E. Alessandri

Laser speckle contrast analysis: a new method to evaluate peripheral blood perfusion in systemic sclerosis patients

Objective The aim of this pilot study was to assess peripheral blood perfusion (PBP) by a new technique, the laser speckle contrast analysis (LASCA), in systemic sclerosis (SSc) patients showing different patterns of nailfold microangiopathy. Correlations between LASCA and single laser Doppler flowmetry (LDF) analysis were also checked. Methods Sixty-one SSc patients and 61 healthy subjects were enrolled. PBP was evaluated using LASCA and LDF. Scleroderma patterns and microangiopathy evolution score (MES) were assessed by nailfold videocapillaroscopy (NVC). Results As detected by LASCA and LDF, PBP was lower in SSc patients than in healthy subjects (p<0.0001), showing SSc patients with the ‘Early’, ‘Active’ or ‘Late’ NVC pattern a progressively lower PBP (p=0.04 and p=0.002, respectively). There was a negative correlation between PBP and MES values (p=0.006 and p=0.002 for LASCA and LDF, respectively). A positive correlation was detected between LASCA and LDF values, in all subjects (p<0.0001). However, LASCA evaluates larger skin areas, is significantly less time consuming, is more accepted by patients and shows lower intra-operator variability than LDF. Conclusions LASCA detected lower PBP in SSc patients than in healthy subjects, and for the first time, LASCA perfusion values were found correlated with progression of NVC patterns of microangiopathy.

Correlations between nailfold microangiopathy severity, finger dermal thickness and fingertip blood perfusion in systemic sclerosis patients

Objective The aim of this study was to identify possible correlations between nailfold microangiopathy severity, finger dermal thickness (DT) and fingertip blood perfusion (FBP) in systemic sclerosis (SSc) patients. Methods Fifty-seven SSc patients and 37 healthy subjects were enrolled. All patients were evaluated by nailfold videocapillaroscopy (NVC) to classify and score the severity of microangiopathy. Both modified Rodnan skin score (mRss) and skin high-frequency ultrasound were used to detect finger DT. Laser Doppler flowmetry (LDF) was employed to detect FBP. Results A positive correlation was found between nailfold microvascular damage severity and both ultrasound-DT (p=0.028) and mRss values (p<0.0001). In particular, both ultrasound-DT and mRss were found progressively higher in patients with ‘Early’, ‘Active’ or ‘Late’ NVC pattern of microangiopathy. A negative correlation was observed between nailfold microvascular damage severity and FBP (p<0.0001), showing the lowest FBP of the patients with more advanced NVC patterns. A negative correlation was observed between FBP, and both ultrasound-DT (p=0.007) and mRss values (p=0.0002). SSc patients showed a higher ultrasound-DT at the level of the fingers, as well as a lower FBP than healthy subjects (p<0.0001). Conclusions This study demonstrates a relationship between nailfold microangiopathy severity, DT and FBP in SSc patients.

Effects of Macitentan and Its Active Metabolite on Cultured Human Systemic Sclerosis and Control Skin Fibroblasts

Objective. To investigate the effects of the endothelin 1 (ET-1) receptor antagonists (ETRA) macitentan, its active metabolite ACT-132577, and bosentan on myofibroblast activation and extracellular matrix production induced by ET-1 in cultured systemic sclerosis (SSc) and control skin fibroblasts. Methods. Fibroblasts were obtained from skin biopsies of 6 patients with SSc and 5 healthy subjects. Some cultured cells were untreated or treated with macitentan, ACT-132577, or bosentan alone (10 μM). Other cultured cells were treated with ET-1 alone (100 nM) or with ETRA, and after 1 h, also with ET-1. After 48 h of treatment, myofibroblast activation was investigated to evaluate the α-smooth muscle actin (α-SMA) expression by immunofluorescence; type I collagen (COL-1) and fibronectin (FN) were investigated by immunocytochemistry, Western blotting, and quantitative real-time PCR (qRT-PCR). Statistical analysis was performed by the nonparametric Mann-Whitney U test. Results. In cultured SSc skin fibroblasts, only the treatment with macitentan significantly reduced the basal level of α-SMA expression (p = 0.03 vs untreated cells). Macitentan also significantly reduced the basal level of COL-1 synthesis, similarly to bosentan (p < 0.05 vs untreated cells). Macitentan or ACT-132577 antagonized the ability of ET-1 to further induce α-SMA expression (p = 0.03), COL-1, and FN synthesis (p = 0.03, p = 0.005); bosentan showed similar effects. These results obtained by immunofluorescence and immunocytochemistry were confirmed by Western blotting and qRT-PCR. The downregulatory effects exerted by ETRA were observed also in cultured human control skin fibroblasts. Conclusion. Macitentan and ACT-132577 seem to downregulate in vitro the profibrotic myofibroblast phenotype induced by ET-1 in cultured human SSc skin fibroblasts.

Advances in nailfold capillaroscopic analysis in systemic sclerosis

Systemic sclerosis is an autoimmune connective tissue disease characterized by early and persistent microvascular impairment which leads to functional and organic manifestations, with progressive fibrosis of the skin and internal organs. Morphological and functional assessment of the peripheral microvasculature is a must, not only for diagnosis but also for the prognosis and therapeutical follow-up of systemic sclerosis patients, as reported in recent studies. Nailfold videocapillaroscopy is the validated technique for the study of scleroderma microangiopathy as it is able to detect peripheral microvascular morphology and both classify and score the capillary abnormalities into different microangiopathy patterns (‘Early’, ‘Active’ and ‘Late’). Indeed, the possibility to early diagnose and follow the microvascular changes and the safety of the technique have made nailfold videocapillaroscopy a mandatory tool for patient evaluation and included its assessment in the new systemic sclerosis classification criteria. Important links between nailfold videocapillaroscopy patterns and systemic sclerosis clinical manifestations have been described.

Correlation between bone quality and microvascular damage in systemic sclerosis patients

Objectives SSc patients are recognized as presenting an increased risk of altered bone mass. The aim of this study was to assess the bone quality, by trabecular bone score (TBS), in SSc patients in correlation with different levels of microvascular damage, as evaluated by nailfold videocapillaroscopy (NVC), and to compare the results regarding bone quality with RA patients and healthy subjects (CNT). Methods Eighty-four SSc patients, 98 RA patients and 60 CNT, were studied. BMD (g/cm2) of the lumbar spine (L1-L4) was analysed by DXA scan. Lumbar spine bone quality was derived from each spine DXA examination using the TBS analysis. NVC patterns were analysed. Results A total of 56/84 SSc patients (66%) as well as 78/98 RA patients (80%) showed bone loss at DXA and BMD was found to be significantly lower than in the CNT (P < 0.001). Similarly, lumbar spine TBS was found to be significantly lower in SSc and RA patients than in CNT (P < 0.001). TBS values were found to be lower in SSc with a late NVC pattern, compared with the active or early pattern (late vs active and early pattern, P < 0.001). There was no statistically significant difference in the mean lumbar spine TBS between SSc and RA patients (P = 0.238). Conclusion The data obtained showed significantly lower bone quality (lower TBS and BMD) in SSc and RA patients compared with CNT. The bone quality seemed lower in SSc patients with more altered microvasculature (late NVC pattern).

Microvascular damage evaluation in systemic sclerosis: the role of nailfold videocapillaroscopy and laser techniques

Microvascular damage and a decrease in peripheral blood perfusion are typical features of systemic sclerosis (SSc) with serious clinical implications, not only for a very early diagnosis, but also for disease progression. Nailfold videocapillaroscopy is a validated and safe imaging technique able to detect peripheral capillary morphology, as well as to classify and to score any nailfold abnormalities into different microangiopathy patterns. Capillaroscopic analysis is now included in the ACR/EULAR classification criteria for SSc. The decrease in peripheral blood perfusion is usually associated with microvascular damage in SSc, which may be studied by different methods. Several of these make use of safe laser technologies. This paper focuses on these new clinical aspects to assess SSc microvascular impairment.

AB0723 Seroprevalence of epstein-barr virus and cytomegalovirus in systemic sclerosis patients: preliminary results

Background Epstein-Barr Virus (EBV) and cytomegalovirus (CMV) are among the most diffused virus in humans, with prevalence of 90% and 80% respectively in adult immunocompetent population 1,2. Hypothesis for a role of EBV and CMV in systemic sclerosis pathogenesis was reported3. Objectives To evaluate the prevalence of EBV and CMV in a population of patients affected by SSc. As an exploratory aim, correlations with available clinical variables were evaluated. Methods A total of 49 patients (44 females and 5 males, age 65,6±9 years, 34 with limited cutaneous involvement and 14 with diffused cutaneous involvement) diagnosed according to the 2013 ACR/EULAR criteria 4 were randomly enrolled at their follow-up visit for a further more detailed analysis. After consent, all participants undergone serological testing for VCA-IgM, VCA-IgG and EBNA-IgG and CMV IgM and IgG, to determine prior or actual EBV and/or CMV infection. All SSc patients undergone also to clinical examination and instrumental evaluations according to SSc clinical standard follow up. Results Results for EBV seroprevalence were: VCA-IgM negative=43 (87%), positive=4 (8,2%), borderline=2 (4,1%) in all SSc patients. VCA-IgG resulted positive in 48 patients (98%) and EBNA-IgG resulted positive in 40 (81,6%) patients, borderline in 4 (8,2%) and negative in 5 (10,2%) patients. Forty-six (93,9%) patients resulted positive for CMV-IgG and 22 (44,9%) showed detectable CMV IgM. Consequently, EBV and CMV seroprevalence was significantly higher (p=0.01 and p=0.032 respectively) than that calculated in general population (90% and 80% respectively). Interestingly, the presence of higher CMV-IgM correlated linearly with higher pulmonary arterial pressure values (p=0.036) and was different in patients with or without sicca syndrome (p=0.012) and arthralgias (p=0.04). Finally, higher CMV-IgG correlated with diffused cutaneous SSc form (p=0.023) and nephropathy (p=0.036). Conclusions The seroprevalence of EBV and CMV positivity was found significantly higher in the sample of SSc patients. EBV seropositivity (VCA-IgG) was present nearly in the totality of patients (98%). Of relevance, the high presence of CMV-IgG (93%) and CMV-IgM (44.9%) in SSc patients: the last correlated linearly with higher pulmonary arterial pressure values. EBV and CMV infections, among the supposed triggers in several autoimmune diseases, might also play a role in SSc patients, at least with progressive disease. References [1] Rickinson AB, et al. Fields virology. Philadelphia: Lippincott Williams and Wilkins; 2001. pp. 2575–2627 [2] Kano Y1, et al. Dermatol Sci. 2000;22:196–204 [3] Arnson Y, et al. Ann N Y Acad Sci. 2009Sep;1173:627–32. [4] van den Hoogen F, et al. Ann Rheum Dis. 2013;72:1747–1755. Disclosure of Interest None declared

OP0037 Evaluation of skin involvement in systemic sclerosis patients by using two ultrasound transducers with different frequency

Background The modified Rodnan skin score (mRSS) is the validated method to evaluate the extension of skin involvement in systemic sclerosis (SSc) and to distinguish between patients with limited cutaneous skin involvement (lcSSc, skin involvement is confined to the extremities) or diffuse (dcSSc) (1,2). Recently several studies have demonstrated that skin high frequency ultrasound (US) is a valid and reproducible technique to measure dermal thickness (DT) in patients with SSc (3–6). Objectives To compare the values of DT obtained by two ultrasound transducers with different frequency (18 MHz and 22 MHz) in evaluating the DT in lcSSc patients and healthy controls. Methods Thirty-seven lcSSc patients (mean age 62±13SD years, mean disease duration 5±5SD years) and 37 healthy controls (CNT) sex and age matched were enrolled after informed consent. Both US transducers of 18 and 22 MHz (Esaote, Genova) were used to evaluate DT in the seventeen areas of the skin (zygoma, fingers, dorsum of hands, forearms, arms, chest, abdomen, thighs, legs, feet) of SSc patients where Rodnan skin score (mRSS) is usually assessed. Skin US was also performed in the same seventeen areas of CNT, looking for DT differences in comparison with lcSSc patients. Statistical analysis was carried out by non parametric tests. Results DT evaluated with the 22 MHz probe was found significantly higher in all body areas in comparison with the 18 MHz transducer, both in lcSSc patients (p<0.01) and in CNT (p=0.05). The median difference of DT values between the two probes was of 0.11 millimetres in lcSSc patients (minimum 0.0023, maximum 0.28 mm) and 0.01 millimetres in CNT (minimum 0.0029, maximum 0.03 mm). Of interest, in lcSSc DT evaluated by 18 MHz transducer was recognized significantly higher (p<0.001) also in four out of six skin areas where the mRSS was found normal (score=0) (upper-arms, chest and abdomen), with exclusion of thighs (p=0.08), in contrast with the classification of lcSSc. However, by using the 22 MHz transducer a statistically significantly higher median DT was showed in all skin areas, included thighs (p<0.01). Finally, a positive statistically significant correlation was observed between the two transducers in the evaluation of DT (p<0.0001), as well as between both probes and mRSS (p<0.0001 for both). Conclusions This study suggests that subclinical dermal involvement may be detectable by skin high frequency US already in patients with limited cutaneous SSc. This study confirms that DT can be better assessed in SSc patients by using a 22 MHz US probe, and suggests that DT might be underestimated by using US probes of lower frequency (18 MHz). However, the DT values obtained using both probes resulted significantly correlated together and with the mRSS. References Clements PJ, et al. Arthritis Rheum 2000;43:2445–54. Moore TL, et al. Rheumatology 2003;42:1559–63. Sulli A, et al. Ann Rheum Dis. 2014;73:247–51. Czirják L, et al. Ann Rheum Dis. 2007;66:966–9. Hesselstrand R, et al. Rheumatology 2008;47:84–7. Kaloudi O, et al. AnnRheum Dis.2010;69:1140–3. Disclosure of Interest None declared

SAT0381 Correlation between three different methods to assess dermal thickness in systemic sclerosis patients with different patterns of nailfold microangiopathy

Background Systemic sclerosis (SSc) is characterized by the increase of dermal thickness (DT) (1). The modified Rodnan skin score (mRss) is the validated method to evaluate the severity of skin impairment (2,3). Several studies have reported the capability of high frequency skin ultrasound (US) to reflect the overall severity of the skin damage in SSc patients (4–5). The plicometer skin test (PST) is another method to evaluated cutaneous involvement in SSc patients (6). Objectives The aim of this study was to identify possible correlations between US, mRss and PST to evaluate DT in SSc patients with different patterns of nailfold microangiopathy. Methods Sixty-three SSc patients (mean age 64±11SD years, mean disease duration 7±6 years, 43 lcSSc and 20 dcSSc) and 63 sex and age matched healthy subjects were enrolled after written informed consent. All subjects were assessed by mRss, US and PST to evaluate the DT in the seventeen skin areas of the body usually evaluated by mRss (zygoma, fingers, hands, dorsum of hands, forearms, arms, chest, abdomen, thighs, legs, feet) (1–6). Nailfold videocapillaroscopy (NVC) was used to assess the proper pattern of microangiopathy and to calculate the microangiopathy evolution score (MES) (7–8). Statistical evaluation was performed by non-parametric tests. Results As expected, the group of SSc patients had a statistically significant higher DT, as evaluated by the three methods, at level of all areas when compared to the control group (p=0.0001). All methods demonstrated a progressively higher DT in patients with “Early”, vs. “Active” and vs “Late” pattern of nailfold microangiopathy (p<0.005), and a positive correlation was observed with MES (r=0.71 p<0.001). A positive correlation was observed in SSc patients between the three method to evaluate DT (PST vs mRss r=0.98, p<0.0001; PST vs US r=0.53, p<0.0001; US vs mRss r=0.53, p<0.0001). Conclusions This study demonstrates a relationship between different methods to assess DT (US, mRss and PST) in SSc patients and a relationship between skin damage and microangiopathy impairment. References Moore TL, et al. Rheumatology 2003;42:1559–63. Sulli A, et al. Ann Rheum Dis. 2014;69:1140–3. Kaldas M, et al. Rheumatology 2009;48:1143–6. Hesselstrand R, et al. Rheumatology 2008;47:84–7. Kaloudi O, et al. Ann Rheum Dis. 2010;69:1140–3. Parodi MN, et al. Br JRheumatol. 1997;36:244–50. Sulli A, et al. Ann Rheum Dis 2008;67:885–7. Cutolo et al. J Rheumatol 2000; 27;155.60. Disclosure of Interest None declared

FRI0569 Evaluation of bone microarchitecture in systemic sclerosis patients: relationships between trabecular bone score (TBS) and disease severity

Background Systemic sclerosis (SSc) is a rare connective tissue disorder characterized by an increased synthesis and deposition of extracellular matrix in the skin and internal organs (1). Several studies described SSc as potential risk factor for osteoporosis, however, to date the bone quality in SSc is unclear (2). Trabecular bone score (TBS) has been recently proposed as an indirect measure of bone microarchitecture (3). Objectives The aim of this study was to assess bone microarchitecture in SSc patients and possible association with disease severity and microangiopathy. Methods Twenty-three female SSc patients (mean age 63.2±12.8 SD years, mean disease duration 92.8±66 SD months, mean Raynauds Phenomenon duration 142.6±126.1 SD months) were enrolled after written informed consent. The assessment of disease severity was performed using the Medsgers severity scale (4). Bone Mineral Density (BMD) measurements at L1-L4, femoral neck and total hip, were performed using DXA Prodigy Densitometer (GE Lunar). TBS was derived for each spine DXA examination using the TBS index (TBS iNsight Medimaps). Nailfold videocapillaroscopy (NVC) was used to assess the microangiopathy based on nailfold video capillaroscopic pattern (NVC) analysis and the microangiopathy evolution score (MES) (5–6). Using the FRAX (Fracture Risk Assessment Tool) we also evaluate the 10-year risk of hip and major joints osteoporotic fracture. Results A positive correlation was observed between TBS and Medsgers general organ score (r=0.5; p=0.01); no other correlations were found between TBS and Medsgers score. Interestingly, TBS was positively and significantly correlated with modified Rodnam skin score (mRss) (p=0.01). When the patients were divided in two groups considering skin involvement by mRss, TBS was found significantly higher into the group with mRss >15 compared to the group with mRss <15 (1.255±0.08 vs 1.163±0.03; p=0.01) No correlations were found between NVC patterns/MES and bone quality assessment (TBS) or bone density assessment (BMD), only a significant correlation, as expected, was observed between MES and skin involvement (mRss) (p=0.05). On the other hands, FRAX, the major osteoporotic fracture risk, positively correlates with Medsgers kidney disease severity (p=0.04) and Medsgers lung disease severity (p=0.04); in addition, FRAX, for hip fracture risk, seems to correlate significantly with Medsgers lung involvement severity (p=0.04). Conclusions This study demonstrates in SSc patients a relationship between clinical disease severity (organ fibrosis/failure) and altered bone microarchitecture (TBS). In addition, skin involvement was found significantly correlated with altered quality of the trabecular bone architecture (TBS) and a significant increase of osteoporotic fracture risk (FRAX) was found correlated with kindey and lung involvement. References Varga J, Abraham D. J Clin Invest. 2007 Mar;117(3):557–67. Loucks J, Pope JE. Semin Arthritis Rheum. 2005 Feb;34(4):678–82. Silva BC et al. J Bone Miner Res. 2014 Mar;29(3):518–30. Medsger TA et al. Clin Exp Rheumatol. 2003;21(3 Suppl 29):S42–6. Sulli A, et al. Ann Rheum Dis 2008;67:885–7. Cutolo et al. J Rheumatol 2000; 27;155.60. Disclosure of Interest None declared