Giovanni Barausse

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. Methodology Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. Principal Findings Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. Conclusions The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage.

Digital Amputation in Systemic Sclerosis: Prevalence and Clinical Associations. A Retrospective Longitudinal Study

Objective. To evaluate the prevalence of digital necrosis requiring surgical amputation in a single-center group of patients with systemic sclerosis (SSc) and to compare the characteristics of patients with and those without this severe complication. Methods. We reviewed the medical records of 188 patients with SSc [162 women, 26 men, mean age 59.2 yrs, mean disease duration 8.0 yrs, mean time from onset of Raynaud’s phenomenon (RP) 11.7 yrs, median followup duration 92 mo] enrolled in the Rheumatology Unit since 2004. Demographic and clinical features were collected, as well as the presence of the typical risk factors for atherosclerosis. Results. Nine patients (4.8%) underwent partial or total surgical digital amputation because of necrotic process; all these patients except 1 had a long history of multiple and persisting digital ulcers. All 9 patients had concomitant large-vessel involvement. Comparison of cases with and without digital amputation showed that this complication was associated with older age, long history of RP, long disease duration, presence of anticentromere antibody, and coexistence of peripheral artery disease and hypercholesterolemia. Discussion. We noted that 4.8% of patients with SSc underwent digital amputation. Our retrospective analysis suggests that peripheral artery disease is strongly associated with digital amputation. The preventive strategy for digital ulcers and amputation associated with SSc should include an extensive diagnostic and preventive investigation for peripheral atherosclerosis.

Vitamin D and autoimmune rheumatic diseases.

Sir, We have read the letter by Belloli L et al. [1], who commented on our paper concerning low levels of vitamin D in patients affected by systemic sclerosis (SSc) [2] and showed further data on hypovitaminosis D in SSc. We agree with Belloli L et al. that in Italy, hypovitaminosis D is very frequent not only in systemic rheumatic diseases as SSc but also in very different unrelated diseases like osteoarthritis [1] as well as in elderly women [3], but not in premenopusal women, in whom low concentrations of vitamin D were found in 27.8% and 3.4% of the cases in winter and in summer, respectively [4]. But in our opinion, the interest concerning hypovitaminosis D in SSc and in other connective tissue diseases was that low levels of vitamin D may influence clinical manifestations; in SLE hypovitaminosis D has been associated with disease activity and severity [5–7] and in undifferentiated connective tissue diseases with a high risk of developing a well-defined connective tissue disease [8]. In rheumatoid arthritis, the serum concentration of vitamin D correlated negatively with disease activity (DAS28) and disability as evaluated by Health Assessment Questionnaire and mobility activities of daily living score [9]. In SSc, an inverse correlation between the serum level of vitamin D has been reported with disease activity and with acutephase reactants [10]; vitamin D deficiency was also associated with more severe diseases [2], supporting both the immunomodulatory properties [11] and the antifibrotic action of the vitamin [12, 13]. Moreover, very recently, it has been reported that vitamin D plays an antiproliferative effect on mesenchymal multipotent cells [14] revealing possible new perspectives on the pleiotropic properties of the molecule. As recently reviewed, the development of autoimmune rheumatic diseases is dependent on the interaction between genetic background and many environmental factors, which include hypovitaminosis D [15]. Therefore, low vitamin D status may represent an easily modifiable factor, whereby treatment may have a beneficial impact on both the development and the clinical phenotype of connective tissue diseases.

AB0684 Bone Metabolism in a Large Cohort of Patients with Systemic Sclerosis

Background Patients with systemic sclerosis (SSc) may have classical and disease-related risk factors for osteoporosis (OP)(1) but there is no agreement about the association between SSc and OP (2). Objectives The aim of this study was to evaluate in a large size cohort of SSc patients bone mineral density (BMD) and to analyze its possible determinants. Methods 106 consecutive outpatients affected by SSc were enrolled for the present study. All patients underwent evaluation of BMD and vertebral fractures (VFA) using DXA (GE Lunar iDXA), pulmonary function test, nailfold videocapillaroscopy, blood samples. For the statistical analysis we preferred to use Z-score to BMD or T-score since the population was composed by patients with different ages and of both sexes. Results Most patients were female with a limited disease pattern (84,9% and 651,% respectively). Mean age was 58.8±12.6, mean BMI was 25.3±4.4 and disease duration was 10 years ±5.8. 25.7 had interstitial lung disease. Parameters correlated with bone metabolism are shown in table 1. 31.1% and 26.4% respectively were or had been smokers or steroid-treated. 65.1% were taking vitamin D supplementation and 18.9% bisphosphonates. 74.4% of women were in menopause. Mean neck Z-score was significantly lower than 0. No significant differences were found for other sites. Female patients were shown to have a total femur and neck Z-score significantly lower than 0 (p=0.028 and p<0.001, respectively). This data was not confirmed for other sites. Thirteen patients out of 96 with a good quality VFA (13.4%) had at least one morphometric non-clinical vertebral fracture. In univariate analysis total femur Z-score was lower in female (p=0.050) and in smokers (p=0.063) and positively correlates with BMI (p=0.001), neck Z-score positively correlates with age (p=0.016) and whole body Z-score positively correlates with BMI (p<0.001). No correlations were found for lumbar Z-score. The multivariate analysis confirmed the positive correlation between BMI and total femur and whole body Z-score and between age and neck femur Z-score (p=0.01, p<0.001 and p=0.019, respectively). Lung fibrosis was shown to correlate with a lower whole body Z-score in multivariate analysis (p=0.049). All data were confirmed even if corrected for vitamin D serum levels and vitamin D supplementation.Table 1 Mean (SD) N (%) Neck Z-score −0,4 (0,835) Total hip Z-score −0,2 (0,962) Lumbar Z-score 0,2 (1,4) Whole body Z-score 0,2 (0,9) Osteoporosis according to T-score  normal 32 (29,9)  osteopenia 50 (46,7)  osteoporosis 26 (24,4) 25OH-vitamin D (ng/ml) 32,4 (17,4) Conclusions Our study evaluates a large sized and well characterized SSc population by a quite complete multivariate analysis aiming to control the most important confounding factors for OP. We found a modest risk of low BMD in patients with SSc and the important protective role of BMI. References Loucks J, Pope JE. Osteoporosis in scleroderma. Semin Arthritis Rheum 2005;34:678-82 Omair MA. Low Bone Density in Systemic Sclerosis. A Systematic Review. J Rheumatol 2013;40:1881-90. Disclosure of Interest None declared

AB1196 Paediatric autoimmune neuropsychiatric disorder associated with group a streptococcal infection (PANDAS): Cardiac and articular involvement

Background PANDAS is a syndrome characterized by obsessive-compulsive disorder (OCD) and/or tic disorder with symptom exacerbations in children after group A β-hemolytic streptococcal (GAS) infections. No cardiac nor articular involvement is present as in the case of Sydenham Chorea. Antinuclear antibodies and nuclear basal involvement at brain MR may be encountered. Objectives We evaluated the possible misdiagnosed cardiac and articular involvement in 30 children affected by PANDAS and regularly assessed at an out-patient ambulatory in the period from 2007 to 2011. Methods 30 children affected by PANDAS according the Swedo SA diagnostic criteria were evaluated at the out-patient ambulatory of the Rhematologic and Neuropsichiatric departments of Verona’s University Hospital in the period from 2007 to 2011; thorough cardiologic evaluation encompassing ECG and echocardiogram was performed Results mean age of the 30 young patients (M:F 23:7) was 7±3 years; all had sierologic evidence of previous streptococcal infection; ECG was normal in all the cases as well as cardiac echogram with the exception of 2 cases of trivial mitral regurgitation without structural valvular alterations; no patient was affected by perferic arthritis, arthargias nor morning stiffness. Conclusions the absence of cardiac or articular involvement in PANDAS patients, as evidenced in this study, confirms its differential diagnosis with Sydenham Chorea Disclosure of Interest None Declared

THU0339 Power doppler ultrasonograpic findings in healthy pediatric enthesises

Background In Rheumatic Ultrasonographic evaluation interest has been raised about the use of power Doppler ultrasonography (PDUS) in the assessment of inflammatory changes in the articular and periarticular structures of children.Nevertheless, there are methodological and interpretative problems due to a lack of ultrasound definition of both the normal joint components and their physiological vascularization in the young population. Objectives To perform PDUS examination of the principal enthesises of healthy children, focusing particularly on their vascularization. Methods We enrolled 33 children (age between 2 and 12 years) Children were assessed by an expert pediatric rheumatologist in order to exclude subjects with clinical history or evidence of muscoloskeletal diseases. The same day a rheumatologist, performed a PDUS examination of the enthesises. The following enthesises were scanned: distal quadriceps tendon, distal and proximal patellar ligament, distal Achilles tendon, medial and lateral epicondyle tendons of the elbow. According to the OMERACT the following elements were evaluated: enthesis thickness, structure or calcifications, bone proliferation, erosions.Vascularization at enthesis level was judged as present or absent in three areas: 1) at the outer edge of the enthesis (peripheral vessel), 2) inside the enthesis (intratendinous vessel), 3) inside the epiphyseal cartilage at the insertion of the enthesis (cartilaginous vessel). Results The remaining 31 subjects were 18 girls and 13 boys, average age 8 years (±2.9). 256 enthesises were evaluated: 180 enthesises of the knee, 40 Achilles enthesises, 36 elbow enthesises. In the gray scale no abnormalities in the enthesises, in the cortical bone or epiphyseal cartilage were idientified. 1) The power Doppler examination showed at least a peripheral vessel around the quadriceps enthesis in 43% of the subjects, around the proximal patellar enthesis in 20%, around the distal patellar enthesis in the 10%, around the Achilles enthesis in the 5%, none around the lateral epicondyle enthesis and in the 22% of subjects around the medial epicondyle enthesis of the elbow. The quadriceps and superior patellar peripheral vessels usually go into the middle part of the patella.2) An intratendinous power Doppler signal was found only in the quadriceps enthesises and it was detected in the 23% of the subjects; this vascular signal appeared always in the same area, that was the distal part of the enthesis, near to the superior pole of the patellar cartilage.3) Cartilagineous vessels adjacent to the enthesis were found close to the quadriceps tendon in the 20% of subjects, close to the proximal patellar tendon in the 30% of the cases, close to the distal patellar tendon in the 47% of the children, close to the Achilles tendon in the 10% of the entheses, close to the lateral epicondyle entheses of the elbow in 67% of the subjects and in the 47% of subjects close to the medial epicondyle entheses of the elbow. Conclusions Power Doppler examination of pediatric enthesis in healthy children can show physiological vessels. These vessels are mainly peritendineus or cartilaginous.Intratendineous vascularization is absent in healthy enthesis, except for the distal part of quadriceps tendon and this finding could represent a pitfall if detected within a context of disease Disclosure of Interest None Declared