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IntroductionDigital diagnostic decision support tools promise to accelerate diagnosis and increase health care efficiency in rheumatology. Rheumatic? is an online tool developed by specialists in rheumatology and general medicine together with patients and patient organizations. It calculates a risk score for several rheumatic diseases. We ran a pilot study retrospectively testing Rheumatic? for its ability to differentiate symptoms from existing or emerging immune-mediated rheumatic diseases from other rheumatic and musculoskeletal complaints and disorders in patients visiting rheumatology clinics.Materials and MethodsThe performance of Rheumatic? was tested using in three university rheumatology centers: (A) patients at Risk for RA (Karolinska Institutet, n = 50 individuals with musculoskeletal complaints and anti-citrullinated protein antibody positivity) (B) patients with early joint swelling [dataset B (Erlangen) n = 52]. (C) Patients with early arthritis where the clinician considered it likely to be of auto-immune origin [dataset C (Leiden) n = 73]. In dataset A we tested whether Rheumatic? could predict the development of arthritis. In dataset B and C we tested whether Rheumatic? could predict the development of an immune-mediated rheumatic diseases. We examined the discriminative power of the total score with the Wilcoxon rank test and the area-under-the-receiver-operating-characteristic curve (AUC-ROC). Next, we calculated the test characteristics for these patients passing the first or second expert-based Rheumatic? scoring threshold.ResultsThe total test scores differentiated between: (A) Individuals developing arthritis or not, median 245 vs. 163, P < 0.0001, AUC-ROC = 75.3; (B) patients with an immune-mediated arthritic disease or not median 191 vs. 107, P < 0.0001, AUC-ROC = 79.0; but less patients with an immune-mediated arthritic disease or not amongst those where the clinician already considered an immune mediated disease most likely (median 262 vs. 212, P < 0.0001, AUC-ROC = 53.6). Threshold-1 (advising to visit primary care doctor) was highly specific in dataset A and B (0.72, 0.87, and 0.23, respectively) and sensitive (0.67, 0.61, and 0.67). Threshold-2 (advising to visit rheumatologic care) was very specific in all three centers but not very sensitive: specificity of 1.0, 0.96, and 0.91, sensitivity 0.05, 0.07, 0.14 in dataset A, B, and C, respectively.ConclusionRheumatic? is a web-based patient-centered multilingual diagnostic tool capable of differentiating immune-mediated rheumatic conditions from other musculoskeletal problems. The current scoring system needs to be further optimized.

Objectives Our knowledge about the effect of tocilizumab (TCZ) on the synovium in rheumatoid arthritis (RA) is limited. The aim of this study was to investigate the effect of TCZ on citrullination and on inflammation in the synovial tissue and in the peripheral blood.Methods 15 patients with RA underwent synovial biopsy before and 8 weeks after TCZ initiation. Clinical evaluation was performed at baseline and at 8 weeks. Using immunohistochemistry, we evaluated the expression of CD68, CD3, CD20, osteoprotegerin (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) before and after treatment with TCZ. We also analysed the expression of protein arginine deiminase (PAD)-2 and PAD-4 enzymes in the synovial tissue and protein citrullination patterns with the help of anticitrullinated protein antibody (ACPA) clones 1325:04C03 and 1325:01B09. Serum levels of interleukin-6 (IL-6), IL-8, RANKL, OPG and C-terminal crosslinked telopeptide type II collagen were measured by ELISA. Paired-wise Wilcoxon signed-rank test was used to compare median values before and after treatment.Results Disease activity in patients was reduced from baseline to 8 weeks. Although PAD-2 and PAD-4 expressions remained unchanged after TCZ treatment, the binding of one ACPA clone decreased in the synovial tissue. TCZ did not affect the number of CD68+ macrophages or CD20+ B cells but induced significant decrease in the number of CD3+ T cells. RANKL and OPG expression remained unchanged in the synovial tissue. A significant increase in the levels of IL-6 and RANKL was observed in the serum. This increase was statistically significant in patients who responded to TCZ (achieving Clinical Disease Activity Index low disease activity or remission) but not in non-responders.Conclusions TCZ reduced synovial T-cell counts but not macrophages. A significant increase of serum IL-6 was observed in responders.

Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target

Background: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. Methods: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. Results: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. Conclusions: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.