Marco Bardelli

Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts

OBJECTIVE It has been demonstrated that the endocannabinoid system is up-regulated in pathologic fibrosis and that modulation of the cannabinoid receptors might limit the progression of uncontrolled fibrogenesis. The aim of this study was to investigate whether the synthetic cannabinoid receptor agonist WIN55,212-2 could modulate fibrogenesis in an in vitro model of dcSSc. METHODS The expression of cannabinoid receptors CB1 and CB2 was assessed in dcSSc fibroblasts and healthy control fibroblasts. To investigate the effect of WIN55,212-2 on dcSSc fibrogenesis, we studied type I collagen, profibrotic cytokines, fibroblast transdifferentiation into myofibroblasts, apoptotic processes and activation of the extracellular signal-related kinase 1/2 pathway prior to and after the treatment with the synthetic cannabinoid at increasing concentrations. RESULTS Both CB1 and CB2 receptors were over-expressed in dcSSc fibroblasts compared with healthy controls. WIN55,212-2 caused a reduction in extracellular matrix deposition and counteracted several behavioural abnormalities of scleroderma fibroblasts including transdifferentiation into myofibroblasts and resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2 was not reverted by selective cannabinoid antagonists. CONCLUSIONS Our preliminary findings suggest that cannabinoids are provided with an anti-fibrotic activity, thereby possibly representing a new class of agents targeting fibrosis diseases.

SAT0196 Dekavil (F8-IL10), A Novel Therapeutic Approach for Rheumatoid Arthritis: Ongoing Phase IB Clinical Trial Results

Background F8-IL10 (Dekavil) is a fully human immunocytokine, composed of the antibody fragment F8, specific to the extradomain A of fibronectin (EDA-FN), fused to the anti-inflammatory cytokine interleukin-10 (IL-10) [1, 2]. F8-IL10 is currently being investigated as a new therapeutic strategy targeting the inflamed tissues in rheumatoid arthritis (RA). Objectives To determine the safety and tolerability of Dekavil when administered in combination with methotrexate (MTX) to patients suffering from RA who had failed at least one anti-TNFα drug therapy. To give a preliminary evaluation of the efficacy of Dekavil when administered in combination with MTX. Methods A Phase Ib non-placebo controlled clinical trial is currently ongoing in order to establish the maximum tolerated dose (MTD) of Dekavil when administered in combination with MTX. Cohorts of 3-6 patients are treated with escalating doses of Dekavil (6, 15, 30, 60, 110, 160, 210, 300, 450 and 600 μg/kg) in combination with a fixed dose of MTX. Dekavil is administered by subcutaneous injection once a week for a maximum of 8 weeks. Results As of today, the first eight cohorts of the study have been completed (dose levels from 6 to 300 μg/kg). Twenty-six patients are evaluable for safety. No dose limiting toxicities nor serious adverse events have been recorded. No MTD has yet been reached; the dose level of 450 μg/kg is currently ongoing. Mild injection site reaction has been reported in 54% of patients. Regarding systemic adverse reactions, one case of progressive anemia was reported in one patient treated at 160 μg/kg. All adverse reactions resolved after the end of treatment with minor or no therapeutic interventions. A therapeutic benefit of Dekavil, in terms of ACR response, has been recorded in the treated patients, even in subjects treated with low drug dosages. To date, 25 patients are evaluable for efficacy. Among this population, ACR20, 50 and 70 responses were achieved by 60%, 32% and 16% of patients, respectively. In addition, two patients treated at 30 μg/kg and 60 μg/kg dose levels achieved a long-lasting remission. Conclusions The promising safety profile, together with the preliminary efficacy responses in this non-placebo controlled trial led to the start of a Phase II placebo-controlled trial within the same patient population to verify the efficacy of low dosages of Dekavil in combination with MTX. The currently available data suggest the targeted delivery of IL-10 to the sites of inflammation is a promising therapeutic approach for RA. References K. Schwager et al. (2009) Arthritis Res. Ther., 11, R142 F. Doll et al (2013) Arthritis Res. Ther., 15, R138 Disclosure of Interest M. Galeazzi: None declared, G. Sebastiani: None declared, L. Bazzichi: None declared, E. Garcia Gonzalez Consultant for: Philogen, N. Ravenni Employee of: Philogen, L. Giovannoni Employee of: Philogen, J. Wilton Consultant for: Philogen, E. Selvi: None declared, M. Bardelli: None declared, C. Baldi: None declared, A. Iuliano: None declared, G. Minisola: None declared, R. Caporali: None declared, S. Bombardieri: None declared, D. Neri Shareholder of: Philogen

Punch biopsy for fat tissue collection in amyloidosis: is it time to stop needle aspiration?

SIR, Amyloidosis is a heterogeneous group of diseases that share the deposition of amyloid fibrils in organs and tissues, with the same characteristic cross-b-sheet secondary structure, independent of their protein primary structure. More than 30 unrelated autologous proteins can produce systemic amyloidoses, either localized or systemic. The various forms differ in pathogenesis and prognosis, but usually show overlapping clinical manifestations, making their differentiation on clinical grounds very difficult. Precise amyloid typing is crucial for adequate treatment of patients because the various forms require different approaches, which can range from autologous stem cell transplantation in amyloid light-chain (AL) amyloidosis to liver transplantation in transthyretin amyloidosis. The diagnosis and classification are based on histological demonstration of amyloid deposits and characterization of the amyloid precursor [1, 2]. Rectal biopsy has traditionally been the recommended method of screening for amyloidosis. However, since 1973, abdominal fat pad aspiration by fine needle has superseded rectal biopsy as the simplest, fastest and most acceptable way to screen for amyloid, when a systemic form is suspected, being a convenient alternative to organ biopsy [3, 4]. The resulting tissue smear is examined by polarized light microscopy after Congo red staining, in order to detect the presence of amyloid. The second step is to identify the amyloidogenic protein, in order to unequivocally establish the type of amyloidosis. Its sensitivity ranges between 54% and 82%. It can be done at outpatient clinic and requires no technical expertise [4]. Abdominal fat tissue can be aspirated with a 16gauge needle connected to a 10-ml syringe [5]. Although it is safe and reliable, fat aspiration by needle does not yield enough tissue for evaluation in some cases, and its low sensitivity could be related to the possible impact of the size of the needle used for aspiration [6]. Furthermore, there is a need for multiple aspirations (at least three) from different places, especially in patients with a thin abdominal wall. Smith et al. [7] first described in 1986 the usefulness, simplicity and reliability of using a 3.0 mm punch biopsy for collecting skin plus s.c. fat, needed for gas chromatographic analysis of fatty acids. Bogov et al. [8] in 2008 described their positive experience of s.c. fat biopsy to diagnose amyloidosis, made by scalpel and not by punch. We describe a single-centre experience of s.c. fat biopsy, performed by punch. Between March 2012 and December 2014, we performed 138 biopsies at outpatient clinic. All patients (84 female and 54 male, mean age 56.2 years) required such a diagnostic procedure, in order to exclude amyloidosis secondary to various rheumatological conditions, including RA, Behçet’s syndrome and autoinflammatory diseases. In addition, the reliability for amyloid detection in fat samples obtained by punch biopsy was evaluated in comparison with fat specimens obtained from May 2010 to March 2012 by fine needle aspiration technique at our clinic. The biopsy site was prepared with three betadine scrubs and covered with a fenestrated sterile drape. The dermis at the biopsy site (at the right anterior axillary line at the level of the umbilicus) was infiltrated with 0.5 ml of 1% lidocaine followed by injection of 4 ml in the very superficial layers of adipose tissue immediately below the skin (no more than 1 cm deep). Punch biopsy was performed using a circular blade (3.0 mm in diameter) attached to a pencil-like handle. The instrument was rotated down through the epidermis and dermis and into the s.c. fat (Fig. 1A). Punch biopsy yields a cylindrical core of tissue that requires gentle handling (usually with a needle) to prevent a crush artefact at the pathological evaluation. The biopsy site was closed with a single absorbable suture (Vicryl 3-0). Because linear closure was performed on the circular defect, stretching the skin before performing the punch biopsy allows the relaxed skin defect to appear more elliptical and makes it easier to close. The skin was stretched perpendicular to the relaxed skin tension lines, so that the resulting elliptical wound and closure were parallel to these skin tension lines. This modified method does not require any special preparations, materials or consumables. It is easily performed and can be applied in all conditions, including patients with disturbances of coagulation. It generally produces only a minimal scar, and no patients experienced adverse events related to the biopsy; only mild bruising was observed in few patients. We did not observe any site infection; only seven patients reported local transient (<48 h) paraesthesia. The fat tissue obtained by the afore-mentioned procedure is significantly richer in volume in comparison with the fat obtained by needle aspiration (Fig. 1B); in addition, this procedure by punch is able to provide an amount of histologically wellpreserved fat tissue. All of the samples obtained by punch biopsy (n = 138) were suitable for pathological evaluation, compared with 74 of 96 samples (78%) obtained by fine needle aspiration. In conclusion, punch biopsy could represent a quick, cheap and simple procedure for fat collection for identification of amyloid deposits.

SAT0232 A Phase IB Clinical Trial in Rheumatoid Arthritis of Dekavil (F8-IL10), A Novel Anti-Inflammatory Immunocytokine

Background Our approach allows the selective delivery and accumulation of IL-10 at sites of inflammation [1,2], using DEKAVIL, an “armed antibody”, composed of the human F8 antibody fragment (specific to the EDA domain of fibronectin) fused to the immunoregulatory cytokine IL-10. This represents a novel therapeutic concept for treatment of chronic inflammation. A potent inhibition of arthritis progression has previously been reported in a model of arthritis [1,2] using DEKAVIL or a murine analogue in combination with MTX. A Phase Ib clinical trial is on-going, featuring weekly administation of DEKAVIL in combination with MTX, to patients with active RA who have failed at least one line of anti-TNF therapy. We now report the results obtained within the first six dose levels. Objectives To establish the MTD of the combined treatment, Dekavil + methotrexate. To study safety and tolerability. To obtain preliminary information on efficacy. Methods Cohorts of 3-4 patients with active RA received escalating doses of DEKAVIL (6, 15, 30, 60, 110 and 160 μg/kg respectively) in combination with fixed dose of MTX (10-15 mg/week). The treatment was given as once weekly sc injection for up to 8 weeks. Safety evaluations based on RCTC v.2.0 [3] on days 1 through 28, including AEs, SAEs and tests were used to determine the DLT, if any. Response was assessed after 4 and 8 weeks of treatment, according to ACR and DAS28 criteria Results Twenty patients have been treated and were evaluable for safety from dose levels between 6 and 160 μg/kg. No MTD has been reached. No DLTs have been encountered or SAEs recorded. Reported adverse reactions were several mild injection site reactions and a single reported systemic adverse reaction, a progressive anaemia at the highest dose level tested so far. Treatment was witheld and the event resolved within 3 weeks. Considering the maximum ACR response achieved: three patients achieved an ACR 70 response, three patients achieved an ACR 50 response and six patients achieved an ACR20 response at various time points during regular or extended follow-up. Variation in the duration of the response has been observed. Of note stand out two patients in the 30 μg/kg cohort and in the 60 μg/kg cohort who achieved long-lasting remission: ACR70s at week 60 (Patient 8) and week 44 (Patient 11), still on-going. A low-titer of anti-fusion protein antibodies was reported for two out of fifteen samples analyzed. Conclusions The promising safety data on the clinical use of DEKAVIL in combination with MTX together with the preliminary positive signs of anti-arthritic activity suggest that the targeted delivery of IL-10 to the site of inflammation may be beneficial to patients with RA. The extended activity periods indicate the possibility for a long lasting therapeutic potential. These results warrant further clinical investigation in the future in randomized trials. References K. Schwager et al. (2009) Arthritis Res. Ther., 11, R142. F. Doll et al (2013) Arthritis Res. Ther., 15, R138. T. Woodworth et al. (2007) J. Rheumatol., 34: 1401-14. Disclosure of Interest M. Galeazzi: None declared, L. Bazzichi: None declared, G. Sebastiani: None declared, D. Neri Shareholder of: Philogen, L. Giovannoni Employee of: Philogen, F. Bacchion Employee of: Philogen, J. Wilton Consultant for: Philogen, E. Garcia Gonzalez Employee of: Philogen, P. Ruffini Employee of: Dompé, M. Bardelli: None declared, C. Baldi: None declared, E. Selvi: None declared, G. Minisola: None declared, R. Caporali: None declared, E. Prisco: None declared, S. Bombardieri: None declared DOI 10.1136/annrheumdis-2014-eular.1179

Arthritis in Melorheostosis. An uncommon feature in a rare disease

A 53-year-old male developed left knee and left ankle arthritis associated with marked induration of the skin and soft tissues distal to the knees. The left knee was swollen, tender, and with a non-reducible contracture and similarly the left ankle and the subtalar joint were stiff and moderately swollen. In addition, diffuse, bluish, small vascular ectasia, hypertrichosis and skin pigmentation of the affected sites were evident (fig1). These symptoms began 20 years before and the patient had been diagnosed with eosinophilic fasciitis and treated with daily prednisone leading to partial improvement of the symptoms, but not of the knee deformity and soft tissue induration. This article is protected by copyright. All rights reserved.

SAT0625 Ultrasound assessment of joints and enthesis in a cohort of patients affected by ochronosis: how common is inflammation?

Background Ochronosis, the musculoskeletal manifestation of alcaptonuria (AKU), is characterized by alterations of the spine and large joints of the limbs similar to those of osteoarthritis. However, some cases of spinal involvement that resembles spondiloarthritis (SpA) have been describe, suggesting a prevalent inflammatory involvement of the joints. Objectives To evaluate the prevalence of inflammatory abnormalities in peripheral joints and enthesis of a cohort of patients affected by AKU. Methods consecutive patients with definite diagnosis of AKU (with or without clinical manifestations) referred at our clinic from 2014 to 2016 were enrolled. All patients underwent a US examination of the following sites bilaterally: metacarpo-phalangeal joints (MCP), proximal interphalangeal joints (PIP), radiocarpal/mid carpal joints, elbow, gleno-humeral, hip, knee, ankle and metatarso-phalangeal (MTP) joints; flexor and extensor tendons of fingers and wrist and the ankle tendons. Further, the enthesis of the rotator cuff of the shoulder, triceps, quadriceps, patellar and Achilles tendon were assessed. Joints and tendons with a synovial sheath were assessed for effusion, synovial hypertrophy and power Doppler (PD) signal while enthesis were evaluated for the presence of PD signal, enthesophytes and calcifications. All the US lesions were scored using a dichotomous scale (presence/absence). All US exams were performed by an expert sonographer blind to clinical history, using an Esaote MyLab70 scanner equipped with high resolution linear probes. Results 11 patients (6 women) were enrolled in this study with a mean age of 57 yo (SD±11,50). the mean number of joints with effusion was 3,9 for each patient (median 3, range 2–8) while the mean number of joints with synovial hypertrophy was of 2,9 (median 2, range 2–7). =0,18 joints (median 0, range 0–2) presented also PD signal. I The mean number of exudative tenosynovitis was0,81 (median 2, range 0–3) while proliferative tenosynovitis (mean 0,54, median 0, range 0–2) and PD in tendons with sheaths (mean 0,27, median 0, range 0–2) were rare. Finally, the mean number of enthesis with PD was 1,27 (median 1, range 0–7), the mean number of enthesophytes was 0,63 (median 0, range 0–3) and for calcifications 4,27 (median 5, range 1–8). Conclusions Ochronotic arthropathy is believed to be characterized by a widespread articular damage, correlated mainly to degenerative processes due to the deposition of Homogentisinic Acid in the joints. The results of this US study showed that joint inflammation is common in ochronotic patients, associated in some cases with peripheral enthesis involvement confirming previously published data (1). The prevalence and the characteristics of the inflammatory manifestations should be further studied in larger cohorts of patients as they could play an important role in the joint damage process in these patients and provide a rationale for the use of new drugs. References Filippou G, Frediani B, Selvi E et al, Tendon involvement in patients with ochronosis: an ultrasonographic study. Ann Rheum Dis 2008 Dec;67(12):1785–6. Disclosure of Interest None declared

THU0499 Neutrophil Extracellular Traps (NETS): A Shared Feature of Acute Microcrystalline Arthritis

Background Neutrophil extracellular traps (NETs) are web-like structures, composed of nucleic acids, histones and granular enzymes, including myeloperoxidase and elastase, that have a physiological crucial role in innate immunity response by trapping microbes. In addition, NETs have been identified as regulators of many inflammatory and autoimmune disorders, including crystal-induced arthritis.[1,2] In particular, a key role in the pathogenesis of gout flares has been postulated for NETs. During gout attacks, NET aggregates should package MSU crystals with a concomitant degradation of proinflammatory cytokines, leading to the rapid resolution of the inflammatory phase.[3] Objectives To evaluate NET expression in synovial fluid from acute CPPD arthritis and to compare NET release between acute MSU and CPPD arthritis. Methods Synovial fluid was collected from patients with active gout or pseudogout attack (n=5 for each group) and immediately centrifuged (805 rad) to separate cells from supernatant. Polymorphonuclear leukocytes were counted and immediately processed for morphological evaluation under transmission electron microscopy (Zeiss EM 109; 4000x). NET expression was evaluated by fluorescence microscopy using SYTOX Green (Life Technologies) and neutrophil elastase specific antibody (ABCAM). Fluorometric quantification of NET components, DNA and neutrophil elastase, was performed in supernatant (Cary Eclipse Varian; exc/em 503/526). Results were normalized to cells/mm3. Peripheral blood neutrophils from healthy donors stimulated with PMA were used as positive controls whereas PMA-untreated neutrophils were used as negative ones. All the experiments were performed according with Helsinki guidelines. Results Extracellular material morphologically compatible with NETs was observed by electron microscopy in samples from both acute MSU and CPPD arthritis. NET structures were confirmed by the presence of extracellular DNA and neutrophil elastase at immunofluorescence. Fluorometric quantification of extracellular DNA and neutrophil elastase showed an increased NET release in both gout and pseudogout samples compared with negative controls (p<0.01). No significant differences in NET release were measured between acute MSU and CPPD arthritis samples. Conclusions Beyond confirming NETs in acute MSU arthritis, we have evidence of NET release in synovial fluid from acute CPPD arthritis. Our preliminary data suggest that the release of NETs could be a common physiophatological pathway to both crystral induced arthropaties. Disclosure of Interest None declared

An acutely swollen knee

A 77 year old retired man presented in a wheelchair to the emergency department with severe, constant pain and progressive swelling of the left knee. His symptoms had suddenly occurred two days before at night after he had gone to bed feeling well. He could not recall any injury to the knee. There was no history of joint disease or joint surgery. His medical history was unremarkable, and he was not taking any regular medication. He was not aware of any family history of arthritis. He smoked five cigarettes a day and drank two glasses of wine a day. On examination, the patient did not have a fever and had a heart rate of 82 beats/min and a blood pressure of 130/75 mm Hg. His body mass index was within normal limits. His left knee was red, hot, swollen, and tender with minimal range of motion. No other joint involvement was detected and there were no extra-articular findings. Synovial fluid was taken from the joint (fig 1⇓) and studied under compensated polarised light microscopy (fig 2⇓). A radiograph of the knee was also taken (fig 3⇓). All other system examinations were normal. Blood tests showed an erythrocyte sedimentation rate of 55 mm/h (normal <25 mm/h) and a C reactive protein concentration of 1.2 mg/dl (normal <0.5 mg/dl). Peripheral white blood cell count, platelet count, and haemoglobin concentration were within normal limits. Kidney and liver function parameters, and electrolytes and serum urate levels, were normal. Fig 1 Macroscopic appearance of the synovial fluid Fig 2 Wet preparation of the synovial fluid under compensated polarised light microscopy Fig 3 Radiograph of the left knee

A whitish-creamy synovial fluid.

A 77-year-old Caucasian man had experienced pain and progressive swelling of the left knee for 2 days prior to admission. Examination revealed an inflamed left knee joint that was aspirated, yielding 10 ml of whitish-creamy synovial fluid (fig 1A). Septic arthritis was suspected. A wet preparation under ordinary light confirmed the presence of numerous leukocytes (25 000 cells/mm3). However, further …

A black femoral head.

A 55-year-old white female complained of progressive pain in the left hip mainly while walking. Since the age of 40 she had suffered from back pain and stiffness. In 2000 she consulted a rheumatologist who noticed brown pigmentation on the sclera and external ear, and diagnosed alkaptonuria. A quantitative essay of …