BackgroundMixed Connective Tissue Disease (MCTD) is characterised by the presence of anti-RNP antibodies with clinical features also found in SSc, SLE and IIM. There is an ongoing debate of MCTD’s position as a CTD. A substancial proportion of MCTD patients develop Interstitial Lung Disease (ILD).ObjectivesThis study was conducted with the aims to explore the value of MCTD diagnosis and risk assessment by developing and validating ILD prediction models.MethodsMultivariable logistic regression analyses were performed in 3 international MCTD cohorts. ILD prediction model development from clinical and laboratory parameters was performed in the Norwegian MCTD cohort (n=119). External validation of the models were performed in the Hungarian MCTD cohort (n=196) and the MCTD cohort from Minnesota, US (n=50). ILD was diagnosed by chest CT examination.ResultsThe cohort characteristics are presented in table 1. An ILD prediction model including Pulmonary Function Test (PFT) results (table 2) and excluding PFT results was developed. The Hosmer-Lemeshow goodness of fit test (HL test) was. 31 and. 71 and the ROC was. 83 and. 78 respectively. The ILD prediction model including DLCO <60% was validated in the Hungarian MCTD cohort and showed good calibration and discrimination (HL test=0.95 and ROC=0.82). The ILD prediction model excluding PFT results showed good calibration and discrimination in both the Hungarian MCTD cohort (HL test=0.72 and ROC=0.80) and the MCTD cohort from Minnesota (HL test=0.96 and ROC=0.67).ConclusionsThe cohorts have different characteristics. Despite these differences the ILD prediction models developed in the Norwegian MCTD cohort have shown external validity when assessed in the Hungarian MCTD cohort and the MCTD cohort from Minnesota. Risk factors of ILD in MCTD patients are high levels of anti-U1 RNP antibodies, absence of arthritis and increasing age. The successive ILD prediction across different MCTD cohorts strengthens the value of MCTD diagnosis and anti-RNP antibody detection in clinical practice.Disclosure of InterestNone declared

Undifferentiated connective tissue disease (UCTD) is a unique clinical entity, a potential forerunner of well-established systemic autoimmune/rheumatic diseases. UCTD is characterized by the presence of various clinical symptoms, as well as a diverse repertoire of autoantibodies, resembling systemic autoimmune diseases. Since approximately one third of these patients consequently transform into a full-blown systemic autoimmune/rheumatic disease, it is of major importance to assess pathogenic factors leading to this progression. In view of the fact that the serological and clinical picture of UCTD and systemic autoimmune diseases are very similar, it is assumed that analogous pathogenic factors perpetuate both disease entities. In systemic autoimmune conditions, a quantitative and qualitative impairment of regulatory T cells has been shown previously, and in parallel, a relative dominance of pro-inflammatory Th17 cells has been introduced. Moreover, the imbalance between regulatory and Th17 cells plays a pivotal role in the initiation and propagation of UCTD. Additionally, we depict a cytokine imbalance, which give raise to a biased T cell homeostasis from the UCTD phase throughout the fully developed systemic autoimmune disease stage. The levels of interleukin (IL)-6, IL-12, IL-17, IL-23, and interferon (IFN)-γ were pathologically increased with a parallel reduction of IL-10. We believe that the assessment of Th17/Treg cell ratio, as well as the simultaneous quantitation of cytokines may give a useful diagnostic tool at the early UCTD stage to identify patients with a higher chance of consecutive disease progression toward serious systemic autoimmune diseases. Moreover, the early targeted immunomodulating therapy in these patients may decelerate, or even stop this progression, before the development of serious autoimmune conditions with organ damage.

Background Heat Shock Proteis (HSP) are intracellular proteins conserved in the course of evolution. Resulting from the antigenic homology of various species, HSP60 and antibodies produced against them can provoke inflammation. Several studies have confirmed that antibodies produced against HSP can facilitate the occurence of atherosclerosis in autoimmune diseases. Objectives The authors analysed the presence of anti-HSP antibodies and their correlation with cardiovascular diseases (CVD) in 30 MCTD patients. Methods The serum level of HSP60 antibodies was measured in the serum of thirty MCTD patients at the Division of Clinical Immunology. Fifteen out of the thirty patients had cardiovascular (CVD) disease. The antibodies were detected by ELISA assay. The lipid parameters, beside the paraoxonase activity, also identified the anti-U1RNP antibody, the antibody against endothelial cell (AECA), the anti-cardiolipin (anti-CL), the endothelin-1 (ET-1) levels and the intima-media (IMT) thickness. Results The level of anti-HSP60 antibodies was higher in the serum of patients with MCTD than in the control group (control and all MCTD: 104.0 (69.0-173.0) OD vs. 173.0 (125.0-265.0) OD, p<0.016). The CVD positive patients had a higher anti-HSP60 level than the CVD negative ones (MCTD CVD positive and CVD negative: 259.0 (176.0-316.0) OD, vs. 137.0 (84.0-157.0) OD, p=0.0013). The MCTD patients9 HDL cholesterol level (p=0.024) and their paraoxonase acitivity were lower than those of the control group. The anti-U1RNP (p<0.0022) serum level and the IMT were also higher in the control group (p=0.002). The CVD positive patients9 serum concentration of anti-HSP60 (p<0.0013), anti-CL IgG (p=0.0005) the ET-1 (p<0.05), and IMT (p<0.001) were significantly higher than that of the CVD negative control group. A close correlation was found between the anti-HSP60 antibody and the AECA (r=0.36, p=0.01) and between the serum levels of anti-HSP60 and ET-1 (r=0.62, p<0.001) Conclusions The presence of anti-HSP60 antibody showed a strong correlation in MCTD with CVD disease, endothelial injury, IMT value and can be considered an atherosclerotic risk factor in MCTD patiens. References Prohaszka Z, Furst G: Immunological aspects of heat-shock proteins-the optimum stress of life. Mol Immunol 2004; 41: 29-44 Bodolay E, Seres I, Szodoray P, Csipo I, Jakab Zs, Vegh J, Szilagyi A, Szegedi Gy, Paragh G: The evaluation of paraoxonase (PON) activity in patients with mixed connective tissue disease. J Rheumatol 2008. 35 (2): 237-43 Zhu J, Quyyumi AA, Rott D: Antibodies the human heat-shock protein 60 are associated with the presence and severity of coronary artery disease: evidence for and autoimmune component of atherogenesis. Circulation 2001; 103: 1071-75 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2894

OBJECTIVE:In this study the alteration of endothelial function, arterial stiffness and autoantibodies was investigated in patients with UCTD.METHODS:Thirty-one patients with UCTD were included in this prospective study. All the patients remained in the UCTD stage during the average 3.8 years follow-up period. The onset of UCTD was denoted as UCTD1, while the end of the follow-up period was called UCTD2. Flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT), autoantibodies [such as anti-SSA, anti-SSB, anti-DNA, anti-RNP, anti-CCP, aCL, anti-oxidized low-density lipoprotein (oxLDL) and AECA], von Willebrand factor antigen, thrombomodulin (TM), endothelin 1 (ET-1) and lipid parameters were measured.RESULTS:In the UCTD1 stage, high-sensitivity CRP (hsCRP) and endothelial cell activation and/or damage markers such as TM, ET-1 and AECA levels were significantly higher compared with controls (controls vs UCTD1: hsCRP, P < 0.0001; TM, P = 0.001; ET-1, P < 0.0001). In the UCTD2 stage, the carotid IMT increased (UCTD1 vs UCTD2, P = 0.01) and FMD further deteriorated (UCTD1 and UCTD2, P = 0.001). In UCTD2 there was a close correlation between the carotid IMT, and duration of the disease (r = 0.612, P < 0.001), the level of TM (r = 0.673, P < 0.001) and anti-oxLDL (r = 0.800, P < 0.001).CONCLUSION:Our data suggest that the presence of inflammation and autoantibodies provoke endothelial cell activation and/or injury in UCTD patients. The persistent endothelial dysfunction may provoke the development of atherosclerosis. FMD was found to be the most sensitive marker for arterial stiffness, and the increase of IMT clearly indicated the existence of preclinical atherosclerosis in UCTD patients.

Heat-shock protein 60 (Hsp60) has been shown to provoke inflammation, and anti-Hsp60 may facilitate the development of atherosclerosis. In this study, we have investigated 30 patients with mixed connective tissue disease (MCTD) and assessed anti-Hsp60 and their relationship to cardiovascular diseases (CVD). Out of 30 patients with MCTD, 15 had CVDs. Anti-Hsp60 antibody was determined by enzyme-linked immunosorbent assay. Since endothelial dysfunction and accelerated atherosclerosis are characteristic to MCTD, a wide array of MCTD-, endothelial dysfunction- and CVD-associated parameters was investigated: serum lipid levels, paraoxonase activity (PON1), rich nuclear ribonucleoprotein U1 (anti-U1RNP), anti-endothelial cell antibodies, anti-cardiolipin and anti-β2-glycoprotein I antibody isotypes (anti-CL and anti-β2GPI), endothelin-1 (ET-1) levels, also intima–media thickness (IMT), a quantitative indicator of atherosclerosis. In MCTD, anti-Hsp60 antibody levels were significantly higher than in healthy individuals (p < 0.02). MCTD patients with CVD had significantly higher levels of anti-Hsp60 compared to MCTD without CVD (p = 0.001). Patients with MCTD had significantly lower high-density lipoprotein cholesterol (p = 0.02) and PON activity (p < 0.001), and significantly increased systolic (p < 0.0002) and diastolic (p < 0.001) blood pressure compared to healthy individuals. Anti-U1RNP levels (p < 0.002) and IMT were higher in patients compared to controls (p = 0.002). The CVD-positive MCTD patients had increased anti-Hsp60 (p < 0.0013), anti-CL IgG (p = 0.0005), ET-1 serum concentration (p < 0.05) and IMT levels (p < 0.001) compared to MCTD patients without CVD. Anti-Hsp60 showed a strong correlation with anti-oxLDL (r = 0.36, p = 0.01) and serum ET-1 (r = 0.62, p < 0.001) and negative correlation with PON activity (r = −0.47, p = 0.01). Anti-Hsp60 indicates endothelial injury, CVD, and can function as a novel atherosclerotic risk factor, also a valuable diagnostic marker in patients with MCTD.

The authors report a rare case of a female patient with mixed connective tissue disease (MCTD) with coexisting antiphospholipid syndrome (APS). Five years after the diagnosis of MCTD high concentrations of anticardiolipin (anti-CL) and anti-β2-glycoprotein (anti-β2GPI) autoantibodies were present in the patient’s serum without thrombotic events. Epstein-Barr virus (EBV) reactivation provoked APS, with the clinical manifestations of livedo reticularis, digital gangrene and leg ulcers. Skin biopsy from the necrotic area showed multiple fibrin microthrombi in the superficial vessels. Corticosteroid pulse therapy, and plasma exchange in combination with synchronized cyclophosphamide was administered, which led to improvement of the digital gangrenes, while no new lesions developed. The number of CD27 high plasma cells decreased, and the previous high levels of autoantibodies also normalized in the peripheral blood. In the case of MCTD with coexisting APS combination therapy, including plasmapheresis has beneficial effects.

A shift in the balance between Th17-cells and regulatory T-cells (Treg) is an important feature of systemic autoimmune diseases (SAID), and may also contribute to their development. Hereby, we assessed the distribution of peripheral Th17 and Treg-cells in patients with undifferentiated connective tissue disease (UCTD), the forerunner of SAIDs and followed these parameters during the development towards definitive SAIDs. Fifty-one UCTD patients were investigated and followed-up for 3 years. Flow cytometry was used to identify and follow three cell-populations: Th17-cells (CD4+IL-17+ T-cells), natural regulatory T-cells (CD4(+)CD25(bright)FoxP3(+); nTregs) and IL-10 producing Type-1 regulatory T-cells (CD4+IL-10+ T-cells; Tr1). Altogether 37.3% of these patients progressed into SAIDs. Th17-cells were increased in UCTD vs. controls, which further increased in those, whom developed SAIDs eventually. The Th17/nTreg ratio gradually increased from controls through UCTD patients, reaching the highest values in SAID-progressed patients. Regarding the Th17/Tr1 ratios, a similar tendency was observed moreover Th17/Tr1 could distinguish between UCTD patients with, or without subsequent SAID progression in a very early UCTD stage. Various immunoserological markers showed association with Th17 and Th17/nTreg at baseline, indicating the consecutive development of a distinct SAID. The derailed Th17/Treg balance may contribute to disease progression therefore could function as a prognostic marker.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disorder, characterized by the presence of antibodies to U1-RNP protein. We aimed to determine phenotypic abnormalities of peripheral B cell subsets in MCTD. Blood samples were obtained from 46 MCTD patients, and 20 controls. Using anti-CD19, anti-CD27, anti-IgD and anti-CD38 monoclonal antibodies, the following B cell subsets were identified by flow cytometry: (1) transitional B cells (CD19+CD27-IgD+CD38(high)); (2) naive B cells (CD19+CD27-IgD+CD38(low)); (3) non-switched memory B cells (CD19+CD27+IgD+); (4) switched memory B cells (CD19+CD27+IgD-); (5) double negative (DN) memory B cells (CD19+CD27-IgD-) and (6) plasma cells (CD19+CD27(high)IgD-). The proportion of transitional B cells, naive B cells and DN B lymphocytes was higher in MCTD than in controls. The DN B cells were positive for CD95 surface marker. This memory B cells population showed a close correlation with disease activity. The number of plasma cells was also increased, and there was an association between the number of plasma cells and the anti-U1RNP levels. Cyclophosphamide, methotrexate, and corticosteroid treatment decreased the number of DN and CD27(high) B cells. In conclusion, several abnormalities were found in the peripheral B-cell subsets in MCTD, which reinforces the role of derailed humoral autoimmune processes in the pathogenesis.

Conductor of regulatory cells: Does vitamin D restore the shifted

Serum and intracytoplasmic cytokines are mandatory in host defense against microbes, but also play a pivotal role in the pathogenesis of autoimmune diseases by initiating and perpetuating various cellular and humoral autoimmune processes. The intricate interplay and fine balance of pro- and anti-inflammatory processes drive, whether inflammation and eventually organ damage will occur, or the inflammatory cascade quenches. In the early and late, as well as inactive and active stages of autoimmune diseases, different cellular and molecular patterns can dominate in these patients. However, the simultaneous assessment of pro- and anti-inflammatory biomarkers aids to define the immunological state of a patient. A group of the most useful inflammatory biomarkers are cytokines, and with increasing knowledge during the last decade their role have been well-defined in patients with autoimmune diseases and immunodeficiencies. Multiple pathological processes drive the development of autoimmunity and immunodeficiencies, most of which involve quantitative and qualitative disturbances in regulatory cells, cytokine synthesis and signaling pathways. The assessment of these biomarkers does not aid only in the mechanistic description of autoimmune diseases and immunodeficiencies, but further helps to subcategorize diseases and to evaluate therapy responses. Here, we provide an overview, how monitoring of cytokines and regulatory cells aid in the diagnosis and follow-up of patients with autoimmune diseases and immunodeficiencies furthermore, we pinpoint novel cellular and molecular diagnostic possibilities in these diseases.

Objective. To study the survival rate and prognostic indicators of mixed connective tissue disease (MCTD) in a Hungarian population. Methods. Two hundred eighty patients with MCTD diagnosed between 1979 and 2011 were followed prospectively. Clinical features, autoantibodies, and mortality data were assessed. Prognostic factors for survival were investigated and survival was calculated from the time of the diagnosis by Kaplan-Meier method. Results. A total of 22 of 280 patients died: the causes of death were pulmonary arterial hypertension (PAH) in 9 patients, thrombotic thrombocytopenic purpura in 3, infections in 3, and cardiovascular events in 7. The 5, 10, and 15-year survival rates after the diagnosis was established were 98%, 96%, and 88%, respectively. The deceased patients were younger at the diagnosis of MCTD compared to patients who survived (35.5 ± 10.4 vs 41.8 ± 10.7 yrs; p < 0.03), while there was no difference in the duration of the disease (p = 0.835). Our cohort study showed that the presence of cardiovascular events (p < 0.0001), esophageal hypomotility (p = 0.04), serositis (p < 0.001), secondary antiphospholipid syndrome (p = 0.039), and malignancy (p < 0.001) was significantly higher in the deceased patients with MCTD. The presence of anticardiolipin (p = 0.019), anti-β2-glycoprotein I (p = 0.002), and antiendothelial cell antibodies (p = 0.002) increased the risk of mortality. Conclusion. Overall, PAH remained the leading cause of death in patients with MCTD. The prevalence of cardiovascular morbidity and mortality, malignancy, and thrombotic events increased during the disease course of MCTD. The presence of antiphospholipid antibodies raised the risk of mortality.

Background Mixed connective tissue disease (MCTD) is a systemic autoimmune disorder, accompanied by chronic inflammation involving several organs. MCTD was first described by “Sharp et al.” in 1972 but there is still little known about the long-term outcome of patients and factors influencing the mortality in MCTD. Objectives To study the survival rate and prognostic indicators of MCTD in a large Hungarian patient population. Methods Two hundred and eighty patients with MCTD diagnosed between 1979 and 2011 were prospectively followed. Clinical features, autoantibodies and mortality data were assessed. Prognostic factors for survival were investigated and survival was calculated from the time of the diagnosis by Kaplan-Meier method. Results A total of 22 of 280 patients died during the follow-up period: the causes of death were pulmonary arterial hypertension (PAH) in 9 patients, thrombotic thrombocytopenic purpura in 3 patients, infections in 3 patients and 7 patients died due to cardiovascular events. The 5, 10, 15 year survival rates after the diagnosis was established were 98%, 96% and 88%. The deceased patients were younger at the diagnosis of MCTD compared to those patients who survived (35.5±10.4 vs. 41.8±10.7 years; p<0.03), while there was no difference in the duration of the disease (p=0.835). Our cohort study showed that the presence of cardiovascular events (p<0.0001), esophageal hypomotility (p=0.04), serositis (p<0.001), secondary antiphospholipid syndrome (p=0.039) and malignancy (p<0.001) was significantly higher in the deceased MCTD patients. The presence of anti-cardiolipin (p=0.019), anti-β2-glycoprotein I (p=0.002) and anti-endothelial cell antibodies (p=0.002) increased the risk of mortality. Conclusions Overall, PAH remained the leading cause of death in patients with MCTD. The prevalence of cardiovascular morbidity and mortality, malignancy and thrombotic events is increasing during the disease course of MCTD and these factors negatively influence the survival. The presence of antiphospholipid antibodies raised the risk of mortality. Disclosure of Interest None Declared

Takács István dr.1 ■ Benkő Ilona dr.2 ■ Toldy Erzsébet dr.3 Wikonkál Norbert dr.4 ■ Szekeres László dr.5 ■ Bodolay Edit dr.6 Kiss Emese dr.7 ■ Jambrik Zoltán dr.8 ■ Szabó Boglárka dr.8 Merkely Béla dr.8 ■ Valkusz Zsuzsa dr.9 ■ Kovács Tibor dr.10 Szabó András dr.11 ■ Grigoreff Orsolya dr.1 ■ Nagy Zsolt dr.1 Demeter Judit dr.1 ■ Horváth Henrik Csaba dr.1 Bittner Nóra dr.12 ■ Várbíró Szabolcs dr.13 ■ Lakatos Péter dr.1

The aim of the present study was to assess the autoantibody profile, dominant clinical symptoms and cluster characteristics of different mixed connective tissue disease (MCTD phenotypes. Two-hundred-and-one patients with MCTD were followed-up longitudinally. Five clinical parameters, Raynaud's phenomenon, pulmonary artery hypertension (PAH), myositis, interstitial lung disease (ILD), erosive arthritis and five auto-antibodies besides anti-U1RNP, antiendothelial cell antibodies (AECA), anti-CCP, anti-cardiolipin (anti-CL), anti-SSA/SSB and IgM rheumatoid factor (RF) were selected for cluster analysis. The mean age of patients was 52.9 ± 12.4 years and the mean follow-up of the disease was 12.5 ± 7.2 years. Patients were classified into three cluster groups. Cluster 1 with 77 patients, cluster 2 with 79 patients and cluster 3 with 45 patients. In cluster 1 the prevalence of PAH (55.8%; p < 0.001), Raynaud's phenomenon (92.2%; p < 0.001) and livedo reticularis (24.6%, p < 0.001) was significantly greater than in cluster 2 and 3. In cluster 2, the incidence of ILD (98.7%; p < 0.001), myositis (77.2%; p < 0.001), and esophageal dysmotility (89.8%; p < 0.001) was significantly greater than that in cluster 1 and 3. In cluster 3, anti-CCP antibodies were present in 31 of 45 patients (68.8%) with erosions. Anti-CCP antibodies were present in 37 of 42 patients (88.0%) with erosions. PAH, angina, venous thrombosis was observed in cluster 1 and pulmonary fibrosis in cluster 2, musculosceletal damage, gastrointestinal symptoms and osteoporotic fractures were most frequent in cluster 3. Cumulative survival assessment indicated cluster 1 patients having the worst prognosis. Cluster analysis is valuable to differentiate among various subsets of MCTD and useful prognostic factor regarding the disease course.

The metabolism of vitamin D is unique in the human body and its diverse effects are present in almost every organ. Vitamin D deficiency is one of the most prominent health issues in the civilized world. For the solution of this concern an extensive collaboration is imperative. Recognizing this necessity the most prominent Hungarian medical associations fighting with the effects of vitamin D deficiency worked out a collective consensus on the importance, diagnosis, prevention and suggested therapy of vitamin D deficiency. Along with the clinical guidelines of the different associations, the result of this consensus could serve as guidance for the practicing doctors in the prevention and therapy of vitamin D deficiency. In addition the consensus aims to direct the attention of decision-makers and the general public on the significance of this issue.