospital, Falls Church, Virginia; 46Department of Pulmonary Medicine, Gazi University School of Medicine, Ankara, Turkey; 47Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; 48Ruhrlandklinik, University Hospital, University of Duisburg-Essen, Essen, Germany; 49Department of Medicine, Tallaght University Hospital, Trinity College Dublin, Dublin, Ireland; 50CardioPulmonary Reserach Center, Alliance Pulmonary Group, Guaynabo, Puerto Rico; 51Department of Internal Medicine, University of Genoa, Genoa, Italy; 52Biomedical Genomics Center, University of Minnesota; Minneapolis, Minnesota; and 53Northwest Genomics Center, University of Washington, Seattle, Washington -- This research was supported by grants from the NHLBI (R01-HL097163, P01-HL092870, and UH3-HL123442) and the Department of Defense (W81XWH-17-1-0597). The COPDGene project was supported by Award Number U01 HL089897 and Award Number U01 HL089856 from the NHLBI. The COPDGene project was also supported by the COPD Foundation through contributions made to an industry advisory board comprised of AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Pfizer, Siemens, and Sunovion.

Background Rheumatoid arthritis–associated interstitial lung disease (RA-ILD) and idiopathic pulmonary fibrosis (IPF) share phenotypic similarities. The gain-of-function MUC5B promoter variant rs35705950 is the strongest risk factor for development of IPF Objectives We hypothesised that rs35705950 would also contribute to the risk of ILD in RA patients. Methods Using a French discovery population and multi-ethnic validation populations from 6 different countries, we tested the association of the MUC5B promoter variant in RA-ILD (n=620), RA without ILD (n=614), and unaffected controls (n=5448). Results The discovery population revealed an association of the MUC5B promoter variant with RA-ILD when compared to unaffected controls (ORadj=3.8 95% CI: 2.8 to 5.2; p=9.7x10–17) (figure 1A). Similar to the discovery cohort, the MUC5B promoter variant was significantly over-represented among the cases of RA-ILD in the multi-ethnic study cohorts when compared to unaffected controls (ORadj=5.5 95% CI: 4.2 to 7.2; p=4.7x10–35) (figure 1A), and when the discovery population and the multi-ethnic cohorts were combined (ORcombined=4.7 95% CI: 3.9 to 5.8; p=1.3x10–49) (figure 1A). Additionally, the MUC5B promoter variant was found to increase the risk of ILD among patients with RA (ORcombined=3.1 95% CI: 1.8 to 5.4; p=7.4x10–5), however, no statistical association with the MUC5B promoter variant was observed for RA without ILD (figure 1B). The association of the MUC5B promoter variant with RA-ILD increased significantly when restricted to the usual interstitial pneumonia (UIP) by high-resolution computed tomography (ORcombined=6.1 95% CI: 2.9 to 13.1; p=2.5x10–6) (figure 1C). Immunohistochemical and in-situ hybridization analysis of RA-ILD lung tissue demonstrated expression of MUC5B by type 2 alveolar epithelial cells undergoing endoplasmic reticulum stress. Conclusions Our findings demonstrate that MUC5B promoter variant rs35705950 is a risk factor for RA-ILD specifically associated with radiologic evidence of UIP. Disclosure of Interest None declared

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31–1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15–2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20–2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23– 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

Objective To identify the cause of disease in an adult patient presenting with recent-onset fevers, chills, urticaria, fatigue, and profound myalgia, who was found to be negative for cryopyrin-associated periodic syndrome (CAPS) NLRP3 mutations by conventional Sanger DNA sequencing. Methods We performed whole-exome sequencing and targeted deep sequencing using DNA from the patient's whole blood to identify a possible NLRP3 somatic mutation. We then screened for this mutation in subcloned NLRP3 amplicons from fibroblasts, buccal cells, granulocytes, negatively selected monocytes, and T and B lymphocytes and further confirmed the somatic mutation by targeted sequencing of exon 3. Results We identified a previously reported CAPS-associated mutation, p.Tyr570Cys, with a mutant allele frequency of 15% based on exome data. Targeted sequencing and subcloning of NLRP3 amplicons confirmed the presence of the somatic mutation in whole blood at a ratio similar to the exome data. The mutant allele frequency was in the range of 13.3–16.8% in monocytes and 15.2–18% in granulocytes. Notably, this mutation was either absent or present at a very low frequency in B and T lymphocytes, in buccal cells, and in the patient's cultured fibroblasts. Conclusion Our findings indicate the possibility of myeloid-restricted somatic mosaicism in the pathogenesis of CAPS, underscoring the emerging role of massively parallel sequencing in clinical diagnosis.

International audience; BACKGROUND: Given the phenotypic similarities between rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) (hereafter, RA-ILD) and idiopathic pulmonary fibrosis, we hypothesized that the strongest risk factor for the development of idiopathic pulmonary fibrosis, the gain-of-function MUC5B promoter variant rs35705950, would also contribute to the risk of ILD among patients with RA. METHODS: Using a discovery population and multiple validation populations, we tested the association of the MUC5B promoter variant rs35705950 in 620 patients with RA-ILD, 614 patients with RA without ILD, and 5448 unaffected controls. RESULTS: Analysis of the discovery population revealed an association of the minor allele of the MUC5B promoter variant with RA-ILD when patients with RA-ILD were compared with unaffected controls (adjusted odds ratio, 3.8; 95% confidence interval [CI], 2.8 to 5.2; P=9.7x10(-17)). The MUC5B promoter variant was also significantly overrepresented among patients with RA-ILD, as compared with unaffected controls, in an analysis of the multiethnic case series (adjusted odds ratio, 5.5; 95% CI, 4.2 to 7.3; P=4.7x10(-35)) and in a combined analysis of the discovery population and the multiethnic case series (adjusted odds ratio, 4.7; 95% CI, 3.9 to 5.8; P=1.3x10(-49)). In addition, the MUC5B promoter variant was associated with an increased risk of ILD among patients with RA (adjusted odds ratio in combined analysis, 3.1; 95% CI, 1.8 to 5.4; P=7.4x10(-5)), particularly among those with evidence of usual interstitial pneumonia on high-resolution computed tomography (adjusted odds ratio in combined analysis, 6.1; 95% CI, 2.9 to 13.1; P=2.5x10(-6)). However, no significant association with the MUC5B promoter variant was observed for the diagnosis of RA alone. CONCLUSIONS: We found that the MUC5B promoter variant was associated with RA-ILD and more specifically associated with evidence of usual interstitial pneumonia on imaging. (Funded by Societe Francaise de Rhumatologie and others.).

We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface.

ACDC (arterial calcification due to deficiency of CD73) is an autosomal recessive disease resulting from loss-of-function mutations in NT5E, which encodes CD73, a 5'-ectonucleotidase that converts extracellular adenosine monophosphate to adenosine. ACDC patients display progressive calcification of lower extremity arteries, causing limb ischemia. Tissue-nonspecific alkaline phosphatase (TNAP), which converts pyrophosphate (PPi) to inorganic phosphate (Pi), and extracellular purine metabolism play important roles in other inherited forms of vascular calcification. Compared to cells from healthy subjects, induced pluripotent stem cell-derived mesenchymal stromal cells (iMSCs) from ACDC patients displayed accelerated calcification and increased TNAP activity when cultured under conditions that promote osteogenesis. TNAP activity generated adenosine in iMSCs derived from ACDC patients but not in iMSCs from control subjects, which have CD73. In response to osteogenic stimulation, ACDC patient-derived iMSCs had decreased amounts of the TNAP substrate PPi, an inhibitor of extracellular matrix calcification, and exhibited increased activation of AKT, mechanistic target of rapamycin (mTOR), and the 70-kDa ribosomal protein S6 kinase (p70S6K), a pathway that promotes calcification. In vivo, teratomas derived from ACDC patient cells showed extensive calcification and increased TNAP activity. Treating mice bearing these teratomas with an A2b adenosine receptor agonist, the mTOR inhibitor rapamycin, or the bisphosphonate etidronate reduced calcification. These results show that an increase of TNAP activity in ACDC contributes to ectopic calcification by disrupting the extracellular balance of PPi and Pi and identify potential therapeutic targets for ACDC.

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-κB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-α, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-κB–binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-κB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa–/– mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment.

Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage-tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor-β (TGF-β) signaling by TGF-β neutralizing antibody, short hairpin RNA-mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell-specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-β-Smad2/3-Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.

Background: We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. Results: R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48→Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-β-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned into the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. Conclusions: A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5–ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.

To investigate the mechanism of scission of proteins by the chemical cleaving agent 1,10-phenanthroline-copper, the active sites of human carbonic anydrase I and bovine carbonic anhydrase II have been targeted for cleavage by a tight binding sulfonamide inhibitor tethered to the metal complex. The inhibitor-phenanthroline-copper conjugate binds to the carbonic anhydrases with sub-micromolar Kd's and, upon addition of a reducing agent, causes scission specifically within the active site of the enzymes to yield a discrete set of cleavage fragments. N- and C-terminal sequencing and mass spectrometric analysis of several fragments indicate that the C-terminal cleavage fragments have free amino groups at their N termini, thereby allowing facile location of the cut sites through standard Edman degradation. The N-terminal cleavage fragments do not have a free carboxyl group at their C termini. It is proposed that scission occurs by abstraction of H at Calpha, followed by oxidation at Calpha by the neighboring cupric ion and cleavage of the Calpha-C(O) bond to give an N-terminal fragment containing a C-terminal acyl amide, and an unstable C-terminal fragment containing an N-terminal isocyanate group which undergoes hydrolysis to a free amino terminus. Modeling of the inhibitor-phenanthroline-copper conjugate within the active site of human carbonic anhydrase I shows that the sites of cleavage that have been identified are fully consistent with the available structural data.

SummaryType I interferon (IFN-α/β or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNβ-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNβ-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses.