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The term pulmonary arterial hypertension (PAH) identifies a heterogeneous group of diseases characterized by a progressive increase in pulmonary arterial resistance (PVR), which causes a significant burden in terms of quality of life, right heart failure and premature death. The pathogenesis of PAH is not completely clear: the remodeling of the small pulmonary vessels is crucial, causing an increase in the resistance of the pulmonary circle. Its diagnosis is based on cardiac catheterization of the right heart. According to the present hemodynamic definition of pulmonary hypertension (PH) proposed by the Guidelines of the European Society of Cardiology/European Respiratory Society (ESC-ERS), the mean pulmonary arterial pressure (mPAP) values are ≥25 mmHg. In case of PAH, apart from an mPAP value ≥25 mmHg, patients must have a >3 Wood units increase in PVR and normal pressure values of the left heart. PH is a pathophysiological condition observed in more than 40 different diseases, while PAH is a primary disease of the pulmonary bloodstream potentially treatable with specific drugs. PAH is a severe complication of systemic sclerosis (SSc) affecting about 10% of the patients. Due to the devastating nature of SSc-PAH, there is a clear need to systematically adopt appropriate screening programs. In fact, despite awareness of the negative impact of SSc-PAH on quality of life and survival, as well as on the severity of lung function, at the moment standardized and shared guidelines and/or screening programs for the diagnosis and the subsequent early treatment of PAH in SSc are not available. The aim of the present paper is to highlight the lights and shadows of SSc-PAH, unraveling the unmet clinical needs on this topic with a proposal of clinical-diagnostic and therapeutic guidelines.

BackgroundNintedanib (NTD) has been approved for Systemic Sclerosis (SSc)-Interstitial Lung Disease (ILD) following the positive results of the SENSCIS trial.Objectivesto describe the efficacy and safety of NTD in SSc-ILD in a real-life setting.MethodsThe clinical data of SSc-ILD patients treated with NTD from 10 Italian SSc centres were retrospectively evaluated at baseline, 6 and 12 months: SSc clinical features, NTD tolerability, pulmonary function tests (PFTs) and modified Rodnan skin score (mRSS) were recorded.Results69 SSc-ILD patients (22 males [32%], mean age 60±12 years, disease onset 50±13 years, 4 [6%] anti-centromere, 53 [77%] anti-topoisomerase I, 3 [4%] anti-RNA-polimerase III) were identified. The vast majority (84%) was previously treated with immunosuppressants: 27 (39%) cyclophosphamide, 45 (65%) mycophenolate mofetil, 6 (9%) methotrexate, 9 (13%) azathioprine, 6 (9%) tocilizumab and 22 (32%) rituximab. In 11 (16%) patients, NTD was the first treatment for SSc-ILD. At baseline, 57 patients (83%) were on corticosteroids (mean daily prednisone dose 6±5 mg), 58 (84%) on immunosuppressants, 47 (68%) on mycophenolate mofetil, 14 (20%) on rituximab, 3 (4%) on tocilizumab, 2 on methotrexate (3%) and 1 (1%) on azathioprine. At baseline HRCT showed UIP pattern in 27 (39%) and NSIP pattern in 42 (61%) patients. The modifications of PFTs and mRSS over time are shown in Table 1. Since NTD introduction, gastro-intestinal (GI) side effects were recorded in 34 (49%) patients, with diarrhoea being the most common complaint (35%), followed by nausea/vomiting (23%) and weight loss (16%). In 21 (30%) patients, after a mean time of 2.6±3.4 months, NTD was maintained after dose adjustment. In 5 (7%) patients NTD was stopped after a median time of 5 (1-6) months due to subocclusion and persistent diarrhoea in 3 patients, untreatable nausea and vomiting in one patient and liver toxicity in 1 patient. During the follow-up after a median time of 10 (6 – 33) months, 4 patients died.Table 1.Pulmonary function tests and mRSS at baseline, 6 and 12 months in SSc-ILD on NTD.Baseline6 monthsP valueBaseline12 monthsP valueFVC (% predicted)64 ± 1865 ± 18 (33 pts)0.63870 ± 1969 ± 18 (20 pts)0.586TLC (% predicted)64 ± 1561 ± 14 (27 pts)0.15464 ± 1465 ± 18 (16 pts)0.944DLCO (% predicted)40 ± 1741 ± 18 (29 pts)0.66040 ± 1838 ± 18 (20 pts)0.304mRSS9 ± 68 ± 6 (26 pts)0.0027 ± 48 ± 6 (15 pts)0.334pts= patientsConclusionOur preliminary data confirm that in a real-life clinical scenario NTD, in combination with immunosuppressants, may stabilize PFT. However, despite the fact that GI side effects are frequent, they may be controlled with NTD dose adjustment thus retaining the drug in SSc-ILD patients. The NTD efficacy on skin involvement needs to be thoroughly evaluated on a larger SSc population.Disclosure of InterestsCorrado Campochiaro Speakers bureau: Boeboehringer ingelheim, Giacomo De Luca Speakers bureau: boehringer ingelheim, Maria Grazia Lazzaroni Grant/research support from: boehringer ingelheim, Giuseppe Armentaro: None declared, Amelia Spinella: None declared, Barbara Vigone: None declared, Barbara Ruaro: None declared, Anna Stanziola: None declared, Devis Benfaremo: None declared, Enrico De Lorenzis: None declared, Francesco Benvenuti: None declared, Silvia Laura Bosello Speakers bureau: boehringer ingelheim, Gianluca Moroncini: None declared, Giovanna Cuomo: None declared, Marco Confalonieri: None declared, Lorenzo Beretta: None declared, Elisabetta Zanatta: None declared, Dilia Giuggioli: None declared, Nicoletta Del Papa: None declared, Paolo Airò: None declared, Lorenzo Dagna: None declared, Marco Matucci-Cerinic Speakers bureau: boehringer ingelheim

Systemic sclerosis (SSc) is a rare disabling autoimmune disease with a similar mortality to many cancers. Two randomized controlled trials of autologous hematopoietic stem cell transplantation (AHSCT) for SSc have shown significant improvement in organ function, quality of life and long-term survival compared to standard therapy. However, transplant-related mortality (TRM) ranged from 3–10% in patients undergoing HSCT. In SSc, the main cause of non-transplant and TRM is cardiac related. We therefore updated the previously published guidelines for cardiac evaluation, which should be performed in dedicated centers with expertize in HSCT for SSc. The current recommendations are based on pre-transplant cardiopulmonary evaluations combining pulmonary function tests, echocardiography, cardiac magnetic resonance imaging and invasive hemodynamic testing, initiated at Northwestern University (Chicago) and subsequently discussed and endorsed within the EBMT ADWP in 2016.

Background:Coronavirus disease-19 (COVID-19) has been a major clinical challenge worldwide. Sex, age and comorbidities have been associated with worse outcome in the general population. Systemic sclerosis (SSc) is a severe, autoimmune disease with frequent multi-organ involvement.Objectives:To assess the impact of COVID-19 and to determine factors associated with worse outcome in SSc patients from the European Scleroderma Trial and Research (EUSTAR) database.Methods:SSc patients from the EUSTAR database with COVID-19 were prospectively collected between 15.03.-31.12.2020. Two outcomes were chosen: (1) hospitalization; and (2) severe outcome defined as either non-invasive ventilation, mechanical ventilation/extracorporeal membrane oxygenation (ECMO) or death. General risk factors assessed were sex, age and number of comorbidities. SSc related risk factors were SSc subtype, autoantibodies, disease duration, SSc associated organ manifestations including interstitial lung disease (ILD), pulmonary arterial hypertension (PAH), cardiac, gastrointestinal (GI), and musculoskeletal involvement; digital ulcers (DU), CRP at last visit, renal disease (scleroderma renal crisis and SSc associated renal insufficiency), modified Rodnan skin score (mRSS) and immunosuppressive treatment. Descriptive statistics and logistic regression models were applied.Results:In total, 178 European SSc patients with COVID-19 were registered with a median observation time of 5.5 weeks (Table 1). 95 patients (53%) could recall SAR-Cov-2 contact, while 47 (26%) had no contact. 156 (88%) were symptomatic at COVID-19 onset with fever, cough, malaise and dyspnea being most prevalent. Over the disease course, 63 (36%) developed pneumonia. In total, 67/176 (38%) were hospitalized which were in 84% due to COVID-19. 41/170 (24%) had a severe outcome including 21 (12%) deaths. 128 (72%) recovered completely, while 14 (8%) complained of sequela, with 7 (50%) stating respiratory complications. Age, non-SSc comorbidities, presence of ILD, PAH and SSc associated renal or cardiac disease were numerically associated with hospitalization and severe outcome (Table 1). Univariable logistic analyses for hospitalization and severe outcome are shown in Figure 1. In multivariable logistic regression, age (OR 1.03, 95%CI 1.01-1.07, p=0.019), presence of non-SSc comorbidities (OR 2.52, 95%CI 1.16-5.47, p=0.019) and SSc-related renal disease (predicting success perfectly) were associated with hospitalization and for severe outcome age (OR 1.05, 95%CI 1.01-1.08).Conclusion:SSc patients at older age, with non-SSc comorbidities, SSc related renal disease or ILD are at risk of a more severe outcome and should follow precautions to avoid COVID-19 infections and need careful monitoring in case of COVID-19.Table 1.SSc disease characteristics of COVID-19 patientsAll(n=172)Hospitalized(n=67)Severe outcome(n=41)Age at COVID-19, yrs (SD)57 (14.0)63 (13.8)65 (12.2)Male sex, n (%)38 (21)18 (27)12 (29)≥1 comorbidity, n (%)63/176 (36)37 (55)30 (58)SSc disease duration at COVID, yrs (SD)11.5 (8.8)13.3 (9.7)12.7 (10.2)Diffuse cutaneous SSc, n (%)74 (42)29 (43)19 (46)mRSS, median (IQR)5 (8)5 (9)5 (7)ILD, n (%)90/175 (51)36/65 (55)26/40 (65)PAH, n (%)21/175 (12)11/65 (17)8/40 (20)GI disease, n (%)112/176 (64)45 (67)30 (73)Cardiac disease, n (%)37/166 (22)19/59 (32)16/36 (44)Musculoskeletal disease, n (%)40/175 (23)15/65 (23)6/40 (15)Renal disease, n (%)8/175 (5)7/65 (11)5/40 (13)Ever DU, n (%)69/175 (39)27/65 (42)14/40 (35)CRP, ng/ml (SD)35/177 (20)14 (21)9 (22)Immunosuppressive treatment, n (%)104/177 (59)41/66 (62)26 (63)Figure 1.Univariable logistic analyses for hospitalization and severe outcomeDisclosure of Interests:Anna-Maria Hoffmann-Vold Speakers bureau: Actelion, Boehringer Ingelheim, Roche, Merck Sharp & Dohme, ARXX, Lilly and Medscape, Consultant of: Actelion, Boehringer Ingelheim, Bayer, ARXX, and Medscape, Grant/research support from: Boehringer Ingelheim, Cathrine Brunborg: None declared, Francesca Tirelli: None declared, Patricia Carreira: None declared, Nicoletta Del Papa: None declared, Arsene Mekinian: None declared, Madelon Vonk: None declared, Alessandro Giollo: None declared, Giacomo De Luca: None declared, Maria De Santis: None declared, Corrado Campochiaro: None declared, Carina Mihai: None declared, Paolo Airò Speakers bureau: Bristol Myers Squibb, Bohringer Ingelheim, Consultant of: Bristol Myers Squibb, Bohringer Ingelheim, non-financial support from CSL Behring, SOBI, Janssen, Roche, Sanofi, Pfizer, Maria Grazia Lazzaroni: None declared, Elisabetta Zanatta: None declared, Rosario Foti: None declared, Yannick Allanore: None declared, Daniel Furst: None declared, Marco Matucci-Cerinic: None declared, Armando Gabrielli: None declared, Oliver Distler Speakers bureau: Actelion, Kymera Therapeutics, Mitsubishi Tanabe Pharma, Abbvie, Acceleron, Alexion, Amgen, AnaMar, Arxx Therapeutics, Baecon, Discovery, Blade Therapeutics, Corbus Pharmaceuticals, Drug Development International, Ltd, CSL Behring, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, iQvia, Kymera Therapeutics, Lilly, Novartis, Pfizer, Topadur and UCB, Consultant of: Actelion, Kymera Therapeutics, Mitsubishi Tanabe Pharma, Abbvie, Acceleron, Alexion, Amgen, AnaMar, Arxx Therapeutics, Baecon, Discovery, Blade Therapeutics, Corbus Pharmaceuticals, Drug Development International, Ltd, CSL Behring, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, iQvia, Kymera Therapeutics, Lilly, Novartis, Pfizer, Topadur and UCB, Grant/research support from: Boehringer Ingelheim.

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.References:[1]Del Papa N, Pignataro F. Front Immunol. 2018 Jun 18;9:1383[2]De Simone C et al. J Eur Acad Dermatol Venereol. 2014 May;28(5):590-6[3]Del Papa N et al. Arthritis Rheum. 2004 Apr;50(4):1296-304Disclosure of Interests:Francesca Pignataro: None declared, Laura Zorzino: None declared, Wanda Maglione: None declared, Antonina Minniti: None declared, Giulia Clericuzio: None declared, Marco Picozzi: None declared, Cecilia Simonelli Employee of: Menarini Silicon Biosystems, Francesco Picardo Employee of: Menarini Silicon Biosystems, Roberto Caporali: None declared, Nicoletta Del Papa: None declared

We retrospectively evaluated the efficacy of autologous hematopoietic stem cell transplantation (AHSCT) in 18 patients with rapidly progressive diffuse cutaneous systemic sclerosis (rp-dcSSc), and compared their disease outcomes with those of 36 demographically- and clinically-matched patients treated with conventional therapies. Cutaneous involvement, by performing modified Rodnan skin score (mRss), lung diffusion capacity, by measuring diffusing capacity of lung for carbon monoxide (DLCO), and disease activity, by applying the European Scleroderma Study Group (ESSG) scoring system, were the outcome variables measured at the baseline time and then every 12 months for the following 60 months in both the AHSCT-treated patients and the control group. In the AHSCT group, treatment-related mortality was 5.6%. In this group, both mRss and ESSG scores showed a significant reduction 1 year after AHSCT (P

Background:Nailfold videocapillaroscopy (NVC) is a feasible method that allows the observation and follow-up of the microvascular changes that mark the course of Systemic Sclerosis (SSc). In previous studies, we demonstrated that the NEMO score, namely the cumulative Number of microhaEMOrrhages and microthromboses, is a good indicator of the steady state level and over time changes of disease activity (DA) in SSc (1-3).Objectives:To verify whether a high NEMO score, and then a high level of active microvascular derangement in the fingers may be predictive of the subsequent development of ischemic digital ulcers (IDUs).Methods:The NEMO score was assessed at baseline (T0) in 98 patients affected by SSc, according to the ACR-EULAR criteria. Out of them, 90 were females, 48 had the limited form and 50 the diffuse cutaneous variant of SSc. ACA and anti-Scl-70 antibodies were positive in 42 and 50 patients, respectively. The NVC pattern was early, active and late in 16, 42 and 40 patients, respectively.Afterwards, patients were closely followed up for 3 years, and the appearance of new IDUs was recorded in any time of the follow up.The T0-NEMO score values of patients who developed IDUs were compared to those of patients who did not. A receiver operating curve (ROC) was constructed, and the area under the curve (AUC) calculated, by plotting the sensitivity and 1-specificity of the different NEMO score values in predicting the development of IDUs.Results:During the follow up, 38 out of 98 patients developed one or more DUs. The NEMO score at T0 was significantly higher in those who developed IDUs with respect to those who did not [median 14.5 (CI 11.0-21.5), and 4.5 (CI 4.0-6.0), respectively, pConclusion:NEMO score is not only a valid tool to assess the level of DA in the course of SSc, but this NVC parameter could also be used as a good predictor of the future development of IDUs in patients with this disease.References:[1]Sambataro et al. Arthritis Res Ther 2014;16:462-69[2]Andracco et al. Arthritis Res Ther 2017;19:133-41[3]Pignataro et al. Arthritis Res Ther. 2019;21(1):258Disclosure of Interests:Nicoletta Del Papa: None declared, Francesca Pignataro: None declared, Wanda Maglione: None declared, Antonina Minniti: None declared, Domenico Sambataro: None declared, Gianluca Sambataro: None declared, Gabriele Valentini Grant/research support from: BMS, MSD, NOVARTIS, LILLY, PFIZER, ABBVIE, CELGENE, Claudio Vitali: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB

Background:Nailfold Videocapillaroscopy (NVC) is an essential tool for the assessment of Raynaud’s Phenomenon (RP) among the Scleroderma Spectrum Disorders (SSDs). Recently, NVC abnormalities have been associated with a diagnosis of Idiopathic Inflammatory Myopathies (IIMs), independently of the presence of RP (1). Moreover, both SSDs and IIMs are commonly associated with Interstitial Lung Disease (ILD), which is the main cause of mortality in these conditions.Objectives:To verify whether NVC may allow a better diagnostic classification in a cohort of patients with ILD followed by pulmonologists.Methods:361 patients affected by ILD were prospectively enrolled in a 30-months observational study. All these patients were clinically evaluated by rheumatologists and pulmonologists together and performed general blood tests, autoantibody research, chest High-Resolution Computed Tomography and NVC. The latter was considered positive in the presence of avascular areas or giant capillaries, and also the presence of Bushy Capillaries (BCs) was recorded.Results:NVC was positive in 17.7% of ILD patients, and a third of these patients did not present RP. Patients with NVC abnormalities had a diagnosis of definite Connective Tissue Disease in 78.1% of cases. NVC resulted decisive in 25% of patients with a final diagnosis of Systemic Sclerosis according to the ACR/EULAR 2013 criteria. The presence of BCs and/or NVC+ in ILD patients with normal serum levels of muscular enzymes was associated with amyopathic IIM (BCs= OR 3.9, 95CI 1.05-14.38, p=0.04; NVC+= OR 5, 95CI 1.29-19.3 p=0.02; BCs and/or NVC+= OR 5.41 95CI 1.24-23.48 p=0.02), regardless the presence of RP.Conclusion:NVC proved to be a valid tool in the correct assessment of ILD patients secondary to SSDs and amyopathic IIMs. Therefore, it could be considered in the diagnostic evaluation of patients affected by ILD, regardless of the presence of RP.References:[1]Sebastiani M et al. Nailfold Capillaroscopy Characteristics of Antisynthetase Syndrome and possible clinical associations: results of a multicenter International Study.J Rheumatol2019; 46: 279-284Table 1.Comparison between ILD patients with and without NVC positivity..ItemsNVC+ patientsNVC- patientspNumber64297Mean Age (±SD)61.4±13.367.8±9.80.001Female%65.649.10.01RP%68.717.1HRCT patterns%NSIP51.536.80.02OP3.18n.s.UIP-like31.1544.8n.s.DIP1.53.4n.s.LIP1.50.4n.s.Indeterminate10.96.7n.s.Final Diagnosis%SSDs45.31IIMs256.4Other CTDs6.213.5n.s.IPAF10.919.5n.s.IPF7.827.90.0007Legend:DIP: Desquamative Interstitial Pneumonia; HRCT: High Resolution Computed Tomography; IIMs: Idiopathic Inflammatory Myopathies, Poly/dermatomyositis, antisynthetase syndrome; IPAF: Interstitial Pneumonia with Autoimmune Features; IPF: Idiopathic Pulmonary Fibrosis; LIP: Lymphocytic Interstitial Pneumonia; NSIP: Nonspecific Interstitial Pneumonia; OP: Organising Pneumonia; RP: Raynaud’s Phenomenon; SSDs: Scleroderma Spectrum Disorders, Systemic Sclerosis + Mixed Connective Tissue Disease; UIP: Usual Interstitial Pneumonia.Disclosure of Interests:Gianluca Sambataro: None declared, Domenico Sambataro: None declared, Francesca Pignataro: None declared, Nicoletta Del Papa: None declared, Michele Colaci: None declared, Lorenzo Malatino: None declared, Alessandro Libra: None declared, Fabio Pino: None declared, Sebastiano Emanuele Torrisi Speakers bureau: Boehringer Ingelheim; F. Hoffmann-La Roche Ltd., Stefano Palmucci Consultant of: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Delphi International Srl, and F. Hoffmann-La Roche Ltd., Lorenzo Cavagna: None declared, Carlo Vanchieri Grant/research support from: F. Hoffmann-La Roche Ltd., Consultant of: AstraZeneca, Boehringer Ingelheim, Chiesi, F. Hoffmann-La Roche Ltd., and Menarini, Speakers bureau: AstraZeneca, Boehringer Ingelheim, Chiesi, F. Hoffmann-La Roche Ltd., and Menarini

Background:Adipose-derived stromal/stem cells (ADSCs) are multipotential non-hematopoietic progenitor cells with anti-inflammatory, immunomodulatory and regenerative effects. They have the advantage of accessibility and potent pro-angiogenic effects when compared with other stem cells, such as bone-marrow derived stem cells. Recent studies have shown that autologous fat grafting may be effective in the treatment of fibrotic and vascular complications in systemic sclerosis (SSc), despite a pro-fibrotic signature.Objectives:Aim of the study was to better characterize the proliferative and secretive profile of ADSCs in normoxic and hypoxic conditions, and to evaluate the mechanisms by which ADSCs exert these observed clinical effects.Methods:Adipose tissue samples were obtained by liposuction from 12 SSc patients and 10 healthy donors (HD). ADSCs were purified according to their adherence to the plastic and characterized to express specific MSC surface antigens by flow cytometry analysis. Proliferation of ADSCs from SSc patients and normal controls was evaluated in normoxic and hypoxic conditions. Fibroblasts and ADSCs derived from SSc patients were co-cultured in direct and indirect culture systems and compared to HD. Fibroblasts proliferation, mRNA expression and protein secretion of VEGF and known fibrotic mediators including TGF β-1, TGFR, CTGF, Collagen type I (Col I) were analyzed in the same conditions.Results:Normoxic and hypoxic culture conditions did not modify the proliferation rate of both normal and SSc ADSCs. Hypoxia significantly increased mRNA expression of VEGF by HD and SSc ADSCs but had no effect on the mRNA expression of pro-fibrotic mediators, ie TGFβ and TGFR. Normal and SSc fibroblast proliferation was significantly reduced in both co-culture systems (p < 0.001) and by treatment with normoxic and hypoxic conditioned medium (CM) (p=0.001 and p=0.002). In the same conditions, mRNA expression and protein secretion of TGF-β1, CTGF and Col I were significantly reduced (p = 0.003, p =0.02, p=0.04). These results were confirmed when normal and SSc fibroblasts were cultured in the presence of ADSCs normoxic and hypoxic CM (p=0.02 and p=0.01). Furthermore, a significant increase in mRNA expression and production of VEGF was observed in SSc fibroblasts cultured in the presence of normoxic and hypoxic CM (p = 0.002 and p< 0.0001, respectively).Conclusion:We found that treatment with the medium from normoxic and hypoxic preconditioned ADSCs has an anti-fibrotic effect through both the inhibition of fibroblast proliferation and key mediators of fibrosis. The increased expression of VEGF by SSc fibroblasts in the presence of normoxic and, even more, hypoxic ADSCs CM, suggests that ADSCs can induce a paracrine pro-angiogenic phenotype, even in fibroblasts with a pro-fibrotic signature. Altogether these data show that ADSCs may exert their anti-fibrotic and pro-angiogenetic effects on SSc fibroblasts by the secretion of paracrine factors, partly explaining the mechanisms underlining beneficial clinical results of fat graft in SSc patients.Disclosure of Interests:Nicoletta Del Papa: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Antonina Minniti: None declared, Francesca Pignataro: None declared, Wanda Maglione: None declared, Gabriele Di Luca: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB

Background:It is well known that Sjögren’s syndrome (SjS) is characterized by an upregulation of interferon (IFN)-induced genes. Namely, IFN type I signature has been reported in peripheral blood mononuclear cells (PBMCs) and in salivary glands of patients with this disease. However, few data are available on possible variability of IFN-induced gene upregulation in different clinical phenotypes of SjS.Objectives:To verify whether upregulation of IFN-induced genes is comparable in patients with SjS characterized by different clinical phenotypes, i.e., patients with systemic extraglandular manifestations (EGMs) versus patients with a disease limited to glandular features (GFs) and with widespread pain (WP).Methods:The study population was composed by 11 patients with SjS and EGMs (1 male, age range 18-78 years), and 10 patients with only GFs and WP (all females, age range 46-81 years), all classified according to ACR-EULAR criteria. The prevalence of anti-SSA(Ro) antibodies was 11/11 and 8/10, respectively. Lip biopsy was positive in all cases. Six healthy normal subjects were also included in the study as control population.Four IFN type I- and 5 IFN type II-induced genes were chosen for the study on the basis of previous literature data. Total RNA from each patient and control was isolated from purified PBMCs, followed by cDNA preparation and real time quantitative-PCR (RQ-PCR) analysis, using specific primer/probe sets. For calculation of relative expression, all samples were normalised against expression of a household gene (beta actin). A further normalization was performed against the mean value of relative expression obtained in the normal controls. Final fold change values were determined from the double-normalised values using the 2−ΔΔCT method (Applied Biosystems).Results:Fold change values of gene expression of both IFN type I- and type II-induced genes in PBMCs were different in the two clinical phenotypes of SjS. Fold change values of IFN type I-induced genes appeared strongly higher in patients with EGM, and some of them only moderately increased in those with only GF and WP. The expression of some of IFN type II-induced genes were slightly increased in patients belonging to both clinical phenotypes. Results are detailed in the table.Table.Fold change values of gene expression in patients with SjS plus EGMs, in patients with disease limited to GF and WP, and in controls.GeneMX1IFIT1IFT3IFI44IDO1GRP1MIGIP-10P2RY14SjS-EGMs85.938.524.440.425.18.34.51.55.5SJS-GF-WP4.21.72.04.84.11.20.60.31.3Controls2.11.61.11.51.41.31.51.31.4Legend. IFN type I-induced genes: MIX, IFN-induced GTP binding protein 1; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; IFIT3, IFN-induced protein with tetratricopeptide repeats 3; IFI44, IFN-induced protein 44.IFN type II-induced genes: IDO1, indolamine-deoxygenase 1; GBP1, guanylate binding protein 1; MIG, C-X-C chemokine 9 (CXCL9); IP-10, C-X-C chemokine 10 (CXCL10); P2RY14, purinergic receptor 14.Conclusion:The present data indicate that IFN type I- and, to a lesser degree, type II-induced genes are upregulated in patients with SjS, but this phenomenon is consistently stronger in patients with systemic EGMs. In patients with only GFs IFN-induced gene upregulation is milder in PBMCs, and then probably more restricted to the exocrine target tissues.Disclosure of Interests:Nicoletta Del Papa: None declared, Claudio Vitali: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Wanda Maglione: None declared, Francesca Pignataro: None declared, Antonina Minniti: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UCB

Background Different phenotypes characterise the clinical spectrum of primary Sjogren’s syndrome (SjS). Patients with a clinical expression limited to glandular features (GFs) are classically distinguished from patients with extra-glandular manifestations (EGMs). The former patients often complain higher level of fatigue and widespread pain (WP). (Segal et al. 2013) This suggests that gene expression pattern may be different in the two subgroups. Objectives To investigate the differences of gene expression in SjS patients with GFs and in those with EGMs. Methods Nineteen patients with SjS were selected for the study. Gene expression in peripheral blood mononuclear cells (PBMCs) was analysed in 4 patients with EGMs and 4 patients with GFs alone using Clariom D human Affymetrix gene chip (Affymetrix, Santa Clara, CA, USA), and compared to that found in healthy controls. Differences in gene expression were evaluated by analysis of variance (ANOVA) and Step-Up FDR-controlling procedure, being FDR corrected p value≤0.01 and fold change >2 considered as statistically significant. Validation of the gene overexpression was performed by quantitative Real Time(qRT)-PCR in PBMCs from all the selected SjS patients, using the ΔΔCt method for comparing relative fold expression differences. Results All the enrolled SjS patients (18 females and 1 male) had a positive lip biopsy, while anti-SSA/Ro antibodies were detected in 10/11 and 6/8 of the patients with EGMs and with GFs alone, respectively. ESSDAI value ranged from 7 to 55 in patients with EGMs (median 17), and from 0 to 2 in patients with GFs alone (median 1). In both types of patients, the functional analysis of the two transcriptomes showed a large number (>1000) of modulated genes that are involved in the well-known pathological processes of SjS, i.e., apoptosis, inflammatory response, immune response, type I and type II interferons, and Toll-like receptors signalling. Genes modulated only in patient with EGMs showed a significant enrichment of the biological processes associated with immune response (79% of all enriched processes) and, namely, of the molecular pathways related to B cell activation. The analysis of the transcripts expressed only in patients with GFs alone showed instead a preponderant enrichment in different metabolic processes (43%) and in processes associated with the central perception of the stimuli. Indeed, genes involved in sensory perception and in nociceptive signals (i.e., ANPEP, TNRF1, P2RY1, IFNG) were modulated exclusively in patients with GFs alone. The significant differential expression of selected genes in the two SjS subgroups was confirmed by the qRT-PCR analysis. Conclusions These data indicate that in SjS patients with GFs alone a dysregulation of pain pathways (namely beta-adrenergic receptor and Notch signalling) may play a role in the development of WP that is common in this subset of patients. The biological mechanisms triggering the activation of these genes remain to be completely clarified. Disclosure of Interest None declared

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) finely regulates both innate and adaptive immune responses through the degradation of the essential amino acid tryptophan into kynurenine and other downstream metabolites, which suppress effector T-cell function and promote the differentiation of regulatory T cells. A novel role for IDO1 as a signaling molecule and a modifier of innate inflammatory responses is now emerging. In particular, IDO1 can either support or antagonize inflammation in a context- and tissuedependent manner. Studies in experimental arthritis have unravelled a previously unappreciated role for IDO in controlling B-cell activation and autoantibody production. IDO dysregulation has been documented in patients with systemic lupus erythematosus, systemic sclerosis and Sjogren's syndrome, as well as in severe sepsis and chronic kidney disease. This article summarizes the contribution of IDO to the pathophysiology of inflammatory/autoimmune disorders, and discusses whether strategies to restore metabolic equilibrium in the kynurenine pathway might be pursued in diseases states such as rheumatoid arthritis and systemic sclerosis.

Different classification criteria for Sjogren’s syndrome (SS) were proposed by different groups of experts before the 1990s. In the 1990s, a European group of investigators carried out a multicenter study and defined the European classification criteria. In 2002, a reanalysis of the European database and consensus among American and European experts allowed the formulation of the American–European Consensus Group (AECG) criteria, which represented the gold standard for classification of patients with SS in the following decades. In 2012, a group of American experts proposed another set of classification criteria that received the preliminary endorsement of the American College of Rheumatology (ACR). To overcome the difficulties, generated by the different classification criteria, a number of American and European investigators decided to form a working group whose objective was to develop a single set of classification criteria. The result of this joint effort is going to be reached in a short time.

Pulmonary arterial hypertension in primary myelofibrosis is common and associated with an altered angiogenic status

Summary Systemic sclerosis (SSc) is often complicated by severe skin ulcers that are unresponsive to traditional treatments. Vascular alterations are responsible for the ischaemic features of the disease in both the skin and visceral organs. Defective neoangiogenesis correlates with an abnormally reduced quantity of circulating endothelial progenitor cells (EPCs) caused by impaired maturation potential and proliferative capacity of bone-marrow endothelial stem cells. We report a patient with nonhealing cutaneous ulcers successfully treated with recombinant human erythropoietin (rHuEPO). The possible biological effects of this drug were also investigated. Before rHuEPO treatment, the bone-marrow sample contained reduced numbers of EPCs, which were functionally impaired. After a 6-month rHuEPO cycle, a marked increase in endothelial progenitor markers was seen, along with a significant reduction in their apoptotic rates. The clinical and laboratory data variations before and after rHuEPO treatment give new insights into the pathogenetic role of impaired endothelial stem-cell maturation and defective neoangiogenesis in patients with SSc.

SUMMARYThe aim of this study was to characterize the antigen specificity and to evaluate the diagnostic and prognostic value of anti-mitochondrial M5 type antibodies (AMA M5). Fifty-eight patients selected on the basis of their AMA M5 positivity were investigated in relationship to their clinical and serological profile. Cross-absorption studies, Western blotting and immunoprecipitation analysis were carried out for AMA M5 antigen specificity characterization. Most patients had a diagnosis of systemic lupus erythematosus (SLE) (65.5%) or of primary anti-phospholipid syndrome (PAPS) (24%); all the patients were positive for IgG or IgM anti-cardiolipin (anti-CL) antibodies and 49% of them also displayed lupus anticoagulant (LA) activity. Anti-β2-glycoprotein I (β2-GPI) IgG were detectable in 30/38 sera (78.9%) and IgM in 34/38 (89.4%). While anti-CL and anti-β2-GPI IgG antibodies were significantly associated with history of thrombosis and fetal loss, AMA M5 displayed a statistical association only for thrombocytopenia and recurrent fetal loss. Absorption with human β2-GPI both in free solution or in solid phase as well as with CL liposomes or CL/β2-GPI liposome complexes did not affect AMA M5 fluorescence. While AMA M5 activity is absorbed by whole mitochondrial preparations, no specific reactivities against several human, bovine and rat mitochondrial proteins could be detected in Western blotting and immunoprecipitation studies. AMA M5 appear to be detectable in both primary and secondary APS, displaying a strong association with the presence of thrombocytopenia and fetal loss. Although strictly related to anti-phospholipid antibodies, AMA M5, anti-CL and anti-β2-GPI antibodies represent distinct serological markers of the APS.

Beta2-glycoprotein I (beta2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)-coated plates. Beta2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274-Cys288 of the CL-binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic beta2GPI interacts, as supported by studies with heparitinase-treated EC. Beta2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of beta2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation

Background In a previous study it has been shown shown that, in patients with systemic sclerosis (SSc), the cumulative number of micro-haemorrhages (MH) and micro-thrombosis (MT), computed in eight finger nailfold videocapillaroscopy (NVC), and called NEMO score, was strongly predictive of disease activity (DA) measured by means of European Scleroderma Study Group (ESSG) score. Even the number of giant capillaries (GC) was correlated to a lesser extent with the ESSG score, and then a modified (m) NEMO score was proposed in which the combined presence of a MH/MT and GC was considered [1]. Objectives The present study was aimed at validating NEMO score in different cohorts of patients. Methods NEMO score and the total number of GC were assessed by eight finger NVC in 122 patients with SSc referring to the Rheumatic Disease Unit of the “Istituto G.Pini” of Milan, where the preliminary study had been carried out (internal validation). The same procedure was performed in 99 patients collected in a different centre, i.e., the Rheumatic Dept. of the II University of Naples (external validation). In all of the patients DA, measured by ESSG score, was assessed at the same time of NVC performance. Results Demographic and clinical characteristics of the patients were summarised in Table 1. Table 2 shows the main statistical results obtained by the analysis of the two cohorts. The mNEMO score does not add anything in terms of sensitivity and specificity to the performance of the simple NEMO score in predicting DA. Conclusions This validation study confirms that the presence of a certain number of MH plus that of MT in eight finger NVC may represent a simple screening method to predict the presence of DA in patients with SSc. References Sambataro D, et al. Arthritis Res & Ther 2014;16:462. Disclosure of Interest None declared

Anticardiolipin (aCL) and anti-beta2-glycoprotein I(antiβ2GPI) antibodies have been shown in animal models as not cross-reacting antibody populations. This observation prompted us to prove if anti-β2GPI exist in human sera by using a reliable method and then to investigate if these are independent from aCl antibodies. We have developed a new ELISA for the detection of anti-β2GPI antibodies employing the coating of the protein in carbonate buffer to irradiated microtitre plates and the filtration of serum samples, that makes irrelevant the binding to the uncoated wells. IgG F(ab)2 fragments from IgG positive sera were shown bind β2GPI, providing that the binding was a specific antibody binding, mediated by the antigen binding site of the antibody molecule; moreover the antibodies were not able to differentiate native and delipidated β2GPI coated plates, making a possible role of a phospholipid contaminant unlikely. On the other hand, the phosphorus content of native as well as delipitated β2GPI was undetectable. IgG, but not IgM, anti-β2GPI antibodies were classically inhibited by the addition of soluble β2GPI, while cardiolipin liposomes appear to modify the reaction in a completely different way, possibly by the described interaction between cardiolipin and β2GPI. The application of the new ELISA to the study of patients has shown that: (1) the presence of anti-β2GPI is statistically associated with the presence of aCL antibodies (P < 0.0001), (2) anti-β2GPI antibodies are related to the classical features of antiphospholipid syndrome (thrombosis: P < 0.001; fetal loss: P < 0.001) while, in this series of patients, aCl antibodies are not (thrombosis: P < 0.126; fetal loss: P < 0.061).

ObjectiveTo identify a core set of preliminary items considered as important for the very early diagnosis of systemic sclerosis (SSc).MethodsA list of items provided by European League Against Rheumatism (EULAR) Scleroderma Trial and Research(EUSTAR) centres were subjected to a Delphi exercise among 110 experts in the field of SSc. In round 1, experts were asked to choose the items they considered as the most important for the very early diagnosis of SSc. In round 2, experts were asked to reconsider the items accepted after the first stage. In round 3, the clinical relevance of selected items and their importance as measures that would lead to an early referral process were rated using appropriateness scores.ResultsPhysicians from 85 EUSTAR centres participated in the study and provided an initial list of 121 items. After three Delphi rounds, the steering committee, with input from external experts, collapsed the 121 items into three domains containing seven items, developed as follows: skin domain (puffy fingers/puffy swollen digits turning into sclerodactily); vascular domain (Raynaud's phenomenon, abnormal capillaroscopy with scleroderma pattern) and laboratory domain (antinuclear, anticentromere and antitopoisomerase-I antibodies). Finally, the whole assembly of EUSTAR centres ratified with a majority vote the results in a final face-to-face meeting.ConclusionThe three Delphi rounds allowed us to identify the items considered by experts as necessary for the very early diagnosis of SSc. The validation of these items to establish diagnostic criteria is currently ongoing in a prospective observational cohort.

Objectives:To measure the prevalence of, and factors associated with, left ventricular (LV) dysfunction in systemic sclerosis (SSc).Methods:The EUSTAR database was first searched. A case-control study of a patient subset was then performed to further identify independent factors associated with LV dysfunction by simple and multiple regression.Results:Of 7073 patients, 383 (5.4%) had an LV ejection fraction (EF) of Conclusion:The prevalence of LV dysfunction in SSc is 5.4%. Age, male gender, digital ulcerations, myositis and lung involvement are independently associated with an increased prevalence of LV dysfunction. Conversely, the use of calcium channel blockers may be protective.

Leflunomide is an immunosuppressive agent that acts by inhibiting pyrimidine synthesis in lymphocytes and other rapidly proliferating cells, as well as by suppressing tumor necrosis factor-α–induced cellular responses. A number of leflunomide-related adverse events have been reported. Among cutaneous side effects, a few cases of subacute cutaneous lupus erythematosus have been described. We report a previously undocumented reaction to leflunomide, manifesting as subacute cutaneous lupus erythematosus and erythema multiforme-like lesions, in a young woman treated with this drug for ankylosing spondylitis. Withdrawal of leflunomide combined with a short cycle of systemic corticosteroid led to the resolution of the patient’s rash, indicating this drug as being responsible for the development of the disease. We conclude that leflunomide might have triggered the occurrence of both subacute cutaneous lupus erythematosus and erythema multiforme in a patient with pre-existing autoimmune diathesis. The suppressive effect of this drug on tumor necrosis factor-α–related mechanisms might have played a role in the induction of such a unique reaction to leflunomide.

Background Autologous fat tissue grafting (AFTG) has been successfully used in the treatment of different sclerotic conditions, including scleroderma. Objectives We evaluated in patients with SSc who complained of a reduced mouth opening, the long-term efficacy of AFTG of the lips in improving mouth opening in comparison with hyaluronic acid (HA) filler. We also investigated whether these procedures may induce some changes in the microvascular architecture and dermal structure of the treated skin area. Methods We studied 30 patients with dcSSc, (median age 37±12 yrs, disease duration 11±8yrs): 15 were treated by topical perioral AFTG according to Coleman technique and 15 by HA filler. The filler was repeated after 3 months. Baseline and after treatment (at months 3, 6, and 12) mouth opening changes were assessed by measuring inter-incisal distance and oral perimeter. Pre- and post-treatment modifications of microvascular architecture were assessed by counting capillaries in the inferior lip videocapillaroscopy (VC) images. Similarly, histological sections of perioral skin biopsy were examined at baseline and 3 months to evaluate dermo-epidermic junction (DEJ), the collagen content (by Masson9s Trichrome staining) and microvessel density (MVD) (by anti-CD34/CD31staining). Results 3 months after treatment both the inter-incisal distance and oral perimeter significantly increased (p Conclusions The present study shows that, in patients with SSc, AFTG can improve mouth opening, induce a neovascularization, and partially restore the skin structure. All these effects were confirmed in the long-term observation. The lack of functional and biological effects in the control group treated by HA filler, suggests that the observed therapeutic effect of lipostructure may be specifically ascribed to the on site transplantation of fat tissue. Our study may open new perspectives for the local and general therapeutic approach to SSc. Disclosure of Interest None declared

Background Autologous haematopoietic stem cell transplantation (AHSCT) has shown to be an effective therapeutic option to prolong survival of patients (pts) suffering from rapidly progressive diffuse cutaneous systemic sclerosis (rp-dcSSc). Objectives This study was aimed at retrospectively comparing the disease outcome of pts with rp-dcSSc treated with AHSCT to that of age-, and sex-matched group of clinically similar pts selected from our cohort and treated with conventional therapies. Methods Eighteen pts (5 M, 13F; median age 40, 20-62 yrs) that underwent AHSCT were compared to 36 age- and sex-matched pts (10 M, 26 F; median age 44, 19-62 yrs), all of them suffering from rp-dcSSc. At the starting evaluation point the two groups were also exactly comparable for disease-duration and clinical findings, in term of skin involvement (mRSS), DLCO as % of predicted value and disease activity (ESSG scoring system). AHSCT was performed by mobilisation with cyclophosphamide (CTX) and G-CSF, selection of CD34+ cells and conditioning regimen with CTX and rabbit ATG. Twenty-five pts in the control group received 6 monthly pulses of intravenous CTX (750mg/m 2 ), 11 received steroids, DMARDS (Methotrexate and Azathioprine) and vasoactive therapies (noCTX). The clinical course in AHSCT and control groups was evaluated from time 0 to 5 years by using Kaplan Meyer survival curves, computing the Hazard Ratio (HR) and Chi square distribution. Results Results of the survival curve analysis of different groups, expressed as HR (95%CI) are reported in the table. Conclusions This retrospective study confirms that AHSCT approach is more effective to induce a longer survival (p=0.0004), and shows that is also able to more rapidly reduce the mRss (p Disclosure of Interest None declared

The prostanoids iloprost and alprostadil are widely used to treat ischaemic changes in patients with Raynaud's phenomenon (RP), but the optimal regimen is poorly defined. We evaluated whether there are differences between iloprost and alprostadil, in terms of either clinical efficacy or of laboratory data, with the aim of assisting in the treatment of connective tissue disease (CTD)-associated RP.Twenty-one women with CTD-associated RP were given intravenous iloprost (11 patients) or alprostadil (10 patients) cyclically (5 consecutive days, followed by 1 day every 30 days). Clinical efficacy (RP symptoms, skin score, digital ulcers) and circulating levels of von Willebrand factor (VWf), tissue plasminogen activator (tPA), thrombomodulin (TM) and Type III procollagen N-terminal propeptide (PIIINP) were evaluated by enzyme-linked immunoassay at different intervals.The overall benefits of iloprost and alprostadil were similar. RP improved in 45% versus 90% of patients; ulcers in 60% versus 40% of patients (iloprost versus alprostadil). Skin score did not significantly change with either drug. Circulating VWf decreased with either drug (iloprost -6.2%, alprostadil -9.4%), while tPA, TM, and PIIINP remained unchanged. Side effects were only minor and less frequent with alprostadil.Iloprost and alprostadil were both of benefit in CTD-associated RP, without significant differences in either clinical efficacy or circulating markers. However, ease of handling and the lower price favours alprostadil.

To determine the prevalence of anti-chromatin antibodies (Abs) in juvenile rheumatoid arthritis (JRA) and to assess any association between the presence of anti-chromatin Abs and clinical subsets of the disease.IgG anti-chromatin Abs and anti-extractable nuclear antigens (ENA) Abs were detected by an enzyme-linked immunosorbent assay (ELISA), and antinuclear Abs (ANA) by indirect immunofluorescence in sera of 89 children with JRA. Ten children with systemic, 32 with polyarticular and 47 with pauciarticular disease onset (uveitis occurred in 17/47 children) were studied. As a control group, 12 sera of patients suffering from idiopathic uveitis and 31 age- and-sex-matched healthy children (HC) were examined.Abs to chromatin were detected in 14/47 (29.8%) of children suffering from pauciarticular onset JRA and in this group the higher prevalence of anti-chromatin Abs has been found in children with chronic uveitis (p = 0.002). Anti-chromatin positivity was observed in 2/10 (20%) of systemic and in 3/32 (9.3%) of polyarticular onset JRA. Furthermore, none of the patients with idiopathic uveitis and HC had Abs to chromatin. anti-chromatin Abs titers remained relatively stable over a 6-month control period.Our results confirm previous data about the presence of circulating anti-chromatin Abs in juvenile arthritis. Interestingly, anti-chromatin Abs were significantly higher in the group of patients with pauciarticular onset with past or present history of uveitis, than in patients without ocular involvement. A long-term follow-up study could be useful to demonstrate the potential utility of these autoantibodies in diagnosing, classifying and treating children affected.

Objective: to evaluate the prevalence and clinical significance of anti-chromatin antibodies (Abs) in juvenile rheumatoid arthritis (JRA). Methods: IgG anti-chromatin Abs were detected by an enzyme-linked immunosorbent assay (ELISA), in sera of 94 children with JRA (10 children with systemic, 38 with polyarticular and 46 with oligoarticular disease onset). As control group, 33 age- and-sex-matched healthy children (HC) were also examined. Results: Abs to chromatin were detected in 24/94 (25,5%) of children suffering from JRA. Particularly, the higher prevalence of anti-chromatin Abs has been found in children with oligoarticular (30,4%) and polyarticular (23,7%) onset JRA. In these groups Abs titers were significantly higher compared to systemic JRA and HC (p=0.003). Anti-chromatin Abs were observed more frequently in patients with oligoarticular disease and chronic uveitis (21,7%). Furthermore, higher levels of anti-chromatin Abs has been found in all the patients treated with anti-TNFα therapy (p

Background As in other autoimmune diseases, even in systemic sclerosis (SSc) the assessment of disease activity (DA) is essential to detect the window of opportunity for a successful treatment. The so far proposed composite indexes for DA assessment imply a long and expensive patient9s work up. Nailfold videocapillaroscopy (NVC) is commonly regarded as an instrument useful for SSc diagnosis and subsetting, and the presence of megacapillaries is considered the hallmark of an active disease. Objectives Since microvascular endothelial activation represents the key mechanism of the disease progression, the present study was specifically designed to evaluate whether the presence and number of micro-hemorrhages (MH) and micro-thrombosis (MT) in NVC could be considered potential predictors of DA in SSc. Methods The study population was constituted by 107 patients with SSc, 50 with the diffuse cutaneous (dc) and 57 with the limited cutaneous (lc) variants of the disease. European Scleroderma Study Group (ESSG) index (Valentini G, et al. Ann Rheum Dis 2001; 60:592-8)) was assumed as the gold standard for DA assessment, and patients having a score ≥3.5 classified as active. NVC was performed at the same time of DA evaluation. The sum of MH and MT detected by NVC examination in 2° to 5° fingers of both hands (named as NEMO score) was then obtained. Results The enrolled patients were predominantly females (97/107), had a mean age of 55.8 yrs. (range 18-84 yrs.) and mean disease duration of 6.1 yrs. (range 0-30 yrs.). According with the defined cut-off value of ESSG index, 32/107 patients were active (14 with dcSSc and 18 with lcSSc). NEMO values were significantly correlated with ESSG index scores (r=0.56, p 0.001), but not with patients9 age and disease duration. Considering the other components of ESSG index, NEMO score was significantly higher in patients having scleredema (p Conclusions The presence of MH and MT appears to be a good predictor for DA in patients with SSc, and enhances the role of NVC as a quick and easy technique to select the patients potentially candidate to be more aggressively treated. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3167

Background A therapy really successful in modifying the natural history of Systemic Sclerosis (SSc) has not so far been described. Conversely, autologous haematopoietic stem cell transplantation (AHSCT) has demonstrated to be sometimes an effective treatment in patients (pts) with severe (s) diffuse cutaneous (dc) SSc. Objectives We report a retrospective analysis of our experience with AHSCT in severe cutaneous SSc evaluating the efficacy on skin and major organ involvement. In addition, we focused on the results of AHSCT on SSc clinical activity by using a validated activity score. Methods Rodnan skin score (mRSS), assessment of heart and lung organ involvement (OI) by echocardiography, respiratory functional parameters (VC, DLCO), and disease activity level, by the European Scleroderma Study Group (ESSG) scoring system, represented the tools used for selecting and monitoring the pts. Pts were eligible if they had a rapidly progressive disease, a mRSS>14, and ESSG score>3, in the absence of severe advanced OI. AHSCT was performed using positively selected CD34+ cells. Mobilisation was performed with CTX 4000 mg/m 2 and G-CSF 10 μg/m 2 /day. Conditioning regimen included CTX 50 mg/kg/day and rabbit ATG 2.5 mg/kg/day. Results Since 2003, 17 pts s-dcSSC were enrolled (4 M, 13 F; median age 40 yrs., ranging 20–62 yrs.), The median follow-up was of 41 months (range 6–136). A beneficial effect was observed in 16/17 pts (94%).The mean mRSS was 20.9 (SD ± 4.0) at baseline (T0), 9.8 (± 4.6) at month 6 (T6), 6.8 (± 2) at month 12 (T12), and 3.0 (± 1.6) at month 60 (T60), (all p Conclusions This study confirms that AHSCT in selected patients with severe dcSSc results in sustained improvement of skin thickening and stabilizations of organ function up to 10 years after transplantation, so leading to a global clinical improvement, as showed by the persistent reduction in the ESSG clinical activity score. According to other experiences, TRM resulted reasonable, as a possible result of patients selection. These findings are in keeping with the view that AHSCT is effective in improving the inflammatory and active phase of SSc, while letting unchanged and stable the fibrosclerotic one. Further studies are needed to evaluate the importance of CD34 selection, the need of immunosuppressive therapy post-AHSCT and the best timing of HSCT in the treatment of SSc patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4375

Background Indoleamine 2,3 dioxygenase 1 and 2 (IDO1-IDO2) enzymes catabolise tryptophan (TR) to its metabolite kyurenine (KNR), and are competitively inhibited by 1-Methyl-TR (1M-TR). IDO1-IDO2, once activated in dendritic cells (DCs) by IFN-type 1, may induce immune-tolerance by suppressing antigen-specific T-cell response directly or by activating T-reg cells [1]. The role of IDO in autoimmune diseases has not been completely elucidated. In collagen-induced arthritis of DBA/1 mice, IDO1 limits the arthritis phenotype, as shown by the detrimental effect of 1M-TR treatment. Conversely, in auto-reactive B-cell-mediated arthritis of K/BxN transgenic mice, IDO1 sustains the autoimmune aggression, as demonstrated by arthritis improvement and auto-reactive B-cell reduction in draining lymph nodes after 1M-TR treatment [2]. Finally, significantly increased plasma KNR levels and KNR/TR ratio have been reported in patients with systemic lupus and Sjogren9s syndrome (SS) [2]. Objectives To verify whether IDO1 is activated in SS focal sialoadenitis (FS), and investigate the relationships between IDO1 expression and the main cellular components in inflammatory foci. Methods Minor salivary gland biopsies from 22 patients (1 male, 21 females) with SS were examined for IDO1 expression using anti-IDO1 antibodies (Abs), and immuno-histochemistry (IHC). Prevalence of B-cells, T-cells, and plasmacytoid (p) DCs was investigated in FS infiltrates using monoclonal anti-CD20, -CD3, and -CD123 Abs, respectively. Immuno-fluorescence (IF) was used to evaluate the co-localization of IDO1+ cells with the immune cells within SS FS. Results Patients were aged 15-77 yrs. (mean 53.4 yrs), and their disease duration ranged from 1 to 16 yrs. (mean 8.9 yrs.). Anti-Ro/La Abs and extra-glandular features were recorded in 15 and 14 patients, respectively. IHC analysis demonstrated consistent expression of IDO1 within the focal infiltrates. A semi-quantitative IHC score showed significant correlation between IDO1 expression and FS scores (Spearman R=0.52, p Conclusions IDO1 activity is present in SS FS, and appears to be related to the amount of pDCs and B-cells. Further investigations are needed to clarify the cell type(s) expressing IDO1 within the SS FS infiltrates and the immuno-regulatory role of this enzyme in SS. References Johnson BA, et al. Immunotherapy 2009;1:645. Filippini P, et al. Curr Med Chem. 2012;19:5381. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3150

The popliteal artery entrapment syndrome represents a complex clinical picture that is generally considered a rare clinical observation. On the contrary, based on clinical signs and diagnostic results, involving different medical (rheumatology and physiatry) and surgical (orthopaedics and angiology) fields, it looks an important disease involving especially young people. The possibility of serious ischemic (thrombotic and/or embolic) complications makes this syndrome a hard challenge for the clinician and the surgeon from a diagnostic and preventive point of view, respectively.

Patients suffering from a major orthopaedic pathology, meaning those diseases or syndromes that involve large bodily segments as the spine, the hip and the knee, often show different associated diseases that may complicate the normal course of surgical treatment. Beyond the usual and normal preoperative procedures, these patients need a careful study of the co-morbidity factors, which even in physiological terms — these subjects are often elderly — complicate the surgical procedure and the post-operative course. Specifically, we will discuss associated vascular pathologies and the available diagnostic tools (Doppler c. w., eco-color-Doppler, power Doppler), used not only to assess the pathologies in question, if present, but to perform a follow-up during the post-operative course, until implementation of the rehabilitation programmes.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that involves skin and internal organs. Vascular abnormalities play a key role in the pathogenesis of the disease. Specifically, endothelial derangement with disruption of the normal architecture of the vessels is a specific finding in patients with SSc and is responsible for the tissue chronic ischemia which can manifest clinically as skin ulcers. Although an extensive body of literature supports the pathogenic importance of SSc, the real mechanisms leading to the scleroderma vasculopathy are not known. Besides defective angiogenesis, recent studies suggest that vasculogenesis is also impaired in SSc patients, with decreased numbers of endothelial progenitor cells. Further studies are needed to confirm these pathogenic hypotheses and to suggest new therapeutic approaches for the treatment of SSc.

Antiphospholipid antibodies (aPL) are associated with poor obstetric outcome, such as recurrent abortions, fetal death, growth retardation, and early preeclampsia (1). Passive transfer of whole immunoglobulin fractions from aPL-positive sera has been found to induce fetal loss and growth retardation in pregnant naive mice, suggesting a direct pathogenetic role (2–4). Although it has been assumed that aPL are directed against anionic PL, current advances in the field suggest that antibodies to PL-binding plasma proteins, such as β2-glycoprotein I (β2GPI), can be detected in standard aPL assays (5). Antibodies specific for β2GPI have been identified and found to be associated with the clinical manifestations of the antiphospholipid syndrome (APS) (6–22). The in vivo immunohistologic demonstration of β2GPI on trophoblast surfaces (23,24) and the induction of fetal loss by anti-β2GPI antibodies in experimental animal models (25,26) suggested a role of anti-β2GPI antibodies in fetal loss. Moreover, even murine and human aPL monoclonal antibodies (mAb) specifically reacting with anionic PL in the absence of any plasma cofactor have been shown to produce fetal loss, growth retardation, placental deposition, and necrosis in experimental animal models (3,27,28). Although experimental models have emphasized the role of thrombotic phenomena in placental tissue (4,27), studies in humans have shown that thrombotic events cannot account for all of the histopathologic findings in placentae from women with the APS (29,30). The possibility of direct villous and extravillous trophoblastic damage by aPL through the recognition of phosphatidylserine (PS) exposed during syncytium formation has been suggested (31). Reported direct effects of aPL on trophoblasts have included inhibition of the intercytotrophoblast fusion process (31), of human chorionic gonadotropin (hCG) or placental lactogen secretion (31,32), and/or of trophoblast invasiveness (31). Furthermore, whole IgG fractions from APS patient sera or xenogenic murine anti-PS mAb have been shown to displace annexin V from trophoblasts (and endothelial cell surfaces in the case of human IgG), thus creating conditions favorable to procoagulant state in vitro (31,33). The purpose of the present study was to investigate the in vitro ability of IgG from sera containing high levels of aPL to bind human trophoblast cells and to affect hCG secretion and invasiveness. Furthermore, to identify whether specific effects were related to individual antibody subpopulations, human mAb reacting with β2GPI or with anionic PL in the absence of any plasma cofactor were investigated for their ability to reproduce the binding to trophoblast cell membranes and the modulation of hormone secretion as well as invasiveness. From our results, it appears that trophoblast cells might represent one target for circulating aPL reacting with β2GPI and/or with “pure” anionic PL (whose binding is independent of any plasma cofactor) and that such antibodies affect trophoblast differentiation–related activities.

SUMMARYAntibody to carbonic anhydrase II, an enzyme abundantly present in biliary epithelium, has been proposed as a diagnostic marker for antimitochondrial antibody-negative PBC. In this study we determine its prevalence and clinical significance in a large series of patients with antimitochondrial antibody-positive and -negative PBC. Reactivity to carbonic anhydrase II was sought by Western immunoblotting in sera from 215 consecutive patients with PBC (26 antimitochondrial antibody-negative), 13 with autoimmune hepatitis, 25 with primary Sjögren's syndrome (pSS), 12 with systemic sclerosis, 19 with systemic lupus erythematosus and 73 healthy subjects. The prevalence of antibody to carbonic anhydrase II (titre 1:100) in PBC was 8%. No specific reactivity to carbonic anhydrase II was found in antimitochondrial antibody-negative PBC (7% versus 8% in antimitochondrial antibody-positive PBC). Ascites (P = 0.006) and Sjögren's syndrome (SS) (P = 0.022) in PBC were significantly associated with presence of the antibody. In patients with SS associated with PBC, the prevalence (19%) was similar to that observed in pSS (16%). At a serum dilution of 1:40, the prevalence of positive sera in PBC rose to 27% but disease specificity was reduced. Our findings in a large population of PBC patients rule out a relation between presence of antibody to carbonic anhydrase II and lack of antimitochondrial antibody. The higher prevalence of ascites found in positive patients warrants further evaluation.

The IgG fraction of human anti-endothelial cell antibodies (AECA) obtained from a patient with Wegener's granulomatosis was used as immunogen to raise AECA mAb in mice selected among those which developed vasculitis-like lesions after immunization. Three mAb (BGM, 3C8 and 7G2), selected by cyto-ELISA and flow cytometry analyses, featured a specific reactivity with human umbilical vein endothelial cells (HUVEC) and the mouse endothelial cell line H5V; on the contrary, HEp2 cells, the murine melanoma B16 cell line, the extracellular matrix as well as several other antigens tested were not recognized. BGM mAb, an IgG3 precipitating a 70 kDa structure from HUVEC, was able to induce endothelial cells to secrete amounts of IL-6 significantly higher than irrelevant controls or mAb binding different endothelial antigens (i.e. CD31, CD29, ICAM-1 and HLA class I). BGM mAb induced significant levels of antibody-dependent cell cytotoxicity (13 +/- 2.5 versus 0.6 +/- 0.03%). To the best of our knowledge, BGM is the first murine mAb specific for human endothelial cells generated by idiotypic manipulation; secondly, its biological properties further support the notion of a pathogenic role for AECA in autoimmune-mediated diseases.

Objective To investigate the ability of human anti-beta 2-glycoprotein I (anti-beta 2 GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. Methods Six human affinity-purified polyclonal anti-beta 2 GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphospholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent beta 2 GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) secretion. Results The affinity-purified IgG and the MAb with anti-beta 2 GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human beta 2 GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-beta 2 GPI MAb also significantly increased IL-6 and 6-keto-PGF1 alpha secretion. Conclusion These findings support the hypothesis that anti-beta 2 GPI antibodies bind and activate EC through the adherent cofactor beta 2 GPI, likely leading to a procoagulant state.

Background Bone marrow (BM)-derived mesenchymal stem cells (MSC) are currently under investigation for their potential role in the management of some autoimmune diseases. Besides their regenerative effects and ability to induce tissue repair, MSC seem to possess potent immunomodulatory and anti-inflammatory effects, either by direct cell-cell interaction or by secreting different molecules able to exert a paracrine effects on local environment. Objectives Aim of the study was to evaluate the paracrine effects of BM-MSCs mesenchymal stem cells (MSC) isolated from normal individuals and patients with systemic sclerosis (SSc), on proliferation and cytokine production of cutaneous fibroblasts. Methods Fibroblasts obtained from skin punch biopsies (10 dcSSc patients and 5 healthy donors -HD) were cultured in vitro . MSC were isolated by plastic adherence from SSc and HD bone marrow (BM). Co-cultures were assessed with HD or SSc fibroblasts with HD or SSc MSC. Fibroblast proliferation was assessed by WST-1 assay and data were expressed as median percentage of 3 replicates. Levels of TGF-β, Stem Cell Factor (SCF), and metalloproteinase MMP-9 were detected in the co-culture supernatants by commercial ELISA Kits. Results Co-culture assays confirmed our previous results: an inhibition of SSc fibroblast proliferation, higher when fibroblasts were cultured with nBM MSCs (% of fibroblast proliferation vs fibroblasts without MSCs (100%): 75.8±9.9% with SSc MSCs vs 64.7±8.3% with nBM MSCs; P =0.038). In the presence of fibroblasts from healthy subjects, MSCs from normal and SSc BM equally inhibited proliferation. In comparison with basal values, the levels of the cytokines showed only in the presence of SSc fibroblasts and HD MSC a significant reduction in the content of SCF (mean±SD: 14.68±3.4 pg/ml vs 11.48±4.4; P =0.01) and TGF-β (3169.8±810 pg/ml vs 2450±900; P =0.0098), while no significant changes were observed in the presence of SSc MSC. As regards MMP-9, the basal value (mean±SD: 0.008±0.01 ng/ml) significantly increased in the presence of both HD (0.035±0.02; P =0.003) and SSc (0.094±0.072 ng/ml; P =0.01) MSC. Conclusions Our findings demonstrate that normal MSC (more than SSc-MSC) may provide an important signal for skin fibroblasts, and particularly for SSc fibroblasts, by both exerting anti-proliferative effects and down-regulating their activation state. The present results may reinforce the perspective for a future use of BM-MSC as a therapeutic option in SSc, namely for their anti-fibrotic potential. Disclosure of Interest None Declared

Background Pulmonary involvement represents a major cause of death of scleroderma patients. Cyclophosphamide showed a small beneficial effect in RCTs. There is a rational for the use of tyrosine kinase inhibitors as potential therapy for systemic sclerosis (SSc) and other fibrosing disorders. Preliminary published clinical trials are inconclusive about imatinib efficacy and showed relevant side effects. Objectives To verify efficacy and tolerability of low dose of imatinib on lung and skin fibrosis in a cohort of scleroderma patients refractory to conventional therapy. Methods A phase II multicentric open trial, which included 30 consecutive patients affected by SSc, with active pulmonary involvement, refractory to cyclophosphamide. The study followed a ‘Simon’s two-stage design’. Patients were treated with oral Imatinib 200 mg/day for 6 months. A “good response” was defined as the improvement both of pulmonary function tests and HRCT-scan pattern. Secondary outcomes were cutaneous involvement (mRSS) and quality of life. Results Seventy-two patients were screened, 42 were excluded. The mean age was 50.83 ± SD 13.8 years and mean disease duration was 5.5 ± SD 4.1 years, 9 (30%) were males and 21 (70%) were females, all Caucasian. According to cutaneous involvement 14 (46%) had limited and 16 (53.3%) had diffuse cutaneous SSc; 93.3% were anti-SCL-70 positives. At the end of the study (month 12) we had 27 completers, 3 patients were lost prematurely because of death. Data refer to 26 patients. Seven of 26 (27%) met the criteria for response. As for mean changes in pulmonary function tests and PaO2 in the whole group, after therapy we observed a worsening of FVC (mean delta -2,74 ± SD 8,13%) and PaO2 (a mean delta of -2.27 ± SD 1.3), with a further worsening after stopping therapy. TLC and DLCO remained substantially stable. The analysis of thoracic HRCT showed a significant reduction in the number of pulmonary segments interested by a ground glass pattern, which persisted after completion of therapy. No changes were detected in the areas interested by a honeycombing pattern. We didn’t observe any significant improvement either in the skin fibrosis or in quality of life. Among serious adverse events we registered 4 cases not related with the experimental drug. Three patients died prematurely and one developed a severe pneumonia in the follow up phase. Concerning minor adverse events they were present in less than 20% of patients, mainly transient and well tolerated. Conclusions According to Simon’s protocol 27% of patients responded, instead of 30% of expected rate, nevertheless, this trial demonstrated that low dose of imatinib stabilized lung involvement in scleroderma patients after failure of conventional therapy with cyclophosphamide, with radiographic improvement of the alveolitis. There was no significant improvement of skin involvement. Except for the 4 SAE the low dose was well tolerated. Together with other published trials these data will provide useful suggestions to design future RCTs. Acknowledgements This study was partially funded by AIFA (Agenzia Italiana del Farmaco). Disclosure of Interest None Declared