Objectives(1) Hypothesis testing of the potency of rituximab (RTX) in preventing fibrotic complications and (2) assessing acceptability and feasibility of RTX in early systemic sclerosis (SSc).MethodsA small, 24-month, randomised, double-blind, placebo-controlled, single-centre trial in patients with SSc diagnosed <2 years was conducted. Patients received RTX or placebo infusions at t=0, t=15 days and t=6 months. Patients were clinically evaluated every 3 months, with lung function tests and high-resolution CT every other visit. Skin biopsies were taken at baseline and month 3. Immunophenotyping of peripheral blood mononuclear cells was performed at every visit, except at months 9 and 18. Adverse events, course of skin and pulmonary involvement and B cell populations in skin and peripheral blood were evaluated.ResultsIn total 16, patients (rituximab n=8, placebo n=8) were included. Twelve patients had diffuse cutaneous SSc. Eighty-eight adverse events (RTX n=53, placebo n=35, p=0.22) and 11 serious adverse events (RTX n=7, placebo n=4, p=0.36) occurred. No unexpected RTX-related events were observed. Mean skin score over time did not differ between the groups. Over time, forced vital capacity and extent of lung involvement slightly improved with RTX, but this difference was insignificant. In peripheral blood B cells depletion was demonstrated.ConclusionsNo unexpected safety issues were observed with RTX in early SSc. Although this small trial could not confirm or reject potential efficacy of RTX in these patients, future placebo-controlled trials are warranted, specifically in the subgroup of patients with pulmonary involvement.Trial registration numberEudraCT 2008-07180-16; Results.

Granulomas in sarcoidosis express high levels of 1a-hydroxylase, an enzyme that catalyzes the hydroxylation of 25-OH vitamin D to its active form, 1,25(OH) 2 vitamin D. Overproduction of 1a-hydroxylase is held responsible for the development of hypercalcemia in sarcoidosis patients. Corticosteroids are used as first-line treatment in organ-threatening sarcoidosis. In this light, osteoporosis prevention with calcium and vitamin D (CAD) supplementation is often warranted. However, sarcoidosis patients are at risk for hypercalcemia, and CAD supplementation affects the calcium metabolism. We studied calcium and vitamin D disorders in a large cohort of sarcoidosis patients and investigated if CAD supplementation is safe. Retrospectively, data of 301 sarcoidosis patients from July 1986 to June 2009 were analyzed for serum calcium, 25-hydroxy vitamin D (25-(OH)D), 1,25-dihydroxy vitamin D (1,25(OH) 2 D), and use of CAD supplementation. Disease activity of sarcoidosis was compared with serum levels of vitamin D. Hypercalcemia occurred in 8%. A significant negative correlation was found between 25-(OH)D and disease activity of sarcoidosis measured by somatostatin receptor scintigraphy. In our study, 5 of the 104 CAD-supplemented patients developed hypercalcemia, but CAD supplementation was not the cause of hypercalcemia. Patients without CAD supplementation were at higher risk for developing hypercalcemia. During CAD supplementation, no hypercalcemia developed as a result of supplementation. Hypovitaminosis D seems to be related with more disease activity of sarcoidosis and, therefore, could be a potential risk factor for disease activity of sarcoidosis. Thus, vitamin D-deficient sarcoidosis patients should be supplemented.

Background and objectiveSystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes.MethodsChromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression.Results1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression.ConclusionSSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

Objective To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc). Methods Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme‐linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6–7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5–7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay. Results Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up‐regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors. Conclusion Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.

IntroductionSystemic sclerosis (SSc) is an autoimmune disease characterised by inflammation, fibrosis and vasculopathy. Digital ulcers (DUs) are a frequent manifestation of vasculopathy in patients with SSc. Despite recent advances in pharmacological treatments, DU still have major health and economic implications. As there is currently no proven therapeutic strategy to promote DU healing, new treatments are urgently needed. Mesenchymal stem or stromal cells (MSCs) may provide a novel therapy for DU in SSc, because of their immunomodulatory and vasculoregenerative properties. Allogeneic MSC therapy involves functionally competent MSCs from healthy donors and may be used as ‘off-the-shelf’ available treatment. This study will evaluate whether allogeneic MSC therapy is a safe and potentially efficacious treatment for DU of SSc.Methods and analysisThe MANUS (Mesenchymal stromal cells for Angiogenesis and Neovascularization in digital Ulcers of Systemic Sclerosis) Trial is a double-blind randomised placebo-controlled trial. 20 patients with SSc with refractory DU will be randomised to receive eight intramuscular injections with either placebo or 50*106 MSCs. The primary outcome is the toxicity of the treatment at 12 weeks after administration. Secondary outcomes include (serious) adverse events, number and time to healing of DU, pain, reported hand function, quality of life and SSc disease activity. We will also evaluate changes in nailfold capillaroscopy pattern, as well as biochemical parameters and biomarkers in peripheral blood and skin biopsies. Follow-up visits will be scheduled at 48 hours and 2, 4, 8, 12, 24 and 52 weeks post-treatment. If the results confirm safety, feasibility and potential efficacy, a large multicentre randomised controlled trial with longer follow-up will be initiated focusing on efficacy.Ethics and disseminationThe study has been approved by the Dutch Central Committee on Research Concerning Human Subjects (protocol no: NL51705.000.15). The results will be disseminated through patient associations and conventional scientific channels.Trial registration numberNCT03211793; Pre-results.