11. Recent Advances in Therapy

P.309

Hit Hard and Early

B.E. Kersten¹, W.H.T. van den Hombergh¹, H.K.A. Knaapen¹, P.M. van der Kraan², C.H.M. van den Ende¹, F.H.J. van den Hoogen¹, M.C. Vonk¹

¹ Department of Rheumatology, Radboud University Medical Center, Nijmegen, THE NETHERLANDS, ² Experimental Rheumatology, Radboud University Medical Center, Nijmegen, THE NETHERLANDS

Introduction: Design of a 12-week explorative, monocentre, randomized, double-blinded, placebo controlled trial protocol to analyze the effect of high dose methylprednisolone on nailfold capillary changes and biomarkers in very early systemic sclerosis.

Accumulating evidence indicates that inflammatory mechanisms drive systemic sclerosis (SSc) vasculopathy and fibrosis, especially early in the disease. SSc patients with very early disease could profit most of early treatment targeting the inflammation. Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. Several studies have shown a mixed response to treatment with glucocorticoids in SSc, which is probably due to the fact that it is seldom initiated very early in the disease. We hypothesize that by inhibition of the inflammatory process driving the disease, treatment with glucocorticoids will induce remission in patients with very early systemic sclerosis.

Material and Methods: This study is a 12-week, randomized, double-blind, placebo-controlled trial to analyze the effect of high dose intravenous methylprednisolone in very early SSc. Thirty patients who fulfill the criteria for very early SSc are randomized in a 2:1 fashion to either methylprednisolone or placebo. Patients receive intravenous infusion on 3 consecutive days over three consecutive months. In this study, the primary endpoint will be the change in capillary density between baseline and 12 weeks. The secondary outcomes of this study are: change in selected biomarkers, change in nail fold capillary changes other than capillary density, signs of an established SSc and change in physical function, general health and utilities questionnaires.

Results:

Conclusions: This trial is the first aiming to treat very early SSc, targeting the inflammatoiry mechanism of the disease. By combining vasculopathy markers, inflammatory biomarkers and clinical signs and symptoms as endpoints in our study, we expect that the results of this study will contribute to the concept that early treatment in SSc is feasible.

P.310

Vitamin D Deficiency Correlates with Advanced Organ Involvement in European Systemic Sclerosis Patients

A.C. Trombetta¹, V. Smith², E. Gotelli¹, M. Ghio¹, S. Paolino¹, C. Pizzorni¹, A. Vanhaecke², B. Ruaro¹, M. Patane¹, A. Sulli¹, M. Cutolo¹

¹ Research Laboratory And Academic Division Of Clinical Rheumatology, Department Of Internal Medicine, University of Genoa, Genoa, ITALY, ² Department Of Rheumatology, Ghent University Hospital, Ghent University, Ghent, BELGIUM

Introduction: In SSc patients, low 25-hydroxyvitamin D (25(OH)D) serum concentrations have been shown [1]. Primary aim of the study was to find possible correlations between 25(OH)D serum levels and multiple clinical parameters, in patients with systemic sclerosis (SSc). Secondary aim was the evaluation of the effectiveness of standard vitamin D replacement therapy.

Material and Methods: 154 SSc patients were recruited, in all seasons. 25(OH)D serum concentrations were evaluated using LIAISON 25-OH system (Diasorin, Italy). In addition, Medsger’s disease severity scale (DSS), nailfold video capillaroscopy (NVC) and all the instrumental examinations covered by the international guidelines were performed [2]. Assumption of any medication, including oral colecalciferol, was considered in the analysis. Non-parametric tests were used for statistical analysis.

Results: The 25(OH)D mean serum concentration was 18.7 ±9 ng/ml (<20 classified as a deficiency). A statistically significant correlation was found with presence/absence of bi-basal fibrotic abnormalities at lung CT scan (16.1 ±8 ng/ml and 20 ±10 ng/ml respectively, p= 0.04). DSS parameters correlating with serum concentrations of 25(OH)D were: peripheral vascular system (p = 0.03), kidney (p = 0.02), gastrointestinal tract (p = 0.05). No significant correlation was observed with the incidence of digital ulcers, which was instead, closely related to NVC patterns (p <0.0001).

As expected, a statistically significant difference was observed between 25(OH)D serum concentrations in different seasons (winter: 14.6 ±7.8 ng/ml, spring: 17.2 ±7.9 ng/ml, summer: 21.43 ±10 ng/ml, autumn: 20.2 ±10 ng/ml, p= 0.032).

Interestingly, no effect of oral colecalciferol (1000 UI per day for at least 6 months) was observed on serum concentrations of 25(OH)D both in the treated (18.8 ±10 ng/ml) or in untreated patients (18.7 ±9 ng/ml, p= 0.81).

Conclusions: 25(OH)D deficiency resulted correlated with advanced lung, peripheral vascular system, kidney, and gastrointestinal tract involvement (according to the DSS scale). Supplementation with standard doses of oral colecalciferol was not effective in increasing serum concentrations of 25(OH)D. Therefore, for substitutive therapy, higher doses of colecalciferol should be evaluated [3].

References: [1] Cutolo M et al. Nat Rev. Rheumatol 2010; 6: 578-87; [2] Medsger TA Jr et al. J Rheumatol 1999; 26: 2159-67; [3] Tangpricha V et al. J Clin Endocrinol Metab 2012; 97: 1082-93.

P.311

Effect of 6 Months Therapy with Iloprost on Enhanced Liver Fibrosis (ELF) Test And Its Association with Disease Activity and Vascular Manifestations in Patients with Systemic Sclerosis (SSc)

K. Stefanantoni¹, I. Sciarra¹, M. Vasile¹, G. Abignano², C. Corinaldesi³, C. Antinozzi³, C. Crescioli³, F. Del Galdo², G. Valesini¹, V. Riccieri¹

¹ Department of Internal Medicine and Medical Specialties- Rheumatology Unit - Sapienza University of Rome, Rome, ITALY, ² Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UNITED KINGDOM, ³ Department of Human, Movement and Health Sciences-Endocrinology Section-University of Foro Italico, Rome, ITALY

Introduction: Iloprost is a synthetic prostaglandin used for vascular manifestation of SSc, in particular for active digital ulcers (DU) and severe Raynaud Phenomenon (RP). Iloprost is also involved in the regulation of fibrosis and inflammation acting on several receptors. ELF test is an algorithm that includes serum concentration of procollagen-III aminoterminal-propeptide, tissue inhibitor of matrix metalloproteinase-1 and hyaluronic acid that seems to correlate with fibrosis, disease activity, disease severity and quality of life in SSc.The aim of this study is to evaluate the effect of Iloprost on vascular manifestations, on disease activity and on serum levels of ELF at baseline (T0) and after 1 month (T1), 3 months (T3) and 6 months (T6) e.v. treatment.

Material and Methods: 30 patients (M/F = 1/29; mean age = 58.2 yrs; mean disease duration = 12.8 yrs) with established SSc according to ACR/EULAR criteria, were enrolled. At T0, T1, T3 and T6 treatments with Iloprost (standard dose) we measured ELF biomarkers on all patient’s sera and we clinically evaluated RP VAS, number of DU, European Scleroderma Study Group Activity Index (EScSGAI).

Results: Regarding ELF levels, they increased although not significantly at T1 and T3 treatments, while there was a statistically significant reduction comparing T3 to T6 values (p = 0.0095). We also found a statistically significant increase (p = 0.04) of RP VAS and in the number of DU at T3, corresponding to the colder winter period, while both parameters decreased at T6, and these reductions were significant compared to T3 values (p = 0.0008). Besides EScSGAI values also showed a statistically significant reduction (p = 0.03) comparing T3 to T6.

Conclusions: Our study confirms the efficacy of Iloprost on vascular manifestation of SSc and highlights its effect on the disease activity as well as on a specific multiple biomarker of fibrosis such as ELF, thus adding more data on the still not completely known role for this drug on the different manifestations of SSc.

P.312

Iloprost Therapy in Systemic Sclerosis: A Possible Role on Disease Activity Changes and CXCL4 Chemokine Serum Levels

K. Stefanantoni¹, I. Sciarra¹, M. Vasile¹, R. Palazzo², L. Frasca², G. Valesini¹, V. Riccieri¹

¹ Dipartimento di Medicina Interna e Specialità Mediche-UOC di Reumatologia- Sapienza Università di Roma, Rome, ITALY, ² ISS-National center for Research and Pre-clinical and Clinical Evaluation of drugs- Immune-mediated diseases Unit, Rome, ITALY

Introduction: Iloprost is a synthetic prostaglandin used for vascular manifestation of Systemic Sclerosis (SSc), in particular it is indicated for active digital ulcers (DU) and severe Raynaud Phenomenon (RP). Iloprost is also involved in the regulation of fibrosis and inflammation. CXCL4, or Platelet Factor 4, is a 70KDa CXC-chemokine synthesized in megakaryocytes and plasmacytoid dendritic cells, released after platelet activation. In SSc, it seems to be higher in patients with early diffuse SSc and it correlates with mRSS values and presence of PAH. The aim of this study is to evaluate the effect of Iloprost on disease activity and on serum levels of CXCL4 at baseline (T0) and after 1 month (T1), 3 months (T3) and 6 months (T6) of therapy.

Material and Methods: 30 patients (M/F = 1/29; mean age = 58.2 yrs; mean disease duration = 12.8 yrs) with established SSc according to ACR/EULAR criteria, were enrolled. At T0, T1, T3 and T6 treatment with Iloprost (standard dose) we determined European Scleroderma Study Group Activity Index (EScSGAI) and serum levels of CXCL4, measured using commercially available immunoassay kit (Human CXCL4/PF4 R&D SYSTEMS®).

Results: EScSGAI values showed a statistically significant reduction comparing T3 to T6 (p=0.03). Regarding CXCL4, we found significantly higher serum levels in SSc patients respect to a group of healthy controls (HC) (p=0.047), while no significant difference was found at T0, T1, T3 and T6. But when patients with higher levels of CXCL4 at baseline were considered (11/30 patients), we found that 7 subjects had a significant improvement in disease activity at T6 evaluated by EScSGAI (p = 0.015). In these patients we also detected a significant reduction in T3 CXCL4 values (p=0.043) persisting at T6. Besides higher basal levels of CXCL4 were detected in those patients with a disease duration less then 60 months (p=0.05) and in those subjects with pericardial effusion (p=0.037), while significantly lower levels of CXCL4 were found in patients with DU history (p=0.049) and esophageal involvement (p=0.008).

Conclusions: Our study highlights a role for Iloprost on disease activity in SSc, while its effect on CXCL4 serum concentrations seems rather complex and needs to be clarified, considering that the possible role for this chemokine in the disease may depend on the different stages and it can affect different clinical aspects of SSc.

P.313

Autologous Fat Grafting in the Treatment of Patients with Systemic Sclerosis: Our Experience and Future Perspectives

A. Spinella¹, M. Pignatti², F. Lumetti¹, G. Boscaini², M. Colaci¹, E. Cocchiara¹, G. De Santis², C. Ferri¹, D. Giuggioli¹

¹ Reumatology Unit, University of Modena and Reggio Emilia, Policlinico of Modena, Modena, ITALY, ² Department of Plastic and Reconstructive Surgery, University of Modena and Reggio Emilia, Policlinico of Modena, Modena, ITALY

Introduction: Systemic Sclerosis(SSc) is a connective tissue disease characterized by digital ulcers(DU) which are a manifestation of the underlying vasculopathy and fibrosis. They occur in up to 50 % of patients, they are painful, recurring and lead to functional disability, despite systemic and local therapies. Other complications are thickening of the skin of the hands and finger contractures as well as the loss of elasticity and fibrosis of the perioral area. Autologous fat grafting(AFG) is a technique used also to promote tissue regeneration. Recently, AFG has been successfully used to treat a progressively larger number of clinical conditions characterized by skin atrophy or fibrosis. We evaluated our preliminary experience with the use of AFG as innovative potential treatment-prevention for SSc skin complications.

Material and Methods: We treated 25 SSc patients (M/F 4/21, mean age 42.16±11.84-SD-years, mean disease duration 163.52±115.56-SD-months, L/D cutaneous subsets 20/5) suffering from long-lasting and poorly responsive hand involvement with or without DU. Furthermore, patients with severe SSc-related mouth thickening and opening limitation were enrolled for perioral approach. The surgical procedure consisted in the injection of centrifuged and purified autologous fat harvested from trochanteric areas or abdomen. 2ml of fat were grafted in each of the 8 sites around the mouth (total 16ml), 0.5 or 1ml of fat were grafted around the neurovascular bundle at the base of each finger. Patients’ clinical evaluation was performed at baseline and 3 months after the surgical procedure. In particular, we collected clinic-serological SSc features, subjective assessment including patients’ degree of satisfaction, clinimetric measures about hands and mouth.

Results: After 3 months we observed an improvement of perioral skin tension in 55,5% of patients and hands thickening in 33,3%. Modified Rodnan Skin Score(mRSS) showed an improvement trend (from 9.5±6.1 to 8.9±5.7; p=0.083). Raynaud measured with Raynaud Condition Score(RCS) was also significantly ameliorated (from 6.0±1.8 to 3.8±1.6; p<0.0001). These positive clinical changes were mirrored by the subjective improvement of patients’ wellbeing (HAQ from 1.45±0.45 to 0.95±0.35, VAS from 74.5±18.5 to 55.5±8.6).

Conclusions: Preliminary assessments at 3 months suggest potential efficacy of AFG to treat SSc skin complications, both at hands and mouth. In particular, we reported a promising improvement of RCS and fibrosis involvement. Enlargement of the study group and further follow up of our operated patients is planned and ongoing in order to confirm these data with more accuracy and to identify more responsive SSc patients through a better characterization of SSc subsets.

P.314

Topical Nitroglycerine (NTG) vs Matching Vehicle in Secondary Raynaud Phenomenon (RP) – A Double-Blind Crossover Study of Subjective and Physiologic Responses to Controlled Cold Challenge

J.R. Seibold¹², D. Khanna¹, L. Mendez², R. Namas¹, M.E. Csuka³, P. Caldron⁴, J. Molitor⁵, A.J. Kivitz⁶, P. Waller⁷, L. Shapiro⁸, S. Najam⁹, A. Khan⁹, V. Steen¹⁰, A. Chadha¹¹

¹ University of Michigan, Ann Arbor, Michigan, USA, ² Medical Research Center of Miami, Miami, Florida, USA, ³ Medical College of Wisconsin, Milwaukee, Wisconsin, USA, ⁴ Arizona Arthritis & Rheumatology Research, Phoenix, Arizona, USA, ⁵ University of Minnesota, Minneapolis, Minnesota, USA, ⁶ Altoona Center for Clinical Research, Altoona, Pennsylvania, USA, ⁷ Accurate Clinical Research, Houston, Texas, USA, ⁸ Albany Medical College, Albany, New York, USA, ⁹ Accurate Clinical Management, Houston, Texas, USA, ¹⁰ Georgetown University School of Medicine, Washington, D.C., USA, ¹¹ Austin Regional Clinic, Austin, Texas, USA, ¹² Medical College of Georgia, Augusta, Georgia, USA

Introduction: A topical therapy for either prevention or palliation of attacks would offer unique advantages to selected patients with Raynaud phenomenon (RP). This study used 0.9% nitroglycerine (NTG) in a microemulsion formulation compared to vehicle to investigate clinical and physiologic responses to repetitive controlled cold challenge in individuals with RP secondary to connective tissue disease (CTD).

Material and Methods: 65 subjects with CTD including 32 with systemic sclerosis (SSc), agreed to stop ongoing Rx for RP. Routinized assessments included 30 minutes of acclimatization at 22.2° C +/- 2.2 followed by up to 16 minutes in a cold room maintained at 4.4° C +/- 2.2 and 60 minutes of recovery. Finger temperature was monitored at regular intervals and subjects completed 10 cm VAS scales for numbness, tingling and pain as well as for overall RP attack severity. A Main Raynaud Symptom (MRS; most bothersome symptom of pain, numbness, or tingling) was chosen for each individual based on their highest VAS score during a baseline cold challenge. Active Rx or vehicle as placebo was applied in a blinded randomized sequence 30 minutes prior to each cold challenge with each treatment studied twice. An enriched subset of subjects responding to NTG but not to placebo was identified from the results of the first paired cold challenges for a subanalysis of responses in the final two cold challenges.

Results:

	Topical NTG	Vehicle	P Value
Main Raynaud Sx	–20.77 mm	–19.56 mm	0.64
% Responders*	33/6451.6%	32/6450%	0.93
MRS Enriched**	–18.70 mm	–13.76 mm	0.45
AUC Skin Temp***	857.5	848.0	NS

^*Responders – subjects with > 15 mm improvement in MRS VAS

^**Enriched Subset – Vascana response > 10 mm AND vehicle response < 15 mm on VAS during first paired cold challenge

^***Area Under Curve -Integral of skin temperature across time during cold exposure (degree-minutes)

Conclusions: Topical NTG was not superior to vehicle placebo in reducing symptoms of RP during controlled cold challenge. Although the VAS response in Main Raynaud Symptom was meaningful (-20.77 mm), response to placebo was of similar magnitude. This was also true of an enriched subset excluding placebo responders. Skin temperature during cold challenge was the measure of digital perfusion. The lack of difference in physiologic response mirrors the subjective measures. Placebo response remains a critical issue in RP trials. This is in spite of choice of a uniform population, personalization of definitions of response (self-selected MRS), a cold challenge designed to closely match in-life experiences and repetitive testing.

P.315

Improvement of Dermal Thickness in Systemic Sclerosis Patients Treated with Endothelin Receptor Antagonist: Long Term Study by High Frequency Ultrasound

B. Ruaro, A. Sulli, C. Pizzorni, S. Paolino, V. Tomatis, E. Alessandri, M. Cutolo

Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine,University of Genova, Genova, ITALY

Introduction: Systemic sclerosis (SSc) is a connective tissue disorder characterized by skin involvement, which may be recognized and studied by modified Rodnan skin score (mRSS) and skin high frequency ultrasound (US) (1-4). Endothelin-1 (ET-1) stimulates fibroblast matrix byosinthesis and it seems implicated in the development of dermal fibrosis in SSc (5,6). Bosentan, a dual ET-1 receptor antagonist, seems effective in reducing skin fibrosis in SSc patients (6). The aim of this study was to evaluate long-term effects of bosentan on dermal thickness (DT) in SSc patients, in comparison with long-term cyclic intravenous iloprost.

Material and Methods: Thirty-eight SSc patients were enrolled during their standard treatment for digital ischemia. At baseline (T0), 19 patients already receiving cyclic intravenous infusion of iloprost (5 continuous days, average 80 mcg/day, every three months), continued the treatment for further 4 years (ILO group). The remaining 19 patients, although they continued the same cyclic intravenous iloprost treatment as the previous group, also received bosentan 125 mg twice a day for 4 years (ILO+BOS group), due to digital ulcers. DT was yearly evaluated by both mRSS and US (18 MHz probe, MyLab 25, Esaote, Italy) at the level of the usual seventeen skin areas (zygoma, fingers, dorsum of hands, forearms, upper-arm, chest, abdomen, thighs, legs, and feet) (2-4). Non-parametric tests were used for the statistical analysis.

Results: A statistically significant decrease of DT, measured by US, was observed in the ILO+BOS group from T0 (median DT 19.3 mm) to T4 (median DT 18.5) (p=0.01). No statistical significant variation of mRSS was observed during the follow-up (median DT at T0 12/51 and at T4 11/51, p=0.70). Conversely, in ILO group, a statistically significant increase of DT, measured by both US (median DT at T0 18.2 and at T4 21.4, p<0.0001) and mRSS (median DT at T0 4/51 and at T4 8/51, p<0.0001), was observed after four years. Transient increase of transaminases was managed by temporary bosentan discontinuation.

Conclusions: In this open study, long-term treatment with ET-1 receptor antagonist in combination with iloprost seems to decrease DT in SSc patients, in contrast to the treatment with iloprost alone.

References:

1. Krieg T, et al. Rheumatology 2009;48:iii14–iii18.

2. Clements P, et al. J Rheumatol. 1995;22:1281-5.

3. Sulli A, et al. Ann Rheum Dis. 2014;73:247-51.

4. Ruaro B, et al. Microvasc Res. 2017;115:28-33.

5. Cutolo M, et al. J Rheumatol. 2010;37:1174-80.

6. Kuhn A, et al. Rheumatology. 2010;49:1336-45.

P.316

Therapeutic Effect of MSC-Derived Extracellular Vesicles in Systemic Sclerosis

P. Rozier^1,2, M. Maumus², A. Maria^1,2, C. Jorgensen^2,3, D. Noel^2,3, P. Guilpain^1,2

¹ Department of Internal Medicine, Multiorganic Diseases, Saint-Eloi Hospital, Montpellier, FRANCE, ² Institute for Regenerative Medicine and Biotherapy, Inserm U1183, Montpellier University, Montpellier, FRANCE, ³ Clinical Immunology and Osteoarticular Diseases Therapeutic Unit, Lapeyronie Hospital, Montpellier, FRANCE

Introduction: Systemic sclerosis (SSc) is a rare and potentially lethal autoimmune disease, with no curative treatment available to date. We recently reported the therapeutic effect of mesenchymal stem cells (MSC) in a murine model of SSc induced by hypochlorous acid (HOCl). While several clinical trials based on the systemic injection of MSC are in progress with encouraging results, the question about a safer and cost-reasonable alternative may be debated. Because MSC act primarily by the secretion of soluble factors that can be released within extracellular vesicles (EV), the use of EV-based acellular therapy in SSc could be such alternative. Indeed, the present study aimed at determining the therapeutic effect of MSC-derived EV, notably exosomes (EX) and microparticles (MP), in the murine model of HOCl-induced SSc.

Material and Methods: SSc was induced in 6-week-old BALB/c mice by daily intradermal HOCl injection. On day 21, each group received an intravenous injection of PBS, 250 000 murine MSC or 250 ng EX or MP isolated from IFNγ-activated or non-activated (NA) MSC. Skin thickness was measured once a week. Mice were euthanized on day 42. Blood, lungs and skin were taken for histological and RT-qPCR analysis of fibrotic-related markers (collagens 1, collagens 3, αSma, TGFβ, MMP1, MMP9, TIMP1, IL1β, IL6, TNFα).

Results: Injection of MSC, EX or MP isolated from NA MSC, significantly reduced skin fibrosis and inflammation while improving ECM remodeling, as shown by lower clinical score, histological analysis and gene expression of fibrotic markers. Same trends were observed in lung tissue. However, EX and MP isolated from IFNγ-activated MSC exerted similar effects indicating no major role of priming for the therapeutic function of MSC-EV in SSc.

Conclusions: Systemic injection of MSC-derived EX and MP in mice with SSc is demonstrated to be an effective curative treatment of the disease, with similar efficacy to that obtained with MSC injection. These data suggest that EV cargo may exert the majority of beneficial effects of MSC in SSc.

P.317

Control of Fibroblast Polarization as Potential New Strategy to Terminate Tissue Fibrosis

T. Wohlfahrt, S. Rauber, A.-E. Matei, A. Soare, M. Luber, G. Schett, J.H.W. Distler, A. Ramming

Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander-University (FAU) Erlangen-Nürnberg, Erlangen, GERMANY

Introduction: Fibroblasts play an important role during the preservation of tissue integrity which goes far beyond deposition of extracellular matrix. Phenotypic changes of fibroblasts rather orchestrate a complex multicellular tissue response in the context of inflammatory and neoplastic diseases. In pathological settings, fibroblasts can either acquire a “pro-fibrotic” phenotype with excessive extracellular matrix production or a “pro-inflammatory“ phenotype releasing matrix-degrading enzymes and promoting tissue destruction. Knowledge on the underlying transcriptional programs that drive these opposing functional phenotypes of fibroblasts is scarce to date.

Material and Methods: Primary fibroblasts from different organs were analyzed by RNA sequenzing (RNASeq), real-time PCR, western blot analysis, and multicolor immunofluorescence microscopy (IF). Polarization of fibroblasts was modulated in vitro, in 3D full skin organoids and in different mouse models of fibrosis.

Results: We identified transcriptional checkpoints for the acquisition of a “pro-fibrotic” phenotype in fibroblasts inducing the production of fibrosis-associated genes, the differentiation of myofibroblasts and precipitating the development of fibrotic diseases, such as pulmonary fibrosis, systemic sclerosis, liver cirrhosis, kidney fibrosis and chronic graft versus host disease. Fibroblast polarization was regulated by epigenetic control mechanisms. These control mechanisms are lost in fibrotic diseases, resulting in a phenotype switch from matrix-degrading “inflammatory” into matrix-producing “fibrotic” fibroblasts. In contrast, genetic and pharmacological re-programing of fibrotic fibroblasts into resting fibroblasts led to regression of fibrosis without interference with physiological tissue repair mechanisms.

Conclusions: Targeting transcriptional checkpoints of fibroblast polarization provide a novel therapeutic option to efficiently but also safely interfere with excessive matrix deposition and allow to restore tissue homeostasis in fibrotic diseases.

P.318

Mesenchymal Stem Cell Function is Affected by Serum-Mediated Oxidative Stress from Systemic Sclerosis Patients

G. Fonteneau¹, C. Bony¹, R. Goulabchand², A. Maria², C. Jorgensen^1,3, P. Guilpain², D. Noel^1,3

¹ IRMB, Inserm, Montpellier University, Saint-Eloi Hospital, Montpellier, FRANCE, ² Department of Internal Medicine, Multiorganic Diseases, Montpellier, FRANCE, ³ Clinical Immunology and Osteoarticular Diseases Therapeutic Unit, Lapeyronie Hospital, Montpellier

Introduction: Systemic sclerosis (SSc) is a life-threatening disease where oxidative stress, in particular advanced oxidized protein product (AOPP), plays a central physiopathologic role. In absence of curative treatment, the therapeutic efficacy of mesenchymal stem cells (MSCs) is being evaluated in the clinics. These clinical studies use allogeneic MSCs since MSCs isolated from SSc patients were shown to display altered properties. Indeed, the present study aimed at evaluating whether the oxidative environment of SSc patients may impact the functions of MSCs from healthy subjects.

Material and Methods: Proliferation, apoptosis and senescence rates of human bone marrow-derived MSCs were evaluated after 3, 6 and 10 days in culture in the presence of various sera: control human serum AB (SAB), SAB with HOCl-induced AOPPs at 400 or 1000 µmol/L (SAB 400 or SAB 1000, respectively) or H2O2-induced AOPPs or SSc patient serum (PS). Reactive oxygen species and nitric oxide production were quantified at 24h. T lymphocyte proliferative assays were performed in presence of MSCs and trilineage potential of differentiation was tested after 21 days in specific culture conditions.

Results: In presence of oxidative environment of PS, phenotype of MSCs was not altered and the overall expression of fibrotic markers was similar to control SAB conditions. The proliferation rate of MSCs was unchanged with low levels of serum AOPPs but decreased with AOPPs levels higher than 400 µmol/L at day 6 and 10. This inhibition of proliferation was associated with higher level of apoptosis and senescence rates. Interestingly, expression of the antioxidant gene SOD2 increased in MSCs cultured with PS, suggesting enhanced antioxidant capacity in presence of AOPPs. Finally, the immunosuppressive function of MSCs was slightly reduced with PS whereas their osteoblastic and adipogenic potential was increased.

Conclusions: We report here that some functional properties of MSCs are affected when cultured with SSc sera. However, evidence from pre-clinical studies and the present one suggest that MSCs can adapt to the oxidative environment and exert their therapeutic effect.

P.319

Two Years Follow-Up of an Open-Label Pilot Study of Treatment with Rituximab in Patients with Early Diffuse Cutaneous Systemic Sclerosis

K. Melsens^1,2, E. Vandecasteele³, E. Deschepper⁴, V. Badot⁵, D. Blockmans⁶, G. Brusselle⁷, E. De Langhe⁸, M. De Pauw², C. Debusschere^1,2, S. Decuman², L. Deroo⁹, F. Houssiau¹⁰, J. Lenaerts⁶, Y. Piette¹, K. Thevissen¹, M. Vanthuyne¹⁰, R. Westhovens⁸, S. Wijnant⁹, F. De Keyser^1,2, V. Smith^1,2, A. Vanhaecke¹¹

¹ Department of Rheumatology, Ghent University Hospital, Ghent, BELGIUM, ² Department of Internal Medicine, Ghent University, Ghent, BELGIUM, ³ Department of Cardiology, Ghent University Hospital, Ghent, BELGIUM, ⁴ Department of Public Health, Biostatistics Unit, Ghent University, Ghent, BELGIUM, ⁵ Department of Rheumatology, Hospital Erasme, Université Libre de Bruxelles, Brussels, BELGIUM, ⁶ Department of General Internal Medicine, University Hospitals Leuven, Campus Gasthuisberg, Louvain, BELGIUM, ⁷ Department of Respiratory medicine, Ghent University Hospital, Ghent, BELGIUM, ⁸ Department of Rheumatology, University Hospitals Leuven, Campys Gasthuisberg, Louvain, BELGIUM, ⁹ Faculty of Medicine and Health Sciences, Ghent University, Ghent, BELGIUM, ¹⁰ Department of Rheumatology, Cliniques Universitaires Saint-Luc, Brussels, BELGIUM, 11 Ghent University, BELGIUM

Introduction: Patients with early diffuse cutaneous systemic sclerosis (dcSSc) are prone to severe organ involvement and consequently high mortality. Rituximab has been shown in some open-label studies to stabilize internal organ involvement and to decrease the skin score. Moreover, a study of our study group shows that this decline in clinical skin score is in line with histopathological findings on skin biopsy. Following these results, a Belgian three-centre initiative was launched to explore safety and efficacy of rituximab in a larger cohort of early, diffuse cutaneous systemic sclerosis (dcSSc) patients.

Material and Methods: Seventeen patients with early dcSSc (disease duration of four years or less) were consecutively included in this open-label study. Patients were treated with two courses of rituximab, at month 0 and 6. One course consisted of two infusions of 1000mg of rituximab, administered with a two-week interval. Clinical examination, lung function testing, echocardiography, disease activity score (based on the European Studygroup Disease disease activity index, EScSG-DAI) and functional status were performed at baseline, at month 3, 6, 12, 15, 18 and 24 months of follow-up. Mixed models analyses (MMA) were used to evaluate changes in parameters over time.

Results: Modified Rodnan skin score (MRSS) changed significantly over time, with a mean of 25.5 (standard deviation [SD] 6.0) at baseline to 12.6 (SD 5.1) at month 24 (MMA p<0.0001), which is a decrease of 51% at month 24 versus baseline. The EScSG-DAI showed significant decrease over the total study period, with a score of 4.1 (SD 1.7) at baseline to 1.5 (SD 1.8) at month 24 (MMA p<0.0001), which is a decrease of 63% at month 24 versus baseline. Additionally, this was significant at all time points versus baseline, both for MRSS and EscSG-DAI. Internal organ status remained clinically stable throughout the study period: no statistically significant differences compared to baseline were found at the follow-up time points. Seven serious adverse events took place, all except for one, considered unrelated to study medication.

Conclusions: This is the first multicentre Belgian collaboration investigating potential efficacy of rituximab in early dcSSc. Rituximab appears to be safe and tolerable and it may have beneficial effects on skin involvement, on overall disease activity and on stabilization of internal organ status in early dcSSc. Trial registration. ClinicalTrials.gov Registration, http://clinicaltrials.gov, number NCT00379431

P.320

Fibrosis Development in HOCL-Induced Systemic Sclerosis: a Multistage Process Hampered by Mesenchymal Stem Cell Therapy

A. Maria¹, K. Toupet², M. Maumus², P. Rozier², M.C. Vozenin³, A. Le Quellec¹, C. Jorgensen², D. Noël², P. Guilpain¹

¹ Department of Internal Medicine, Multi-organic Diseases, Saint-Eloi Hospital, Montpellier, FRANCE, ² Institute for Regenerative Medicine and Biotherapy (IRMB), Inserm U 1183, Montpellier University, Saint-Eloi Hospital, Montpellier, FRANCE, ³ Laboratory of Radiation Oncology Department, University Hospital of Lausanne (CHUV), Lausanne, SWITZERLAND

Introduction: Skin fibrosis is the hallmark of systemic sclerosis (SSc) a rare intractable disease with unmet medical need. We previously reported the anti-fibrotic potential of mesenchymal stem cells (MSCs) in a murine model of SSc. This model, based on daily intra-dermal injections of hypochlorite (HOCl) during six weeks, is an inducible model of the disease. In this study, we aimed at characterizing the development of skin fibrosis in HOCl-induced SSc, and evaluating the impact of MSC infusion during the fibrogenesis process.

Material and Methods: In a longitudinal study, we decomposed HOCl-SSc induction in three time-periods and examined skin thickness, histological and biological parameters after three weeks (d21) and six weeks (d42) of HOCl challenge, and three weeks after HOCl discontinuation (d63). Treated-mice received intravenous infusion of 2.5x105 MSC three weeks before sacrifice (d0, d21 or d42).

Results: HOCl injections induced a two-step process of fibrosis development: first, an ‘early inflammatory phase’, characterized at d21 by highly proliferative infiltrates of myofibroblasts, T-lymphocytes and macrophages; second, a phase of ‘established matrix fibrosis’, characterized at d42 by less inflammation, but strong collagen deposition. A third phase of ‘spontaneous tissue remodelling’ was observed after HOCl discontinuation, characterized by fibrosis partial receding, due to enhanced MMP1/TIMP1 balance. MSC treatment reduced skin thickness in the three phases of fibrogenesis, exerting more specialized mechanisms: immunosuppression, abrogation of myofibroblast activation, or further enhancing tissue remodelling.

Conclusions: HOCl-SSc mimics three fibrotic phenotypes of scleroderma, all positively impacted by MSC treatment, which demonstrates the great plasticity of MSC, a promising cure for SSc.

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Regenerative Surgery in Systemic Sclerosis

G. Magalon¹, A. Daumas¹, J. Magalon^2,3, D. Casanova⁴, C. Philandrianos⁴, N. Sautereau¹, P. Guillaume-Jugnot¹, F. Dignat-George^2,5, J. Veran³, F. Sabatier^2,3, B. Granel^1,2

¹ Assistance Publique Hôpitaux Marseille, Internal Medicine, Marseille, FRANCE, ² Aix Marseille University, Marseille, FRANCE, ³ Culture & Cell Therapy Laboratory, Marseille, FRANCE, ⁴ Assistance Publique Hôpitaux Marseille, Plastic Surgery Department, Marseille, FRANCE, ⁵ Assistance Publique Hôpitaux Marseille, Hematology & Vascular Biology, Marseille, FRANCE

Introduction: Over the last few years, adipose tissue-derived stem cell therapy has emerged as a novel therapeutic approach. The adipose tissue contains various cells such as adipose-derived stem/stromal cells, endothelial progenitor cells and immune cells which act together for tissue repair and regeneration. The abundant supply of fat tissue, ease of isolation, the ability to secrete angiogenic growth factors and abundance of stem/progenitor cells make adipose-based therapy an ideal for ischemic and non-healing wounds.

Material and Methods: Systemic sclerosis can benefit from Regenerative Surgery with:

• - injection of the autologous adipose-derived stromal vascular fraction (AD-SVF) in fingers. 12 patients, SCLERADEC (NTC01813279)

• - autologous adipose tissue injection in the treatment of digital ulcer, Del Papa et al. 15 patients

• - autologous adipose tissue injection in the treatment of refractory Raynaud’s. Bank et al. 13 patients

• - microfat injection in the face. 12 patients, SCLEROFACE (NCT02206672) and standard Coleman’s fat grafting, Del Papa et al. 20 patients

• - our team has improved the microfat injection by adding Platelet Rich Plasma (PRP) in the treatment of the face.

Results: - AD-SVF injection in fingers: patients long term outcomes showed from baseline, a 62.5% improvement of the CHFS (Cochin Hand Function Scale) score, a 51.1% improvement of the Scleroderma Health Assessment Questionnaire, a 33.1% improvement of the hand pain and an 88.3% improvement of the Raynaud Condition Score (p<0.001).

• - autologous adipose tissue injection for the treatment of digital ulcer allows rapid healing

• - autologous adipose tissue injection in the treatment of refractory Raynaud’s. Bank et al. reported a significant improvement of their composite score after 30cc of fat grafting by hand.

• - Fat & microfat injection in the face to improve skin pliability and quality with aesthetic benefit and significant decrease in the MHISS score.

• - combined subcutaneous microinjection of autologous fat and platelet-rich plasma (PRP) in face favors adipose tissue maintenance and survival.

Conclusions: These innovative therapies are safe and can be considered cost-effective given the long duration of benefit following a single application and the autologous cells which can easily be harvested.

Concerning future perspectives, the combination of fat grafting with AD-SVF and PRP will be interesting in the aim to have both volumizing and trophic effect for indication in which both effects.

By combining the lipostructure technique with the regenerative cell therapy tissue, graft longevity and positive effect could be enhanced.

Two randomized double blind, placebo-controlled clinical trials with ADSVF are ongoing in USA (NCT02396238) and in France (NCT02558543).

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Anti RNAPOL and TOPOI Autoantibody Reactivity and Disease Manifestations from the Fasscinate Systemic Sclerosis Trial

S.J. Lopez¹, A. Jahreis¹, T. Sornasse¹, J. Pei¹, J. Siegel¹, J. Garg¹, D. Khanna², C.J.F. Lin¹

¹ Genentech, South San Francisco, USA, ² University of Michigan, Ann Arbor, USA

Background: Autoantibody signatures are routinely identified in patients with systemic sclerosis (SSc) and their clinical relevance has been increasingly recognized. Safety and efficacy of subcutaneous tocilizumab (TCZ) in the faSScinate randomized, placebo (PBO)-controlled trial was previously reported. Here, analyses of patient autoantibody reactivity and baseline disease characteristics are presented.

Objective: To identify associations between autoantibodies in SSc patients and disease manifestations

Methods: From the faSScinate trial, 87 patients were assessed by ELISA for reactivity with anti-centromere (>10 U/mL), PM/Scl (>10 U/mL), RNA polymerase III (RNAPOL) (>20 U/mL), topoisomerase I (TOPOI) (>10 U/mL), and U1 SmRNP (>10 U/mL). Association of reactivity with baseline mRSS and ppFVC, as well as disease progression and treatment response at week 48, were analyzed by Welch twosample t-test and mixed model repeated measures analysis.

Results: Autoantibody reactivity was as follows: centromere (0%), PM/Scl (1%), RNAPOL (34%), TOPOI (44%), and U1SmRNP (3%). RNAPOL and TOPOI reactivity was mutually exclusive. At baseline, RNAPOL was associated with a 7.0% higher percent predicted FVC (ppFVC) compared to RNAPOL negative patients (p < 0.05) whereas TOPOI was associated with a 7.2% lower ppFVC compared to those who were TOPOI negative (p < 0.05). Autoantibody reactivity and treatment effects were not significant for mRSS measures at week 48. At week 48, in the TOPOI negative group, patients treated with PBO experienced more of a decline in ppFVC compared to those treated with TCZ (p = 0.034). No significant difference in change in ppFVC was detected in the TOPOI positive group between the TCZ- and PBOtreated patients. Analysis of the RNAPOL positive and negative groups did not yield any significant result for the comparison of change in ppFVC between the TCZ - and PBO-treated patients.

Conclusions: Autoantibody distribution was consistent with previously reported populations. RNAPOL positivity was associated with significantly higher ppFVC at baseline whereas TOPOI was associated with significantly lower ppFVC. The autoantibody profile (RNAPOL and TOPOI) had little clinically meaningful influence on the effect of TCZ on change in mRSS or ppFVC at week 48. These observations are based on a small study and need to be verified in a larger study.

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Tocilizumab Treatment for Patients with Systemic Sclerosis. Description of 14 Patients from Meir Medical Center

Y. Levy, G. Zacay

Meir Medical Center, Kfar Saba, Israel

Introduction: Systemic sclerosis (SSc) is a fibroinflammatory autoimmune disease with severe involvement of skin, lungs and other organs. Until now, no treatment has been proven to provide good results for this disease.

In the last 10 years several studies demonstrated the importance of Interleukin 6 (IL6) as a pivotal cytokine in the development of fibrosis and angiopathy especially in SSc.

It has been shown that sera and different tissues (such as lung) in SSc patients contain high level of IL6. In addition, there is a 30-fold increase in spontaneous IL6 production of fibroblasts from affected SSc skin, compared to normal fibroblasts. IL6 production is also increased in mice with bleomycin induced SSc, whereas naturalization of IL6 in those mice prevents development of skin fibrosis .

Material and Methods: During the last five years, 14 patients with diffuse systemic sclerosis were treated with Tocilizumab in our center. Six of them were in early stage of the disease, within two years from diagnosis. Four had longer duration of the disease. Two patients had rheumatoid arthritis overlapped with SSc while two other had systemic sclerosis with polymyositis overlap.

12 patients received intravenous tocilizumab (8 mg/kg once a month) and the other two were treated with subcutaneous tocilizumab (162mg once a week).

Results: There was a significant improvement in skin, musculoskeletal symptoms and lung involvement in the six patients with early SSc. Modified skin score decreased dramatically, while pulmonary function tests improved or remained stable.

The four patients with long standing disease showed some improvement with arthralgia and myopathy as well as other musculoskeletal symptoms, with modest improvement in quality of life, however no other objective improvement was observed.

Significant improvement was observed with the two patients with systemic sclerosis polymyositis overlap: creatine kinase level normalized, skin fibrosis and lung function tests improved.

Two patients with Rheumatoid Arthritis overlapped with SSc demonstrated no significant improvement following the treatment.

Of interest is a phase 2 RCT study which checked the safety and efficacy of subcutaneous tocilizumab in adults with SSc. The research showed a trend for improvement in skin score and some evidence for less decline in lung function. Due to this successful phase 2 study, a phase 3 study was launched

Conclusions: Our experience in treating SSc patients with tocilizumab is promising especially for early systemic sclerosis.

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Pertinence of Mycophenolate Mofetil’s Area Under the Curve Systemic Sclerosis Associated with Diffuse Skin and/or Pulmonary Involvement

P. Legendre¹, B. Blanchet², R. Porcher³, A. Berezne¹, M. Allard², J. London¹, B. Terrier¹, P. Cohen¹, C. Le Jeunne¹, L. Mouthon¹

¹ Service de Médecine Interne, Hôpital Cochin AP-HP; Université Paris Descartes, Paris, FRANCE, ² UF de Pharmacocinétique et Pharmacochimie, Hôpital Cochin, AP-HP, Paris, FRANCE, ³ Université Paris Descartes, Paris, FRANCE

Introduction: To determine a mycophenolate mofetil (MMF) area under curve (AUC0-12) threshold associated with a clinical response on skin involvement or interstitial lung disease (ILD) in patients with SSc at one year.

Material and Methods: This retrospective, monocentric study was based on «French Scleroderma Database » inpatients treated by MMF and who experienced a blood sampling to calculate an AUC0-12 plus two pulmonary function tests and skin evaluation after one month and one year.

Variation of modified Rodnan skin score (mRSS) at one year and variation of forced vital capacity (FVC and/or diffusing lung capacity for carbon monoxide (DLCO) to evaluate skin and ILD-SSc response, respectively.

Results: We included 52 patients in the study, mean age 49 years (17–79), 36 (6%) were females. Fifty patients had skin sclerosis, 39 (75%) had diffuse skin involvement with a median mRSS of 14 (0 – 38). Thirty-eight (76%) patients had ILD, with median FCV and DLCO of 81% (37 – 127) and DLCO 56% (28 – 103) from predicted values, respectively. Twenty-five patients (51%) had pulmonary fibrosis. MMF was given for 10 months (0 – 173) at a median dose of 2g/day (0.5 – 3). Regarding the entire population, no correlation was found between mRSS (p = 0,085), FVC (p=0,800) or DLCO (p=0,720) variations at one year and AUC0-12 value.

Conclusions: Conclusion: In this retrospective study we found no correlation between AUC0-12 of MMF and skin involvement or ILD evolution. Based on these results we might not recommend performing AUC0-12 of MMF in SSc patients.

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The Selective S1P1 Antagonist Act- 334441 Ameliorates Murine Sclerodermatous Chronic Graft Versus Host Disease

M. Kano, M. Date, T. Matsushita, Y. Hamaguchi, K. Takehara

Department of Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa, JAPAN

Introduction: Sphingosine 1 phosphate (S1P) is a lipid mediator that controls the flow of lymphocytes between the lymphoid tissue and blood. S1P also plays a role in several physiological functions such as angiogenesis, inflammation, regulation of the immune system, and the release of neurotransmitters. Recently, S1P/S1P receptor signaling has been shown to play an important role in the pathogenesis of systemic sclerosis (SSc). In this study, we investigated whether the selective S1P1 antagonist ACT-334441 attenuates murine sclerodermatous chronic graft versus host disease (Scl-cGVHD), which is an animal model for SSc and cGVHD.

Material and Methods: Serum S1P levels were measured in 50 patients with SSc, 18 patients with systemic lupus erythematosus (SLE), and 20 control subjects. Scl-cGVHD was induced by transplantation of the bone marrow and splenocytes from B10.D2 donor mice into sublethally irradiated BALB/c recipient mice. ACT-334441 was orally administered to Scl-cGVHD mice, either from days 0–42 (preventive model) or days 22–42 (therapeutic model) after bone marrow transplantation.

Results: Serum S1P levels were significantly higher in patients with SSc compared with that in patients with SLE and control subjects. Early administration of ACT-33441 (preventive model) inhibited skin and lung fibrosis in Scl-cGVHD mice. In addition, delayed administration of ACT-33441 (therapeutic model) attenuated the severity and fibrosis in Scl-cGVHD mice. Immune genis staining and flow cytometry analysis showed that ACT-334441 suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the inflamed skin of Scl-cGVHD mice compared to the control mice. By contrast, ACT-334441 increased the frequency of myeloid suppressor cells and regulatory T cells in the spleen of Scl-cGVHD mice. In addition, ACT-334441 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines such as IL-1B and IL-6, in the skin of Scl-cGVHD mice.

Conclusions: ACT-33441 attenuated skin and lung fibrosis in Scl-cGVHD mice by inducing regulatory cells and decreasing the skin-infiltrating immune cells. These results suggested that the selective S1P1 antagonist ACT-33441 is a promising candidate for treating patients with SSc and Scl-cGVHD.

P.326

Molecular Mechanism for the Therapeutic Effect of Extracorporeal Shock Wave Therapy on Digital Ulcers of Systemic Sclerosis

Y. Kamogawa, H. Fujii, T. Shirai, T. Ishii, H. Harigae

Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, JAPAN

Introduction: Digital Ulcers (DUs) in systemic sclerosis (SSc) are frequently associated with severe Raynaud’s phenomenon and not usually caused by vasculitis. Since such refractory ulcers are not primarily based on inflammation, immunosuppressants including steroids have little therapeutic effect on digital ulcers, and so there are only palliative treatments. However, the effect of these treatments is insufficient in most cases. Shock wave (SW) is a pressure wave propagating at supersonic velocity through the medium. Extracorporeal shock wave therapy (ESWT) has been applied to the treatment for various diseases such as chronic tendinopathy, ischemic heart disease, and wound healing disorders as well as urinary stones. The therapeutic effect of ESWT is considered to depend on biological responses, not only mechanical injury, however the precise molecular mechanisms remain unclear. Previously, our group reported, for the first time, the effect of ESWT on digital ulcers of SSc. For the purpose to develop more effective therapeutic strategy, we designed in vitro and in vivo SW irradiation system to identify a master regulator for early gene response to SW treatment

Material and Methods: (1) Human Dermal Microvascular Endothelial Cells (HDMECs) were cultured and treated with SW. Total RNA was extracted from HDMECs 2 hours after SW treatment, and gene expression profiling was analyzed using microarray (Agilent). (2) Shock Wave application was performed 100 shots (0.25 mJ/mm2, 4 Hz) on left thigh of rat. 30 minutes and 4 hours after SW treatment, the irradiated skin section of a rat was excised and total RNA was extracted and gene expression profiling was analyzed using microarray.

Results: In vitro microarray analysis of HDMECs revealed that FosB, a component of transcriptional factor, AP-1, was reproducibly increased. FosB was confirmed to be induced with rapid turnover by SW treatment in rat skin tissues. CXCL2, which was known to be as an angiogenic chemokine, was also found to be remarkably increased in skin tissues and we showed that FosB directly regulate the expression of CXCL2 on HDMECs under PMA stimulation.

Conclusions: FosB-CXCL2 axis, which is induced early after SW treatment, may contribute to the following angiogenic response in skin tissues and this finding may provide a clue for developing a more effective ESWT for DUs in SSc.

P.327

Non-Randomized Controlled Trial to Evaluate the Effect of Extracorporeal Shock Wave Therapy on Digital Ulcers in Systemic Sclerosis

T. Ishii, Y. Kamogawa, H. Fujii, Y. Shirota, T. Shirai, Y. Fujita

Department of Hematology and Rheumatology, Tohoku University Hospital, Sendai, JAPAN

Introduction: Patients with systemic sclerosis (SSc) often display Raynaud’s phenomenon, which causes digital skin ulcers. Since these ulcers are not associated with autoimmune factors, conventional immunosuppressive therapies, vasodilators, and anticoagulants are often ineffective. Extracorporeal shock wave therapy (ESWT) at low energy is shown to be effective in stimulating numerous growth factors endogenously, inducing angiogenesis and healing of injuries and wounds. This study evaluated ESWT for treatment of refractory skin ulcers caused by SSc and assessed its efficacy and safety.

Material and Methods: We enrolled 60 patients with SSc and refractory digital ulcers with no response to intravenous prostaglandin E1 therapy at least 4 weeks. Of these, 30 were treated with ESWT and 30 received conventional treatment. Patients in the conventional treatment group were permitted to use any currently available therapies. Patients in the ESWT group continued pre-study treatments.

One ESWT session consisted of 100 impulses at 0.08–0.25 mJ/mm2 applied to 10 areas on both hands. Treatment was performed weekly for 8 weeks. Outcomes were evaluated according to the number of skin ulcers. The primary endpoint was the mean decrease in the number of skin ulcers at 8 weeks after treatment start.

Results: The mean decrease in the number of ulcers at 8 weeks was 4.47 in the ESWT group and 0.83 in the conventional treatment group and the difference was significant (p<0.0001). The proportion of subjects whose total number of ulcers decreased by 70% or more at 8 weeks was 26.7% in the conventional treatment group and 70.0% in the ESWT group and this was also significant (p<0.0008). The average number of new ulcers in the 8 weeks after the start of treatment was 1.57 in the conventional group and 0.23 in the ESWT group. No serious adverse events associated with ESWT were reported during the study period.

Conclusions: After 8 weeks, ESWT demonstrated clinically meaningful improvement in SSc patients with refractory digital ulcers. This treatment is well-tolerated and minimally invasive, can be repeated without adverse effects, and requires no anesthesia. Overall, the results of our study suggest that ESWT is a novel and efficacious treatment that can be added to pharmacologic therapy.

P.328

Rituximab Treatment for Systemic Sclerosis-Associated Interstitial Lung Disease

L.P. Ananieva, O.A. Koneva, O.V. Desinova, S.I. Glukhova, M.N. Starovoytova, L.A. Garzanova, O.B. Ovsyannikova, A.V. Volkov, E.N. Alexandrova

V.A. Nasonova Research Institute of Rheumatology, Moscow, RUSSIA

Introduction: Treatment options for interstitial lung disease associated with systemic sclerosis (SSc) are limited. Evidence from a number of studies has suggested that rituximab (RTX) may improve lung function and skin fibrosis in patients with SSc. The aim of the study was to assess short-term efficacy of RTX treatment in a cohort of patients with SSc-associated interstitial lung disease (ILD).

Material and Methods: We investigated 71 pts. Data on clinical and laboratory response were collected prospectively. The mean follow-up period was 13±2 mo. Mean age was 46 years (17-66), diffuse cutaneous subset of the disease had 42 (59%), Scl-70 positivity-73% of pts. Duration of the disease was 5,6±4,4 yrs. The cumulative mean dose of RTX was 1,43±0,6. Forty eight pts were treated with < 2g RTX (group 1, a mean dose of 1,1±0,1g) and 23 pts received > 2g RTX (group 2, a mean dose of 2,2±0,6g). All pts received concomitant treatment with prednisolone and 45% - with immunosupressants.

Results: A good response was reported in 29 (41%), partial response in 40 (56%), no response in 2 (3%). The significant changes were observed in decreasing of activity index mSSR, hsCRP, IgG, the number of pts with high ANF titer, prednisolone dose and in increasing of aperture of the mouth, left ventricular ejection fraction, 6-minute walk test. There were no differences in pulmonary artery systolic pressure and HAQ-DI. Forced vital capacity (FVC) increased from 77,35±19,9 to 82,59±20,65 (p=0,001). Clinically significant increasing of FVC>10% was observed in 19 (26,7%) pts. In total improvement in FVC (delta FVC) reached 5,24%, but changes were much more in group 2 (delta FVC 8,98%) than in group 1 (delta FVC 3,75%), p=0,01. The diffusing capacity for carbon monoxide (DLCO) remained stable, but there were significant differences between groups: delta DLCO amounted to 3,75% in group 2, on the contrary it showed a decrease -1,6% in group 1 (p=0,005). The absolute number of B-cells at the end of the study was significantly lower in group 2 (mediana 0,001; 25th%-0,00; 75th%-0,012) than in group 1 (mediana 0,005; 25th%-0,0015; 75th%-0,023), p=0,037.

Conclusions: The results of this study confirm the data of other studies, which suggest a favorable effect of RTX in patients with SSc-ILD. Major clinical as well as laboratory efficacy parameters improved substantially. The patients received RTX>2g showed a good response of pulmonary function and the improvement of lung function was associated with more effective depletion of B-cells at one year.

P.329

Adipose-Derived Cell Therapy for Hand Dysfunction in Patients with Systemic Sclerosis: a Randomized, Double-Blind, Placebo-Controlled Trial

D. Khanna¹, C. Derk², R. Domsic³, S. Kafaja⁴, J. Ervin⁵, M. Mayes⁶, S. Chatterjee⁷, A. Shah⁸, J. Varga⁹, R. Simms¹⁰, R. Spiera¹¹, V. Steen¹², S. Shahouri¹³, V. Hsu¹⁴, J. Yocum¹⁵, M. Adams¹⁶, P. Caldron¹⁷, R. Martin¹⁸, D. Furst⁴, L. Hummers¹⁹, M. Marino²⁰, J. Fraser²⁰

¹ University of Michigan, Ann Arbor, USA, ² University of Pennsylvania, Philadelphia, USA, ³ University of Pittsburgh Medical Center, Pittsburgh, USA, ⁴ David Geffen School of Medicine at UCLA, Los Angeles, USA, ⁵ Center For Pharmaceutical Research, Kansas City, USA, ⁶ University of Texas Houston Medical School, Houston, USA, ⁷ Cleveland Clinic, Cleveland, USA, ⁸ Duke University Medical Center, Durham, USA, ⁹ Northwestern University, Chicago, USA, ¹⁰ Boston University Medical Center, Boston, USA, ¹¹ Hospital for Special Surgery, New York, USA, ¹² MedStar Georgetown University Hospital, Washington DC, USA, ¹³ Heartland Research Associates, Wichita, USA, ¹⁴ Robert Wood Johnson Medical School, New Brunswick, USA, ¹⁵ Baptist Health Center for Clinical Research, Little Rock, USA, ¹⁶ Central Kentucky Research Associates Inc, Lexington, USA, ¹⁷ Arizona Arthritis and Rheumatology Research, Phoenix, USA, ¹⁸ West Michigan Rheumatology, Grand Rapids, USA, ¹⁹ Johns Hopkins University, Baltimore, USA, ²⁰ Cytori Therapeutics Inc., San Diego, USA

Introduction: Hand dysfunction is almost universal in systemic sclerosis (SSc). There is no approved treatment for this indication. The STAR Trial was a 19-center prospective, double-blind, randomized, controlled trial to assess safety and efficacy of Adipose Derived Regenerative Cells (ADRCs) in SSc patients with more than minor hand dysfunction (Cochin Hand Function Score [CHFS] >20).

Material and Methods: Adipose tissue collected by a small volume liposuction was processed with the Celution® System to prepare ADRCs for immediate subcutaneous administration under local anesthesia. Subjects were randomized (1:1) to receive ADRCs or a visually-matched placebo into all fingers of each hand. Safety was assessed throughout the trial. Efficacy was evaluated at multiple timepoints over 48 weeks using the CHFS, HAQ-DI, and other patient-reported and quantitative assessments of hand function. The co-primary outcome measure was improvement in CHFS at week 24 and 48. Baseline CHFS was 40.6±11.0; baseline HAQ-DI was 1.66±0.53.

Results: 88 subjects (51 with diffuse SSc) with median disease duration of ~13 years and median age ~53 were enrolled. The procedure was well-tolerated; 6 subjects reported serious adverse events (1 ADRC (2.1%); 5 placebo (12.5%)) with no SAEs related to study procedures and no deaths. The group combining patients with limited and diffuse SSc subtypes exhibited greater numerical improvement over baseline for the ADRC group than placebo for the primary endpoint. This did not achieve statistical significance (11.5 vs. 10.2 week 24; p>0.05: 11.0 vs. 8.9 week 48; p> 0.05). However, pre-specified analysis of subjects with diffuse SSc showed early and sustained improvement in both CHFS and HAQ-DI in the ADRC group that achieved or approached statistical significance (CHFS 13.1 vs 4.6; p=0.008 at 12 weeks; 12.0 vs. 6.6; p=0.069 at 48 weeks; HAQ-DI 0.23 vs 0.10; p=0.096 at 12 weeks; 0.22 vs 0.04; p=0.044 at 48 weeks). This was not evident in subjects with limited SSc. HAQ-DI subscores relevant to hand function (Eating, Grip, etc.) showed treatment efefct while those not related to the hand (Walking and Arising) did not. Improvement in certain other endpoints (new ulcer counts, hand corner distances, EQ-5D) was also evident in subjects with diffuse SSc.

Conclusions: Treatment with ADRCs was safe. While not meeting the primary endpoint for all SSc patients, subjects with diffuse SSc appeared to exhibit clinically meaningful improvement in several hand parameters. Further study is warranted of this therapy in SSc patients with diffuse disease; a subset with significant unmet need.

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Novel Machine Learning Classifier Accurately Predicts the Intrinsic Molecular Subsets for Patients with Systemic Sclerosis in Multiple Independent Cohorts

J. Franks¹, V. Martyanov¹, G. Cai², M. Whitfield¹

¹ Geisel School of Medicine at Dartmouth College - Department of Molecular and Systems Biology, Hanover, USA, ² University of South Carolina - Department of Biostatistics, Columbia, USA

Introduction: High-throughput gene expression profiling of skin biopsies from patients with systemic sclerosis (SSc) has identified four “intrinsic” gene expression subsets conserved across multiple cohorts and tissues: inflammatory, fibroproliferative, normal-like, and limited. In order to consistently classify patients in clinical trials or for diagnostic purposes, supervised methods that can assign a single sample to a molecular subset are required. Here, we introduce a novel machine learning classifier which is a robust predictor of intrinsic subset and test it on multiple patient cohorts.

Material and Methods: Three independent SSc cohorts (Milano et al. 2008, Pendergrass et al. 2012, Hinchcliff et al. 2013) with gene expression data and intrinsic subset assignments were carefully curated and merged to create a training dataset covering a broad set of 297 skin biopsies representing 97 unique patients. Supervised machine learning algorithms were rigorously trained and evaluated using repeated three-fold cross-validation. We performed external validation using two independent SSc datasets: Chakravarty et al. 2015, which contains 16 samples/8 patients and Gordon et al. 2015, which contains 12 samples/6 patients. Additionally, we validated the classifier on a cohort of SSc patients with gene expression data independently generated by Assassi et al. 2015 (102 samples/97 patients). We used weighted gene co-expression network analysis and g:Profiler to functionally characterize gene modules associated with the intrinsic subsets.

Results: Repeated cross-fold validation identified gene expression features using multinomial elastic net and incorporated them into the final model which achieved an average classification accuracy of 88%. All molecular subsets were classified with high average sensitivity and specificity, particularly inflammatory (83.3% sensitivity, 95.8% specificity) and fibroproliferative (89.7% sensitivity, 94.1% specificity). In external validation, the classifier maintained an accuracy ranging from 70% to 85%. In a re-analysis of gene expression data from Assassi et al., we were able to identify patients representing the canonical inflammatory, fibroproliferative, and normal-like subsets. The inflammatory subset showed gene modules with increased expression enriched in biological processes such as inflammatory response, lymphocyte activation, and stress response. Similarly, gene modules enriched for cell cycle processes were increased in the fibroproliferative subset.

Conclusions: We have developed a highly accurate classifier for SSc molecular subsets for single samples trained and tested on diverse cohorts comprised of 427 skin biopsies from 208 independent patients. Machine learning methods provide a robust mechanism for stratifying intrinsic gene expression subsets and can be used to aid clinical decision-making and interpretation for SSc patients and in clinical trials.

P.331

Tocilizumab is a Promising Treatment Option for Therapy Resistent Juvenile Localised Scleroderma Patients

I. Foeldvari¹, J. Anton², M. Friswell³, B. Bica⁴, J. De Inocencio⁵, A. Aquilani⁶, N. Helmus¹

¹ Hamburger Zentrum fuer Kinder- und Jugendrheumatologie, Hamburg, GERMANY, ² Sant Joan de Deu Hospital, Barcelona, SPAIN, ³ Great North Childrens Hospital, Newcastle, UNITED KINGDOM, ⁴ Universidade Federal do Rio de Janeiro, Rio de Janeiro, BRAZIL, ⁵ University Hospital 12 de Octubre, Madrid, SPAIN, ⁶ Ospedale Bambino Gesù, Rome, ITALY

Introduction: Juvenile localised scleroderma (jlSc) is an orphan disease. Most patient respond to treatment ot methotrexate or mycophenolate. In case of nonresponse or partial response, based on the promising tocilizumab (TOC) data of adult systemic sclerosis studies, TOC seems to be a promising option. There is no publication regarding the effectiveness of tocilizumab in jLS.

Material and Methods: Participants of the pediatric rheumatology email board were asked, if they follow patients with jlSc, who are treated with TOC. Clinical characteristics and the response to TOC was assessed.

Results: Six centres responded and reported 11 patients. The mean age at disease onset was 5.5 years. Disease duration at time of the initiation of TOC was 53.5 months. 5 patients had linear subtype, 2 of them Parry Romberg and one of them coup de sabre. Three had generalized subtype, 2 mixed subtype and 1 limited subtype/morphea. Before starting TOC, patients received 10/11 Methotrexate, 7/11 a combination of Methotrexate and Mycophenolate, 1 abatacept and 1 anti-TNF therapy. Reason to start TOC was in 9 patients increase in Localised Scleroderma Activity Index (mLoSSI), in two patients increased extracutaneous activity.

The mean duration of TOC therapy was 14.75 months. 3/11 received TOC as monotherapy. 8/11 as combination therapy. Therapy success was reflected by a decreased mLoSSI in 9/11 patients, No new lesion occurrence and with Parry Romberg subtype, no increase in the facial atrophy. In 8/8 patients physician global and in 8/8 the patients global disease activity decreased. In 3/3 patients, the number of active joints decreased, in one patients the limb discrepancy decreased. The mean modified Rodnan skin score decreased from the mean value of 9.6 to 5.5.

Conclusions: In this small cohort of patients TOC seems to be a promising rescue medication in methotrexate/mycophenolate nonresponsive patients. A prospective controlled study would be important to prove the seen effect in a controlled way.

P.332

Tocilizumab for Systemic Sclerosis-Rheumatoid Arthritis Overlap: a Case Series

I. Filipescu^1,2,3, L. Damian^1,3, I. Felea^1,3, M. Deac-Badarinza¹, C. Pamfil^1,2,3, S. Rednic^1,2,3

¹ Emergency Clinical County Hospital - Rheumatology, Cluj-Napoca, ROMANIA, ² Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, ROMANIA, ³ Centre for Rare Musculoskeletal Autoimmune and Autoinflammatory Diseases, Cluj-Napoca, ROMANIA

Introduction: Background: Tocilizumab, a biologic anti-IL6 therapy approved for rheumatoid arthritis (RA), was recently employed in systemic sclerosis (SSc) cases.

Objective: The aim of this study was to present a real-life experience with tocilizumab in RA/SSc overlap in a tertiary hospital, over the last year.

Material and method: We assessed the records of RA/Ssc cases fulfilling the activity criteria of RA in order to be eligible for biologic therapy (clinical and radiographic changes of RA, at least two DMARDs tried and failed, including MTX and ESR >32 and/or CRP >3 times the normal value), along with diagnosis of SSc, either diffuse or limited. Seropositivity for anti-CCP, RF as well as anti-Scl70, anti-centromere and/ or other SSc antibodies specificity (by immunoblot) and capillaroscopic changes were recorded. The DAS 28, Rodnan scores and the 6-minutes walking test were recorded upon enrollment and at the 6-months assessment. The pulmonary assessment (chest radiograph, functional ventilatory tests and, if clinically required, HCRT and/ or DLCO) were employed. The patients with features of inflammatory myopathy were excluded. The significant side-effects were also recorded.

Results: We identified 5 cases, all females, aged (range 28-62, average 42 years). The RA radiographic stage was 2 in 3 cases, and 1 and 3 in 1 case respectively. All patients were seropositive for anti-CCP antibodies and 4/5 also for RF. The co-existing SSc was diffuse in 3 cases, with anti-Scl70 positive Ab, and limited in 2 cases, with anti-centromere and anti-Ro52 positivity each. The intestitial lung enhancement was noted in 4 cases, with discrete alteration of functional tests in 2. Four patients were on methotrexate, one on azathioprine and 2 also on hydroxychloroquine. At the 6 month assessment, the DAS score decreased from (average values) 5.2 to 4.1. The Rodnan score also climbed down from (average values) 12.5 to 10.8 An average 67.7 m increase from baseline in 6MWD in patients receiving biological treatment was recorded.

Conclusion: In the RA/SSc overlap cases, tocilizumab led to moderate improvement of skin scores, besides of arthritis, with no significant side-effects.

P.334

Intravenous Immunoglobulins Prevents Experimental Fibrosis in a Murine Model of Systemic Sclerosis

M.M. Farhat^1,2,3,4, S. Speca^1,2,3,4, M. Jendoubi^1,2,3,4, C. Hauspie^1,2,3,4, T. Guerrier^1,2,3,4, V. Sobanski^1,2,3,4, S. Sanges^1,2,3,4, E. Hachulla^1,2,3,4, G. Lefevre^1,2,3,4, M. Labalette^1,2,5, S. Dubucquoi^1,2,5, D. Launay^1,2,3,4

¹ Univ. Lille, U995 LIRIC - Lille Inflammation Research International Center, Lille, FRANCE, ² INSERM, U995, Lille, FRANCE, ³ CHU Lille, Département de Médecine Interne et Immunologie Clinique, Lille, FRANCE, ⁴ Centre National de Référence Maladies Systémiques et Auto-immunes Rares (Sclérodermie Systémique), Lille, FRANCE, ⁵ CHU Lille, Institut Immunologie, Lille, FRANCE

Introduction: Systemic sclerosis (SSc) is an autoimmune disease characterized by an extensive multi-organs fibrosis. Immunosuppressants are effective in some extent but their incomplete efficacy is hampered by a higher infection risk. Intravenous immunoglobulins (IVIG) have a good safety profile, exhibit immunomodulatory and antifibrotic properties and hence could be a relevant treatment for SSc. The purpose of this study was to investigate the effects of IVIG in an experimental model of SSc.

Material and Methods: SSc was induced in 6 weeks old Balb/c mice by subcutaneous injections of HOCl five days a week during six weeks (n=20), whereas control mice received subcutaneous injections of PBS (n=20). Human IVIG was administrated intravenously by single retro-orbital injection at a dose of 2g/kg the first day of HOCl administration (n=20). A control group received an injection of 2% Maltose (n=20). Skin thickness was assessed during the protocol until the sacrifice (day 42). Skin tissues were collected in 4% PFA and processed for histological analysis. Dermal thickness was measured by performing a May-Grünwald–Giemsa staining of 4 µm skin sections; collagen deposition was assessed by performing a Picrosirius red-staining and quantified by using a color deconvolution method. In addition, immunostaining of skin sections was performed in order to evaluate the α-smooth muscle actin (α-SMA) expression. Frozen skin tissues were analyzed to also assess the mRNA expression of main inflammatory and pro-fibrotic genes (by quantitative reverse transcription polymerase chain reaction). Collagen deposition was also evaluated by measuring the content of hydroxyproline in 10 mg of frozen tissue.

Results: Mice exposed to HOCl developed a diffuse cutaneous SSc with higher dermal thickness compared to the PBS group. IVIG significantly reduced dermal thickness and collagen deposition in HOCl-receiving mice. The amount of α-SMA positive cells evaluated by immunofluorescence was reduced in the HOCl treated mice receiving IVIG. mRNA expression profile of various markers of fibrosis (fibronectin, TGFβ) or inflammation (TNFα, IL-1β, IL-6) were also significantly decreased in the skin of HOCl mice treated with IVIG compared to HOCl-treated mice receiving 2% maltose.

Conclusions: These results demonstrate the efficacy of IVIG in preventing experimental fibrosis in a HOCl murine model of SSc.

P.335

Effect of Single Course of Ilomedin® in Therapy of Digital Ulsers Due to Systemic Sclerosis (SSc)

M. Starovoytova, O. Desinova, O. Koneva, O. Ovsyannikova, L. Gorzanova

V.A.Nasonova research Rheumatology Institute, Moscow, RUSSIA

Introduction: Systemic sclerosis is a systemic autoimmune disease of a connective tissue. It is characterized by vascular injuries which result in digital ischemic ulcers. According to EUSTAR (Eular Scleroderma Trial and Research group) 36.2% of patients suffering from systemic sclerosis have digital ulcers. We are still in search of the optimal vascular therapy of this complication.

Material and Methods: To assess effectiveness of intravenous iloprost (ilomedin®), tolerance and the time necessary to start its effect on SSc patients with digital ulcers (DU). 21 women have been examined (aged 29 to 66 years old (ME 47,5), 7 patients with diffuse systemic sclerosis, 14 patients with limited systemic sclerosis, duration of the disease from 4 to 36 years (ME 19,7), number of DU from 1 to 10, total score - 64). llomedin was administered intravenously during 5 days, the dosage was 20 mkg a day. Refresher course was undertaken after 3 months. All patients received standart SSc therapy. Methods of evaluation: the account of DU, Health Assessment Questionnaire (HAQ) and SSc-HAQ, visual analogue scale (VAS).

Results: Ilomedin tolerance was mainly satisfactory. 61.9% of patients rated their tolerance as good, 38 % of patients reported stronger pain in ulcers immediately after injections. 32%of patients reported headache and dyspepsia.

There was no any definite improvement immediately after 5th injections. After 3 month of the treatment we observed a decrease of the total number of the DU (from 64 to 52), no new DU, a decrease of the pain status (VAS from 72.38 to 46.61 mm, p<0,001). Minimal improvement of HAQ and SSc-HAQ was not significantly changed .

Conclusions: Intravenous iloprost is effective and well tolerated drug in the treatment of ischemic digital ulcers. Further studies with more patients and the duration of observation would be need to evaluate the potentially beneficial effect and optimal regimens for therapy of digital ulsers due to systemic sclerosis.

P.336

Increased CXCL10 Serum Levels in SSc can be caused by Myocytes Activation and Targeted by Sildenafil. A Novel Therapeutic Opportunity for PDE5 Inhibition

C. Corinaldesi¹, G. Abignano², C. Antinozzi³, V. Riccieri⁴, G. Valesini⁴, M. Vasile³, F. Marampon³, A. Lenzi⁵, K. Ballard⁶, C. Crescioli³, F. Del Galdo⁷

¹ University Of Leeds - Leeds Institute of Rheumatic And Musculoskeletal Medicine, Leeds, United Kingdom, ² Rheumatology Institute of Lucania (Irel), San Carlo Hospital of Potenza And Madonna delle Grazie Hospital of Matera, Potenza, Italy, ³ University of Rome Foro Italico - Department of Movement, Human and Health Sciences, Rome, Italy, ⁴ Sapienza University of Rome - Department of Internal Medicine and Medical Specialties, Rome, Italy, ⁵ Sapienza University of Rome - Department of Experimental Medicine, Rome, Italy, ⁶ Myriad Rbm, Austin, Usa, ⁷ Leeds Teaching Hospital Nhs Trust, Leeds, United Kingdom

Introduction: The IFN signature chemokine CXCL10 correlates with muscle severity, besides overall poor prognosis in Systemic Sclerosis (SSc) (Liu X et al. Arthritis Rheum 2103). A growing body of pre-clinical data indicates that phosphodiesterase type 5 (PDE5) inhibition has extravascular effects by inhibiting inflammation induced chemokine production including CXCL10 in cardiomyocytes and astroglial cells (Di Luigi L et al. Inflammation 2016; Rothhammer V et al. Semin Immunopathol 2015; Nunes AK et al Cytokine. 2012). Here we explored the potential contribution of myocytes to increased CXCL10 in SSc and the potential role of sildenafil on their proinflammatory activation.

Material and Methods: CXCL10 circulating level was analysed in sera of 109 SSc patients fulfilling ACR/EULAR 2013 criteria and 34 healthy subjects, matched for sex and age, by multiplexed, bead-based immunoassay. Written informed consent was collected for all subjects. Human skeletal muscle cells were treated for 24h with IFNγ(1000 U/ml)+TNFα(10 ng/ml) in presence or absence of increased concentrations of sildenafil (from 0.1mM to 10mM) to evaluate CXCL10 protein secretion. Supernatants were collected and evaluated by ELISA.

Results: CXCL10 serum level was significantly higher in SSc patients vs. HC subjects (602.1±155.1 pg/ml vs. 197.5±14.9 pg/ml, P<0.001) independently of diffuse or limited clinical subset (p>0.05); the presence of sildenafil in therapeutic regimen was associated with lower serum CXCL10 compared to subjects with same disease activity/severity (455.2±211.8 pg/ml vs. 633.1±183.02 pg/ml, P<0.05). CXCL10 was significantly higher in patients with increased Creatine Kinase (CK) (2703±2172 pg/ml vs. 454±82.51 pg/ml, P<0.01) and its concentration strongly correlated with the levels of CK (r=0.986, p <0.001) and with Medsger muscle Severity score (r=0.445, p<0.001). In vitro, CXCL10 secretion was strongly upregulated in IFN-stimulated human skeletal muscle cells vs. control cells (1373.68±293.20 pg/mg vs. 12.48±0.82 pg/mg, P<0.05). Treatment with sildenafil suppressed the IFN induced CXCL10 release in a dose dependent manner down to 50% at 1M sildenafil concentration (592.49±121.19 pg/mg, P<0.05).

Conclusions: The strong correlation of CXCL10 with severity of muscle damage as assessed by serum CK and Medsger Muscle severity score, indicates/confirms the involvement of IFN-pathway activation during myositis in SSc. Our in vitro data suggest that myocytes could be directly involved in IFN induced CXCL10 secretion and that they are a suitable direct target of PDE5 inhibition. Further studies are needed to identify the molecular mechanisms of PDE5 inhibition on pro inflammatory cytokine secretion. Notwithstanding, our data pave the way to extend the therapeutic scope of sildenafil to a disease modifying agent in SSc Myositis.

P.337

Imatinib-Loaded Gold Nanoparticles Ameliorate Experimental Lung Fibrosis Induced by Bleomycin

V. Codullo¹, F. Meloni², S. Breda^1,3, S. Inghilleri², E. Cova², C. Montecucco¹, J. Distler³

¹ Rheumatology, IRCCS Policlinico San Matteo, University of Pavia, Pavia, ITALY, ² Respiratory Disease Clinic, IRCCS Policlinico San Matteo, University of Pavia, Pavia, ITALY, ³ Department of Internal Medicine 3 University of Erlangen-Nuremberg, Erlangen, GERMANY

Introduction: Systemic Sclerosis (SSc) is an autoimmune disorder frequently affected by an interstitial lung involvement (ILD) that significantly deteriorates long term outcomes. In previous experiments we proved that specifically engineered GNPs loaded with imatinib (GNP-HCim) significantly inhibited proliferation and induced apoptosis of fibroblast-like cells drived from ILD-SSc patients. In vitro, GNP-HCim showed higher efficacy compared to the drug alone.

AIM To demonstrate in vivo the efficacy of GNP-HCim in ameliorating bleomycin-induced lung fibrosis

Material and Methods: Eight-week-old C57BL6 male mice (n=8/group) were assigned to either: (1) controls receiving intratracheal aereosolization of saline solution and unloaded Gold Nanoparticles (GNP); (2) mice treated with intratracheal instillation of bleomycin (50 uL) on day 0 and GNP; (3) mice treated with bleomycin on day 0 plus imatinib-loaded Gold nanoparticles (GNP-HCim); (4) mice treated with bleomycin plus intraperitoneal (i.p.) Imatinib (50 mg/kg, once daily). GNP or GNP-HCim were administered by aereosol on day 10-15-20-25 and 3 h before culling. All mice were sacrificed on day 28. Lung specimena were analysed by electron microscopy, immunohistochemistry and immunofluorescence. Data were evaluated by 2 blind observers and analysed with GraphPrism software for statistics

Results: The administration of imatinib i.p or via GNP reduced pathologic changes of the lungs as evaluated by the Lung Injury score and the Ashcroft score. Collagen quantification by Picro Sirius Red revealed a significantly reduced staining only in the GNPHCim group (p=0.0135 vs controls). Immunofluorescence revealed a significant reduction in SMA+ myofibroblast counts when mice were inhaled with GNP-HCim (14.36±1.69/hpf) or injected i.p with imatinib (7.64±1.17/hpf) as compared to the bleomycin fibrotic group (24.01±3.58/hpf, p=0.003 and p=0.0013 respectively). Immunofluorescence stainings also revealed significantly reduced counts of CD45+ and CD44+ cells in groups treated with imatinib, either i.p. or within GNP-HC, versus controls. In imatinib i.p. and GNP-HCim-treated groups there was a significant reduction of phosphorylated c-Abl and PDGF-R, two downstream targets of imatinib, to levels comparable to group 1 controls with respect to the bleomycin group. Finally, electron microscopy revealed the presence of GNP in macrophages exclusively inside alveolar macrophages.

Conclusions: In the experimental model of bleomycin-induced lung fibrosis imatinib delivered to lungs through inhaled GNP was as effective as imatinib administered by i.p. route. These data favour the use of GNP in the development of new therapeutic approaches to SSc-ILD, a devastating complication with a very limited number of effective treatments.

P.338

Abatacept in Systemic Sclerosis : Safety and Efficacy Data from the Eustar Network

I. Castellvi¹, M. Elhai², C. Bruni³, P. Airo⁴, L. Beretta⁵, V. Codullo⁶, C. Montecucco⁶, A. Balbir⁷, F. Iannone⁸, M. Matucci-Cerinic³, O. Distler⁹, Y. Allanore²

¹ Hospital Universitari de la Santa Creu i Sant Pau. Department of Rheumatology, Barcelona, SPAIN, ² Cochin Hospital. Department of Rheumatology A, Paris, FRANCE, ³ University of Firenze. Department of Experimental and Clinical Medicine. Division of Rheumatology, Firenze, ITALY, ⁴ Spedali Civili Di Brescia. Department of Rheumatology, Brescia, ITALY, ⁵ Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milano, ITALY, ⁶ Policlinico Sant Matteo. Department of Rheumatology, Pavia, ITALY, ⁷ Rambam Health Care Campus and Rappaport Faculty of Medicine. B Shine Rheumatology Unit, Haifa, ISRAEL, ⁸ Ospedale Policlinico di Bari. Department of Rheumatology, Bari, ITALY, ⁹ University Hospital Zürich. Department of Rheumatology, Zürich, SWITZERLAND

Introduction: Systemic Sclerosis (SSc) is an autoimmune disease that can cause multiorgan involvement. Abatacept ( ABA ) is a biologic therapy that modulates adaptative immunity. Some convincing pre-clinical data and a few observational trials supports that abatacept could be useful in SSc treatment.

Material and Methods: Retrospective multicenter observational study that enrolled patients with SSc treated with ABA. We collected epidemiological and clinical outcomes. Indeed we also collected from 6 months before to last followup the next parameters: modified Rodnan Skin Score (mRSS), joints, lung and gastrointestinal involvement, concomitant medications, and laboratory tests. First we analized the frequency of adverse affects and its relationships. Secondly we compared the evolution of different involvements during ABA treatment. Statistical significance were assumed with p values < 0.05.

Results: Data of 18 SSc patients were collected (100% female) with a mean age of 59,8±11,9 years old and a disease duration of 14±5.6 years.

Limited cutaneous SSc was the most frequent type and 9/18 patients had overlap disease (all Rheumatoid Arthritis) .The most frequent antibody was antitopoisomerase (57%). The main indication to use was arthritis and ABA was prescribed for a mean treatment period of 30±23 months.

During the followup 8/18 patients had AE and 8 presented infections (50% < 3 months after ABA). During first year of followup only 1 patient needed to stop the treatment because of AE.

We observed a relationship between infections at 3 months and ACA positive patients ( p<0.04), the use of high dose prednisone before ABA start (p<0.03) and with high baseline DAS28ESR (p<0.03) also.

Noninfectious AE at month 3 were more frequent in patients that just before starting ABA had higher DAS28ESR score and that were treated with methotrexate and prednisone. The previous use of biologic therapy were not associated with AEs or infections.

ABA demonstrated efficacy in arthritis with DAS improvement from 5.8±2.8 at baseline to 2.7±1.7 at 12 months (p<0.04).

However, no significant change was observed for mRSS (from 12.2±11.4 to 10.5±6.4 at 6 months) or any other involvement.

Conclusions: ABA demonstrated a good safety profile and some effectiveness on joint involvement. ACA patients profile emerged from our study to predict some adverse events and could be useful to stratify SSc patients candidates to receive ABA. Data from RCT are required to evaluate the effects of ABA on skin and lung diseases.

P.339

Treatment with Abatacept Protects Against Experimental Gastrointestinal Tract, Lung Parenchymal and Vessel Fibrosis

G. Boleto¹, S. Pezet¹, A. Cauvet¹, J. Sadoine², C. Guignabert^3,4, C. Nico¹, F. Batteux¹, Y. Allanore^1,5, J. Avouac^1,5

¹ Université Paris Descartes, Sorbonne Paris Cité, INSERM U1016, Institut Cochin, CNRS UMR8104, Paris, FRANCE, ² EA 2496 Pathologie, Imagerie et Biothérapies Orofaciales, UFR Odontologie, Université Paris Descartes and PIDV, PRES Sor, Paris, FRANCE, ³ INSERM UMR_S 999, Le Plessis-Robinson, Paris, FRANCE, ⁴ Université Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, Paris, FRANCE, ⁵ Université Paris Descartes, Sorbonne Paris Cité, Service de Rhumatologie A, Hôpital Cochin, Paris, FRANCE

Introduction: A previous report showed that abatacept (IgG-CTLA-4) prevented and induced regression of inflammation-driven dermal fibrosis in two different mouse models of systemic sclerosis (SSc). We aimed to assess the effects of abatacept in two complementary mouse models reflecting gastrointestinal tract involvement, lung fibrosis and pulmonary hypertension (PH), mimicking severe SSc organ damage.

Material and Methods: Abatacept was given in the chronic graft-versus-host disease (cGvHD) mouse model, characterized by liver and gastrointestinal tract involvement, and in the Fra-2 mouse model characterized by non-specific interstitial pneumonia and pulmonary vascular remodeling leading to PH. Mice were treated by intraperitoneal injections of abatacept (1mg/mL in the cGvHD model for 6 weeks; 1mg/mL or 10mg/mL in the Fra-2 mouse model for 4 weeks) or human IgG1 (negative control).

Results: Treatment with abatacept was well tolerated in all mouse models.

In the cGvHD model, treatment of allogeneically transplanted mice with abatacept led to a significant reduction of ALT (24%, P=0.014) and AST levels (61%, P<0.001). Pathological analysis of colon revealed decreased inflammatory infiltrates and destruction of crypts in allogeneically transplanted mice receiving abatacept.

When assessed by chest micro-CT imaging, Fra-2 transgenic mice treated with abatacept displayed a significant 12% decrease in lung density (10 mg/ml, P=0.037) and a marked increase in functional residual capacity as compared to IgG1-treated mice (16% for 1 mg/ml, P=0.001 and 14% for 10 mg/ml, P=0.005).

Consistently, abatacept 10mg/L decreased histological fibrosis score (Ashcroft score) as well as hydroxyproline content by 79% (P=0.009) and 31% (P=0.044) respectively, as compared to IgG1-treated mice.

Treatment with abatacept 10mg/mL markedly reduced protein levels in the lesional lungs of Fra-2 transgenic mice of the fibrogenic markers MCP1 (79%, P=0.043) and osteopontin (87%, P=0.039). Levels of TGF-β were also reduced with abatacept (61% for 1mg/mL, P=0.037 and 69% for 10mg/mL, P=0.013).

Upon treatment with abatacept a substantial reduction of right ventricular systolic pressure (28.1±1.5 mmHg vs. 36.0±5.1 mmHg, P=0.037 for 10mg/mL) and right ventricular hypertrophy (0.29±0.01 vs. 0.33±0.010, P=0.037 and 29±0.01% vs. 0.33±0.01% for 10mg/mL, P=0.037) was observed compared to IgG1-treated mice. Consistent with these findings, abatacept 10mg/mL was associated with significant decrease in percent medial wall thickness and numbers of muscularized distal pulmonary arteries.

Conclusions: We demonstrate that treatment with abatacept improves gastrointestinal involvement, prevents lung fibrosis and attenuates PH in SSc pre-clinical mice models. These findings suggest that abatacept might be an appealing therapeutic approach for severe internal organ involvement in SSc beyond its already demonstrated effects on skin fibrosis.

P.340

Sodium Thiosulphate is an Effective Treatment for Dystrophic Calcifications

R. Bech, J. Baumgartner, A. Braae-Olesen

Aarhus University Hospital, Department of Dermatology and Venerology, Aarhus, DENMARK

Introduction: Some patients with autoimmune diseases develop dystrophic calcification. The dystrophic calcifications are typically located to extensor sites of the extremities and fingertips. The lesions with dystrophic calcification can be very pain full, ulcerate and become infected. Dystrophic calciphications are present in up to 72% of patients with systemic sclerosis. However, other patient groups can also develop calcifications of the skin, for example patients with renal impairment exposed to gadolinium-based contrast. These patients can develop the severely debilitating disease, nephrogenic systemic fibrosis. In nephrogenic systemic fibrosis the skin thickens like in patients with systemic sclerosis, and ulcerating calcifications can develop in for example fingertips and extensor sites of the extremities. Earlier, the dystrophic calcifications have been treated with surgery. Yet, wound healing is often slow and sometimes incomplete. Intravenous sodium thiosulphate has with some success been used in systemic calciphylaxis. However, intravenous sodium thiosulphate has substantial side effects. We therefore decided to do a clinical trial in which we injected sodium thiosulphate intralesionally to the dystrophic calicfications of patients with systemic sclerosis or nephrogenic systemic fibrosis.

Material and Methods: We consecutively include patients with systemic sclerosis or nephrogenic systemic fibrosis and disabling dystrophic calcifications from July 2014 until September 2018. We treat the patients with intralesional injections of sodium thiosulphate 150 mg/ml, or 250 mg/ml for selected patients injected into the base of the lesion. Patients are evaluated by the same clinician at week 4 and 12. The smallest calcifications are treated with a single injection of sodium thiosulphate. More widespread lesions are treated with repeated sodium thiosulphate injections every week for 4 weeks.

Results: A total of 33 patients have so far been treated with sodium thiosulphate, and in these 110 lesions were treated with 158 injections. The lesions size varied from 2 mm to 50*65 cm. Injection doses varied from 7.5 to 1500 mg per injection. Nine of the patients experienced complete and 22 patients partial remission of the dystrophic calcifications. Most patients have experienced a transient pain related to injection, yet, reduced pain after treatment.

Conclusions: Intralesional injection with sodium thiosulphate is an effective treatment for dystrophic calcifications in the skin of patients with systemic sclerosis and nephrogenic systemic fibrosis. Sodium thiosulphate reduces pain related to the dystrophic calciphications and it reduces the size of the lesions.

P.341

Endovenous Prostanoids Infusions for the Treatment of Systemic Sclerosis Microangiopathy: Comparison Between Two Therapeutic Regimens

S. Barsotti^1,2, S. Bilia¹, A. D’Ascanio¹, A. Della Rossa¹, M. Mosca¹

¹ Rheumatology Unit, Azienda Ospedaliero-Universitaria Pisana, Pisa, ITALY, ² Department of Medical Biotechnology - Università di Siena, Siena, ITALY

Introduction: Endovenous prostaglandins infusions (EPI) represent a traditional therapeutic approach for the treatment of vascular manifestations in patients with systemic sclerosis (SSc). However the data about the best therapeutic scheme in medical literature are contrasting. Aim of our work was to compare two therapeutic approaches with prostaglandins in patients with SSc.

Material and Methods: We enrolled 47 SSc patients according to EULAR 2013 criteria treated with EPI (M:F=3:44, mean age 62±13.9 years, 32 lc-SSc, 15 dc-SSc with mean disease duration of 13.4±9 years from first non-Raynaud symptom; 24/47 patients were ACA+, 18/47 Scl-70+, 5/47 were only ANA+). Each patient was treated monthly with two different therapeutic regimes: from October 2015 to March 2016 with iloprost 100 mcg administered continuously in 24-36 hours according to patients’ tolerability (phase A) and from October 2016 to March 2017 with 25 mcg discontinuously five hours/day for two days (phase B). We collected data about: visual analogue scale for Raynaud’s phenomenon severity (RP-VAS), number of digital ulcers (DUs), side effects (SE), concomitant vasoactive therapy (phosphodiesterase-5 inhibitors, endothelin-receptors antagonists, calcium channel blockers), DUs infections and need for hospitalization for DUs.

Results: The mean RP-VAS at the end of monthly cycle was 3.14±2.64 in phase A, and 3.61±3 in phase B (p=n.s.).

Twenty-five patients presented DUs: 14(30%) in phase A and 20(42.5%) in phase B. Each patient presented a mean DUs number of 0.395±0.98 in phase A and 0.932±1.35 in phase B (p=0.023). No statistical significant difference was observed about the incidence of disease subset, DUs infections, need for hospitalization and/or concomitant vasoactive therapy.

No SE were reported in phase A, while 17 patients reported SE in phase B. The most common SE reported were headache (17 pts), nausea (11), flushing (11), vomiting (5), diarrhea (4). In 4/17 patients the treatment was stopped due SEs, however no SE was so severe to request hospitalization or to prolong hospitalization in inpatients.

Conclusions: Our study is one of the first to analyze the different effects of two EPI therapeutic regimes in the treatment of SSc microangiopathy. Our results suggest that, although both the therapeutic schemes allowed a similar control of Raynaud’s phenomenon in SSc patients, the treatment with a discontinue EPI scheme was associated to a higher number of DUs and higher incidence of SEs. Additional prospective studies are needed to better define the most appropriate therapeutic scheme to obtain the optimal improvement of microangiopathy in SSc patients.

P.342

PAN-PPAR Agonist IVA337 is Effective in the Prevention of Experimental Lung Fibrosis and Pulmonary Hypertension

J. Avouac¹, I. Konstantinova², C. Guignabert³, J. Sadoine⁴, T. Guilbert⁵, S. Pezet⁵, A. Cauvet⁵, L. Tu³, J.M. Luccarini², J.L. Junien², P. Broqua², Y. Allanore⁶

¹ 1Rheumatology A and INSERMU1016, Paris Descartes University, Cochin Hospital, Paris, FRANCE, ² Inventiva, Daix, FRANCE, ³ INSERM UMR_S 999, Plessis Robinson, FRANCE, ⁴ Université Paris Descartes, Montrouge, FRANCE, ⁵ INSERM U1016, Paris, FRANCE, ⁶ 1Rheumatology A and INSERMU1016, Paris Descartes University, Cochin Hospital, Paris, FRANCE

Introduction: Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors known to modulate fibrosis. The pan-PPAR agonist IVA337 recently demonstrated efficacy in prevention and treatment of experimental skin fibrosis. Our objective was to evaluate the antifibrotic effects of IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).

Material and Methods: IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterized by non-specific interstitial pneumonia and severe vascular remodeling of pulmonary arteries leading to PH. Mice received 2 doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.

Results: Both 30 mg/kg and 100 mg/kg doses of IVA337 were well tolerated in all mouse models. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis induced by bleomycin compared to mice receiving 30 mg/kg of IVA337 or vehicle. Indeed, IVA337 (100 mg/kg) strongly reduced by 61% and 28% tissue density on histological measurements and total lung hydroxyproline concentrations, respectively, as compared to vehicle. IVA337 at 100 mg/kg also significantly decreased col1, col3 and fibronectin in lesional lungs.

Fra-2 transgenic mice treated with 100 mg/kg of IVA337 displayed reduced lung density (20% vs. vehicle) and significant increase of functional residual capacity (30% vs. vehicle) when assessed by chest micro-CT imaging. These results were emphasized by a 50% reduction of the Ascroft fibrosis score and by a 48% reduction of hydroxyproline concentrations upon IVA337 (100 mg/kg) compared to vehicle treated mice.

Regarding vessel remodeling and related pulmonary hypertension, treatment with 100 mg/kg of IVA337 led to a substantial attenuation of right ventricular systolic pressure and right ventricular hypertrophy compared to mice receiving the vehicle. Furthermore, IVA337 given at 100 mg/kg markedly reduced medial wall thickness and the number of muscularized distal pulmonary arteries.

Successful targeting of the TGF-b signaling axis and reduction of T cell, B cell and macrophage infiltration were observed in lesional lungs of both mouse models upon treatment with IVA337 100 mg/kg. In addition, IVA337 restored PPARs expression in lesional lungs.

In primary human lung fibroblasts, IVA337engaged PPAR gamma and inhibited in a dose-dependent manner TGFb-mediated fibroblasts to myofibroblasts transition and PDGF-mediated proliferation.

Conclusions: These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for systemic sclerosis both, for skin and key cardiovascular complications.

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Is abatacept a Usefull Treatment for Patients with Localized Scleroderma? A Case Description of all Localized Scleroderma Patients Treated with Abatacept at the Department of Dermatology, Aarhus University Hospital, from 2009 to 2016

S. Wehner Fage, K. Bakke Arvesen, A.B. Olesen

Department of Dermatology, University Hospital of Aarhus, Aarhus, Denmark

Introduction: Localized scleroderma (LoS) is a group of sclerosing skin diseases affecting more females and young adults. Adjacent tissue (fascia, muscle, bone) may also be affected. In 2011 we presented two cases of treatment resistant diffuse deep LoS patients with significant improvement of abatacept on active disease and old fibrotic lesions. Since then we have offered abatacept to treat therapy resistant moderate to severe active LoS among adult patients. Abatacept is a fusion protein that functions as a T-cell co-stimulation blocker that inhibits TNF-? and prevents T-cell activation. In this study we aim to further determine if abatacept may have a positive effect on disease activity in otherwise treatment resistant LoS.

Material and Methods: Through 2009-2016 we followed up on all adult treatment resistant LoS patients treated experimentally with abatacept. The patients were treated with either 500 or 750 mg abatacept intravenously on days 1, 15, 30, and thereafter every 4-6 weeks. Some patients switched to subcutaneous injection of 125 mg abatacept/week during treatment. Evaluations, including either modified Rodnan Skin Score (MRSS) or Localized Scleroderma Cutaneous Assessment Tool (LoSCAT), were performed by well trained doctors. Primary endpoints were changes in MRSS, LoSCAT, and/or change of size of lesions.

Results and Conclusion: A total of 13 adults were treated with abatacept. The treatment was generally well tolerated. The primary and secondary outcomes of the study will be presented at the congress.

Earlier, in the first cases of previous treatment resistant LoS patients, we have observed a reduction in activity. This retrospective descriptive case study has several limitations in the design as well as a well known difficulty in assessing disease severity in various forms of scleroderma. Still, we suggest that abatacept may be an option in treatment resistant LoS of different subtypes, but further studies are needed.