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Objectives: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. Methods: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. Results: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. Conclusions: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc., This work was supported by eU/eFPia/innovative Medicines initiative Joint Undertaking PrecisesaDs Grant no. 115 565.

OBJECTIVE: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. METHODS: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. RESULTS: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. CONCLUSION: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases. ispartof: ARTHRITIS & RHEUMATOLOGY vol:73 issue:6 pages:1073-1085 ispartof: location:United States status: published

Background: Bronchiectasis is the final result of different processes and most of the guidelines advocate for a careful evaluation of those etiologies which might be treated or might change patients’ management, including cystic fibrosis (CF). Main body: CFTR mutations have been reported with higher frequency in bronchiectasis population. Although ruling out CF is considered as a main step for etiological screening in bronchiectasis, CF testing lacks of a standardized approach both from a research and clinical point of view. In this review a list of most widely used tests in CF is provided. Conclusions: Exclusion of CF is imperative for patients with bronchiectasis and CFTR testing should be implemented in usual screening for investigating bronchiectasis etiology. Physicians taking care of bronchiectasis patients should be aware of CFTR testing and its limitations in the adult population. Further studies on CFTR expression in human lung and translational research might elucidate the possible role of CFTR in the pathogenesis of bronchiectasis.

This work was supported by EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant No. 115565.

Background Prokinetics are used to treat enteric dismotility symptoms in systemic sclerosis (SSc) patients, but they often lack adequate efficacy. The most effective prokinetics belonging to the serotonin (5-HT4) receptor agonists class were withdrawn due to cardiac toxicity in relation to modest 5-HT4 receptor affinity. Prucalopride is a high-affinity 5-HT4 receptor agonist with no major cardiac issues, for which the efficacy in SSc has not yet been assessed. Methods Forty patients with self-reported mild to moderately severe enteric symptoms were enrolled in a cross-over 2 × 2 study. Subjects were randomized 1:1 to prucalopride 2 mg/day or no treatment for one month and vice versa after a 2-week washout period. Before and after each sequence the patients compiled the University of California Los Angeles gastrointestinal tract (UCLA GIT) 2.0 questionnaire and the numbers of complete intestinal movements were recorded. Oro-cecal transit time (OCTT) was evaluated by lactulose breath test in a subgroup of patients. Data were evaluated by mixed linear models corrected for the number of laxatives used during the study periods. Results There were 29 subjects who completed the study; 7 subjects withdrew due to side-effects and 4 subjects were not compliant with the study procedures. As compared to dummy treatment, prucalopride was associated with more intestinal evacuations (p

Background:Genome-wide gene expression profiles and pathways analysis may help to discover deregulated processes underlying the pathogenesis of complex diseases or their phenotypic expression. Little or nothing is currently known about pathways associated with disease severity and damage in SSc.Objectives:To perform a whole blood transcriptome analysis and to characterize the individualized functional pathways associated with disease severity scores in SSc patients via a discovery and replication strategy.Methods:Whole blood samples were collected in RNA stabilizers from a discovery and a replication cohort of 67 and 34 patients, respectively. RNAseq data were generated by Illumina sequencing in two independent experiments pathways analysis was conducted according to the Functional Analysis of Individual Microarray Expression (FAIME) protocol (1). FAIME scores from Reactome pathways were correlated with the Scleroderma Clinical Trial Consortium Damage Index (SCTC-DI) total scores or with each of its two components (mortality and morbidity) as calculated from regression coefficient previously published (2). A non-parametric partial correlation analysis correcting the results for the use of steroids, immunosuppressants and disease duration was performed. Results independently associated with damage in both cohorts at the 0.1 level after 1000-fold permutation-testing were considered as significant and replicated.Results:A total of 1116 pathways were analyzed. None of them was associated in both cohorts with the total SCTC-DI; similarly, no association was found with the SCTC mortality component. On the contrary, 26 pathways showed an independent and replicated association with the SCTC morbidity component, including platelet degranulation, the transcriptional activity of SMAD2/SMAD3, Toll Like Receptor 2 (TLR2) cascade and related intracellular signaling events involving MyD88, IRAK and NF-kB, and the deregulation of selected transcription factors (TFAP2, E2F6).Conclusion:Selected molecular events involving the innate immune system, signaling and platelet metabolism are associated with the morbidity component of the SCTC-DI in SSc patients, reflecting an irreversible loss of function in several organs and apparatus. The sensitivity of these associations to individual change in accrual damage is to be determined.References:[1]PMID: 22291585; (2) PMID: 30928903Acknowledgments:This work was partially supported by EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant No. 115565Disclosure of Interests:Chiara Bellocchi: None declared, Marzia Rossato: None declared, Guillermo Barturen: None declared, Giulia Segatto: None declared, Barbara Vigone: None declared, Zuzanna Makowska Employee of: Bayer AG, Anne buttgereit Employee of: Bayer AG, Martin Kerick: None declared, Marta Alarcon-Riquelme: None declared, Javier Martin Ibanez: None declared, Lorenzo Beretta Grant/research support from: Pfizer

Objective MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. Methods The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjogren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. Results 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. Conclusions Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

Dysbiosis has been described in systemic autoimmune diseases (SADs), including systemic lupus erythematosus (SLE), Sj&ouml, gren&rsquo, s syndrome (SjS), and primary anti-phosholipid syndrome (PAPS), however the biological implications of these associations are often elusive. Stool and plasma samples from 114 subjects, including in SLE (n = 27), SjS (n = 23), PAPs (n = 11) and undifferentiated connective tissue (UCTD, n = 26) patients, and geographically-matched healthy controls (HCs, n = 27), were collected for microbiome (16s rRNA gene sequencing) and metabolome (high-performance liquid chromatography coupled to mass spectrometry) analysis to identify shared characteristics across diseases. Out of 130 identified microbial genera, a subset of 29 bacteria was able to differentiate study groups (area under receiver operating characteristics (AUROC) = 0.730 &plusmn, 0.025). A fair classification was obtained with a subset of 41 metabolic peaks out of 254 (AUROC = 0.748 &plusmn, 0.021). In both models, HCs were well separated from SADs, while UCTD largely overlapped with the other diseases. In all of the SADs pro-tolerogenic bacteria were reduced, while pathobiont genera were increased. Metabolic alterations included two clusters comprised of: (a) members of the acylcarnitine family, positively correlating with a Prevotella-enriched cluster and negatively correlating with a butyrate-producing bacteria-enriched cluster, and (b) phospholipids, negatively correlating with butyrate-producing bacteria. These findings demonstrate a strong interaction between intestinal microbiota and metabolic function in patients with SADs.