Regulation of mRNA translation by a photoriboswitch.

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)-a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes

Anti-fibroblast antibodies detected by cell-based ELISA in systemic sclerosis enhance the collagenolytic activity and matrix metalloproteinase-1 production in dermal fibroblasts

Objectives. Antibodies binding to the surface of fibroblasts (anti-fibroblast antibodies: AFA) have been described in systemic sclerosis (SSc). We aimed to assess the effect of AFA on extracellular matrix (ECM) turnover and whether AFA were associated with anti-topoisomerase-I antibody. Methods. IgG were purified from AFA-positive and AFA-negative sera selected within 20 SSc and 20 healthy individuals, and tested on normal dermal fibroblasts, at protein and mRNA level, for their capacity to induce collagen deposition or degradation. Results. Fibroblasts stimulated with AFA-positive but not with AFA-negative and control IgG showed an increased capacity to digest collagen matrix and produce metalloproteinase-1 (MMP-1) while their production of total collagen, type I collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) was unaffected. The steady-state mRNA levels of MMP-1, COL1A1 and TIMP-1 paralleled the protein levels. AFA-positive IgG did not induce Smad 2/3 phosphorylation, indicating that this transforming growth factor-β signalling pathway was not involved. IL-1 and tumour necrosis factor (TNF) neutralization did not reverse the enhanced production of MMP-1, suggesting a direct effect of AFA on fibroblasts. Finally, anti-topoisomerase-I antibodies were present in 11 of 12 AFA-negative IgG, and an anti-topoisomerase-I monoclonal antibody failed to enhance MMP-1 production, thus indicating a lack of correlation between AFA and anti-topoisomerase-I antibody. Conclusions. These results indicate that SSc antibodies binding to fibroblasts enhance matrix degradation and MMP production events that may favour inflammation but do not directly impact on fibrosis developmen

The Swiss Systemic lupus erythematosus Cohort Study (SSCS) - cross-sectional analysis of clinical characteristics and treatments across different medical disciplines in Switzerland.

OBJECTIVES: To describe disease characteristics and treatment modalities in a multidisciplinary cohort of systemic lupus erythematosus (SLE) patients in Switzerland. METHODS: Cross-sectional analysis of 255 patients included in the Swiss SLE Cohort and coming from centres specialised in Clinical Immunology, Internal Medicine, Nephrology and Rheumatology. Clinical data were collected with a standardised form. Disease activity was assessed using the Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI), an integer physician's global assessment score (PGA) ranging from 0 (inactive) to 3 (very active disease) and the erythrocyte sedimentation rate (ESR). The relationship between SLE treatment and activity was assessed by propensity score methods using a mixed-effect logistic regression with a random effect on the contributing centre. RESULTS: Of the 255 patients, 82% were women and 82% were of European ancestry. The mean age at enrolment was 44.8 years and the median SLE duration was 5.2 years. Patients from Rheumatology had a significantly later disease onset. Renal disease was reported in 44% of patients. PGA showed active disease in 49% of patients, median SLEDAI was 4 and median ESR was 14 millimetre/first hour. Prescription rates of anti-malarial drugs ranged from 3% by nephrologists to 76% by rheumatologists. Patients regularly using anti-malarial drugs had significantly lower SELENA-SLEDAI scores and ESR values. CONCLUSION: In our cohort, patients in Rheumatology had a significantly later SLE onset than those in Nephrology. Anti-malarial drugs were mostly prescribed by rheumatologists and internists and less frequently by nephrologists, and appeared to be associated with less active SLE

No association of complement mannose-binding lectin deficiency with cardiovascular disease in patients with Systemic Lupus Erythematosus.

Cardiovascular (CV) morbidity is the major cause of death in patients with Systemic Lupus Erythematosus (SLE). Previous studies on mannose-binding lectin (MBL) gene polymorphisms in SLE patients suggest that low levels of complement MBL are associated with cardiovascular disease (CVD). However, as large studies on MBL deficiency based on resulting MBL plasma concentrations are lacking, the aim of our study was to analyze the association of MBL concentrations with CVD in SLE patients. Plasma MBL levels SLE patients included in the Swiss SLE Cohort Study were quantified by ELISA. Five different CV organ manifestations were documented. Of 373 included patients (85.5% female) 62 patients had at least one CV manifestation. Patients with MBL deficiency (levels below 500 ng/ml or 1000 ng/ml) had no significantly increased frequency of CVD (19.4% vs. 15.2%, P = 0.3 or 17.7% vs. 15.7%, P = 0.7). After adjustment for traditional CV risk factors, MBL levels and positive antiphospholipid serology (APL+) a significant association of CVD with age, hypertension, disease duration and APL+ was demonstrated. In our study of a large cohort of patients with SLE, we could not confirm previous studies suggesting MBL deficiency to be associated with an increased risk for CVD

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-0/IL-113 from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk over¬comes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radia¬tion (1–6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-10, -6, -10, and -27 or TNF-a and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Impor¬tantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-0/IL-10 and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would other¬wise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity. J. Leukoc. Biol. 100: 000–000; 2016

IL-25 participates in keratinocyte-driven dermal matrix turnover and is reduced in systemic sclerosis epidermis

OBJECTIVES: Evidence shows that dysfunctional SSc keratinocytes contribute to fibrosis by altering dermal homeostasis. Whether IL-25, an IL-17 family member regulating many epidermal functions, takes part in skin fibrosis is unknown. Here we address the role of IL-25 in skin fibrosis. METHODS: The expression of IL-25 was evaluated by immunofluorescence and in situ hybridization in 10 SSc and seven healthy donor (HD) skin biopsies. Epidermal equivalents (EE) reconstituted by primary HD keratinocytes were used as a model to study transcriptomic changes induced by IL-25 in the epidermis. RNA expression profile in EEs was characterized by RNAseq. The conditioned medium (CM) from primary SSc and HD keratinocytes primed with IL-25 was used to stimulate fibroblasts. IL-6, IL-8, MMP-1, type-I collagen (Col-I), and fibronectin production by fibroblasts was assessed by ELISA. RESULTS: SSc epidermis expressed lower levels of IL-25 compared with HDs. In EEs, IL-25 regulated several molecular pathways related to wound healing and extracellular matrix remodelling. Compared with control CM, the CM from IL-25-primed keratinocytes enhanced the fibroblast production of MMP-1, IL-6 and IL-8, but not of Col-I nor fibronectin. However, IL-25 significantly reduced the production of Col-I when applied directly to fibroblasts. The activation of keratinocytes by IL-25 was receptor-dependent and evident after a very short incubation time (10 min), largely mediated by IL-1, suggesting enhanced and specific release of preformed mediators. CONCLUSIONS: These results show that IL-25 participates in skin homeostasis, and its decreased expression in SSc may contribute to skin fibrosis by favouring extracellular matrix deposition over degradation

Management of CPAP Follow-up by Telemonitoring in Obstructive Sleep Apnea: The PROTEUS Project

Riccardo Drigo,1 Andrea Ballarin,1 Francesco Menzella,1 Micaela Romagnoli,2 Mauro Salasnich,2 Luigi Marino,3 Paolo Lucernoni,3 Maddalena Chizzolini,4 Pasquale Tondo5 1Department of Critical Care, Unit of Pulmonology, Montebelluna Hospital, Montebelluna, Italy; 2Unit of Pulmonology, Ca’ Foncello Hospital, Treviso, Italy; 3Unit of Pulmonology, Vittorio Veneto Hospital, Vittorio Veneto, Italy; 4Unit of Respiratory Rehabilitation, Motta Di Livenza Rehabilitation Hospital, Motta di Livenza, Italy; 5Department of Medical and Surgical Sciences, University of Foggia, Foggia, ItalyCorrespondence: Pasquale Tondo, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy, Email [email protected]: CPAP is the standard treatment for obstructive sleep apnea (OSA), but as many as 50% of patients discontinue its use, within three years. The PROTEUS project aims to enhance CPAP adherence through telemonitoring.Patients and Methods: OSA patients undergoing CPAP treatment since July 2018, had an in-person reassessment after three months, followed by quarterly telemonitoring by providers, who intervened in cases of poor adherence (less than 4 h·night− 1 or less than 70% of days), excessive mask leakage, or elevated residual apnea-hypopnea index (rAHICPAP).Results: A total of 486 (~87%) out of 558 patients analyzed remained adherent to CPAP after five years. The average rAHICPAP was 3.95 ± 5.25 events·h− 1, the average CPAP usage was 6.35 ± 1.72 hours. Elevated mask leakage occurred in 25% of patients. No significant differences were found between sexes.Conclusion: The PROTEUS project showed promising results in supporting long-term CPAP adherence. However, further research is needed to validate its long-term impact and wider applicability in OSA management.Keywords: CPAP, management, OSA, real-life study, telemonitorin

Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients

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

OP0137 GENOME-WIDE WHOLE-BLOOD TRANSCRIPTOME PROFILING IN A LARGE EUROPEAN COHORT OF SYSTEMIC SCLEROSIS PATIENTS

Background:The analysis of annotated transcripts from genome-wide expression studies data is of paramount importance to understand the molecular phenomena underlying the occurrence of complex diseases, such as systemic sclerosis (SSc).Objectives:To perform whole-blood transcriptome and pathway analysis on whole-blood (WB) RNA collected in two cohorts of European SSc patients. Via a discovery and validation strategy we aimed at characterizing the molecular pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations.Methods:WB samples from 252 controls and 162 SSc patients were collected in RNA stabilizers. Patients were divided into a discovery (n=79; Southern Europe) and validation cohort (n=83; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the FAIME algorithm. In parallel, a immunophenotyping analysis on 28 circulating cell populations was assessed. We then tested: the presence of differentially expressed genes or 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:A total of 15224 genes and 1277 related functional pathways were available for analysis. Among these, 99 genes and 225 pathways were significant in both sets. The heatmap in figure shows the relative expression of replicated pathways and the distribution of cases and controls (red and green bars). Among the significant pathways we found a deregulation in: type-I IFN, TLR-cascade and signalling, function of the tumor suppressor p53 protein, platelet degranulation and activation. Correlation analysis showed that the count of several cell subtypes is jointly associated with RNA transcripts or FAIME scores with strong differences in relation to the geographical origin of samples; neutrophils emerged as the major determinant of gene expression in SSc-whole-blood samples.Conclusion:We discovered a set of differentially expressed genes and pathways that could be validated in two independent sets of SSc patients highlighting a number of deregulated molecular processes that have relevance for the pathogenesis of autoimmunity and SSc.Acknowledgments:This work was supported by EU/EFPIA/Innovative Medicines Initiative Joint Undertaking PRECISESADS grant No. 115565.Disclosure of Interests:Lorenzo Beretta Grant/research support from: Pfizer, Guillermo Barturen: None declared, Barbara Vigone: None declared, Chiara Bellocchi: None declared, Nicolas Hunzelmann: None declared, Ellen Delanghe: None declared, László Kovács: None declared, Ricard Cervera: None declared, Maria Gerosa: None declared, Rafaela Ortega Castro: None declared, Isabel Almeida: None declared, Divi Cornec: None declared, Carlo Chizzolini Consultant of: Boehringer Ingelheim, Roche, Jacques-Olivier Pers: None declared, Zuzanna Makowska Employee of: Bayer AG, Anne buttgereit Employee of: Bayer AG, Ralf Lesche Employee of: Bayer, Martin Kerick: None declared, Marta Alarcon-Riquelme: None declared, Javier Martin Ibanez: None declare