Diána Simon

Construct validity evaluation of the European Scleroderma Study Group activity index, and investigation of possible new disease activity markers in systemic sclerosis

OBJECTIVES To evaluate the construct validity of the European Scleroderma Study Group (EScSG) activity index and to propose modifications if necessary. METHODS One hundred and thirty-one consecutive patients were investigated and re-evaluated 1 year later. Modified Rodnan skin score (MRSS), skin ulcers and joint contracture numbers, hand anatomic index (HAI), BMI, spirometry, carbon monoxide diffusing capacity (DL(CO)), left ventricular ejection fraction, pulmonary arterial hypertension, HAQ Disability Index (HAQ-DI), patient skin self-assessment questionnaire and several biomarkers were recorded, in addition to the data required for the EScSG activity index. Statistical analysis was performed by categorical principal component analysis (CATPCA). RESULTS The EScSG activity index appeared in the same dimension as the HAQ-DI, ulcer score and joint contractures, MRSS, patient-reported skin score and HAI by CATPCA. Parameters of lung involvement appeared in another dimension. We constructed a 12-point activity index that was equally associated with both dimensions, by adding the forced vital capacity/DL(CO), change in DL(CO), change in the ulcer scores, HAQ-DI and patient-reported skin score. Biomarkers including vascular endothelial growth factor, soluble P-selectin glycoprotein ligand-1, CRP and albumin were related to both the EScSG and the 12-point index, though they did not improve the total variance of the model. CONCLUSION The construct validity of the EScSG activity index is good, though the lung-related disease activity may not be sufficiently represented. Further validation steps may be required for both the EScSG and our 12-point activity index.

Anti-topoisomerase I autoantibodies in systemic sclerosis☆

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin, vascular abnormalities and activation of the immune system. The majority of SSc patients have autoantibodies against nuclear antigens. Among these antibodies against topoisomerase I (topo I) are frequently detected in sera of SSc patients. Since the discovery of these antibodies as immunoglobulins reacting with a 70 kDa nuclear protein (Scl-70), a massive body of clinical and experimental data has been generated. In this review we summarize accumulated evidence about anti-topo I autoantibodies in SSc, including results of epitope mapping studies and investigations on the possible pathogenic role of these antibodies.

Increased proportions of functionally impaired regulatory T cell subsets in systemic sclerosis

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Divergence of Vascular Specification in Visceral Lymphoid Organs—Genetic Determinants and Differentiation Checkpoints

Abstract Despite their functional similarities, peripheral lymphoid tissues are remarkably different according to their developmental properties and structural characteristics, including their specified vasculature. Access of leukocytes to these organs critically depends on their interactions with the local endothelium, where endothelial cells are patterned to display a restricted set of adhesion molecules and other regulatory compounds necessary for extravasation. Recent advances in high throughput analyses of highly purified endothelial subsets in various lymphoid tissues as well as the expansion of various transgenic animal models have shed new light on the transcriptional complexities of lymphoid tissue vascular endothelium. This review is aimed at providing a comprehensive analysis linking the functional competence of spleen and intestinal lymphoid tissues with the developmental programming and functional divergence of their vascular specification, with particular emphasis on the transcriptional control of endothelial cells exerted by Nkx2.3 homeodomain transcription factor.

AB0042 Natural Autoantibodies Against Citrate Synthase and DNA Topoisomerase I in Patients with Rheumatoid Arthritis and Spondyloarthritis Receiving Anti-TNF-α Therapy

Background and objectives Natural autoantibodies bind to evolutionarily conserved antigens in organs and tissues of the body and may have regulatory functions in the immune system and play a role in the clearance of apoptotic cells. It has been known that some patients treated with anti-TNF-α agents develop autoantibodies, including antinuclear and anti-double-stranded DNA antibodies. However, in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) treated with TNF-α blockers there have been no studies regarding the presence of natural autoantibodies against citrate synthase (CS), a mitochondrial inner membrane enzyme, and DNA topoisomerase I (topo I), an enzyme that relaxes the superhelical stress in DNA. Therefore, our aim was: (1) to investigate whether patients with RA and SpA receiving anti-TNF-α treatment produce natural autoantibodies to CS and topo I; and (2) to test whether such therapy affects the levels of these autoantibodies. Materials and methods The study included a total of 36 patients (20 with RA, 16 with SpA) who received certolizumab or etanercept therapy. The levels of anti-CS and anti-topo I (immunodominant fragment) autoantibodies (IgM and IgG) were measured in the blood serum using enzyme-linked immunosorbent assay developed in our laboratory. The measurements were done at the starting point (0), 6, or 12 months of therapy. Results In the combined patient group (RA + SpA) we detected significantly increased levels of anti-CS IgM and IgG antibodies after 6 and 12 months of anti-TNF-α therapy. The anti-topo I IgM and IgG were significantly elevated only after 12 months of therapy, when compared to baseline antibody levels before the start of treatment. Conclusions The significance of the presence of anti-CS and anti-topo I natural autoantibodies is still a subject of research. We hypothesise that the increase in the levels of these natural autoantibodies might be a marker for the functional role of the natural immune system in patients treated with anti-TNF-α blockers. Because TNF-α is a pleiotropic cytokine, blocking its function can elicit various side-effects, including haematological, immunological, and others. Natural autoantibodies may function in the clearance of apoptotic cells and they may help restore the immunological milieu by reducing inflammation and protecting against some infections, functions in which the natural immune system has been implicated.

AB0686 Switched Memory B Cell Expansion is Associated with Disease Severity in Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury, autoimmune phenomena, inflammation, and fibrosis of the skin and various internal organs. Several lines of evidence indicate that abnormal B-cell function plays a key role in the development of SSc. The anti-CD20 monoclonal antibody therapy seems to show some clinical efficacy in SSc further emphasizing the importance of B cells in the pathomechanism of the disease. The B-cell compartment in peripheral blood of SSc patients contains an elevated number of naive and a decreased number of memory B cells. Objectives The aim of the present research was to set up an algorithm for the extended analysis of these B-cell subsets and to evaluate the clinical significance of the defined subpopulations. Methods Peripheral blood samples were obtained from SSc patients and healthy controls, PBMCs were isolated using ficoll gradient centrifugation, followed by magnetic bead separation of CD19+ B cells. Multiparametric flow cytometry was performed with antibodies specific for CD27, IgD, CD80, CD95 molecules. Detection of CD27 and IgD was applied to distinguish between naive (CD27-IgD+) and memory (CD27+) B cells. IgD posivity was also used to separate non-switched (CD27+IgD+) and switched (CD27+IgD-) memory subsets. In addition to expression of CD80, which provides a co-stimulatory signal necessary for T cell activation and survival, expression of CD95 – FAS receptor was also examined to investigate the activation state of the previously identified B cell subpopulations. Results The ratio of naive B cells was higher, the proportion of memory B cells, including both subsets, was decreased in SSc patients compared to healthy controls. Among SSc patients the ratio of switched memory and CD95+ memory B cells was higher in diffuse cutaneous SSc and in patients with pulmonary fibrosis. The proportion of switched memory B cells was also elevated in the anti-Scl-70 antibody positive group compared to ACA positive patients. In dcSSc patients the ratio of CD95+ memory B cells was also higher. Conclusions According to our results detailed flow cytometric analysis of naive and memory B-cell subsets could contribute to better distinction between the two SSc subtypes and to the evaluation of disease severity, consequently may be a useful new tool in routine immunological diagnostics. Acknowledgements This work was supported by Hungarian Scientific Research Fund - OTKA 75912 and 112939. Disclosure of Interest None declared

AB0731 Investigation of Regulatory T Cells in Systemic Sclerosis

Background Systemic sclerosis (SSc) is a chronic autoimmune disease affecting multiple organs. It is characterized by excessive extracellular matrix deposition in the skin and internal organs accompanied by vascular damage and alterations of immune function. Regulatory T cells take part in the regulation of the immune system, development of tolerance against self antigens and the inhibition of autoimmunity. The role of Tregs seem to be essential in the pathogenesis of SSc. Objectives The aim of our work was to study the changes in the ratio of Treg populations and their cytokine production in peripheral blood samples of SSc patients compared to healthy controls (HC) and to evaluate the changes in relation to the activity and severity of the disease. Methods Peripheral blood samples were collected from SSc patients regularly visiting the Department of Rheumatology and Immunology not on immunosuppressive therapy and from HCs. PBMCs were isolated using ficoll gradient centrifugation and stimulated with the combination of PMA/ionomycin/brefeldin. Tregs (CD4+CD25+Foxp3+CD127-), Treg subgroups based on CD62L positivity and their cytokine production (IL-10, TGFβ) were investigated using flow cytometry. Results The ratio of Treg cells was increased in SSc compared to HCs while percentage of the IL-10 producing Tregs was lower. The ratio of CD62L+ Treg cells was higher in SSc than in HCs and their TGFβ producing proportion was decreased. The percentage of the IL-10 producing CD62L+ Treg cells was lower in active SSc compared to the inactive group. Conclusions According to our results the alterations of the ratio of Treg populations and their cytokine producing proportion in SSc patients is different from HCs and correlates with the activity of the disease. Consequently our study can help the early diagnostics of SSc and the assessment of its activity. Disclosure of Interest None declared

Natural and Pathologic Autoantibodies

Detection and characterization of autoantibodies reacting with self-antigens is generally used in laboratory diagnostics. However, the presence of different autoantibodies in the blood serum doesn’t mean automatically a pathologic condition. Autoantibodies are present both in different diseases as autoimmune diseases, chronic inflammation or infections, and in healthy individuals without any symptoms. The present paper discusses the detailed analysis of recognition pattern and fine epitope specificity of these autoantibodies to better understand of their occurrence and evolution, and their role in physiologic and pathologic conditions.