Ewa Morgiel

Systemic sclerosis without antinuclear antibodies or Raynaud's phenomenon: a multicentre study in the prospective EULAR Scleroderma Trials and Research (EUSTAR) database

OBJECTIVE To assess patients with SSc who present without circulating ANAs or RP. METHODS Five thousand three hundred and ninety patients who fulfilled the ACR criteria for SSc and were enrolled in the EULAR Scleroderma Trials and Research (EUSTAR) database were screened for the absence of both RP and circulating ANA. To differentiate SSc from its mimics, additional information was gathered using a standardized questionnaire. RESULTS Five thousand three hundred and seventy-eight (99.8%) of the 5390 SSc patients in the EUSTAR database had either detectable ANA or a history of RP. Twelve (0.2%) patients lacked both circulating ANA and RP. Details of the medical history could be obtained for seven patients. Three cases were compatible with ANA-negative and RP-negative SSc and were not typical of any known SSc mimic. Four patients had a malignancy: two had breast cancer, one had multiple myeloma with possible scleromyxoedema and one had bladder carcinoma. There was no temporal relationship between the onset of skin fibrosis and that of the tumour. Although no patient with confirmed nephrogenic systemic fibrosis was identified among the cases of ANA-negative and RP-negative SSc, the presentation of one patient could be compatible with that of nephrogenic systemic fibrosis other than for the absence of chronic kidney disease or of known prior gadolinium exposure. CONCLUSION We have identified a very small subgroup of SSc patients who lack both circulating ANA and RP, none of whom fulfils the diagnostic criteria for any known SSc mimic. Prospective studies are needed to elucidate the clinical presentation, evolution and outcome of such patients.

The role of microRNA-5196 in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterised by tissue fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from patients with SSc play an important role in early stages of SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP‐1), IL‐8 and reactive oxygen species (ROS) induction. However, the exact factors that contribute to chronic inflammation and subsequently fibrosis progression are still unknown.

The Safety of Intravenous Cyclophosphamide in the Treatment of Rheumatic Diseases.

BACKGROUND The therapeutic effects of cyclophosphamide (CP) in the treatment of systemic rheumatic diseases are related to its immune suppressive activity. However effective, the application of CP is restricted due to multiple adverse effects. OBJECTIVES This retrospective study was conducted to determine the frequency of adverse effects attributed to CP toxicity. MATERIAL AND METHODS The study involved 65 patients (17 male; 48 female) receiving intravenous CP between October 2007 and December 2010. The mean age at onset was 51.2 years (range 19-77 years). The most common diagnoses were systemic sclerosis (20), systemic lupus erythematosus (13) and vasculitis (13). The indications for treatment with CP were interstitial lung disease in the course of systemic diseases (33), vasculitis (24), glomerulonephritis (5) and changes in the central nervous system (3). The patients were administered 400-1000 mg CP in intravenous infusions at 2-16 week intervals, with the addition of sodium 2-sulfanylethanesulfonate (mesna) before and after each pulse. RESULTS Out of 65 patients 40 (60%) reported adverse effects: infections in 24 (37%), nausea in 19 (29%), vomiting in 11 (17%), abdominal pain in 7 (11%) and pancytopenia in one, leading to cessation of the therapy. No association was found between the frequency of side effects and the treatment duration (p = 0.632), age (p = 0.852), diagnosis (p = 0.171) or nominal dose (p = 0.321). CONCLUSIONS As knowledge about CP continues to increase, this medication remains a safe way to treat many rheumatic diseases.

A1.20 Epigenetic modificationsand TLR8 signalling contribute to systemic sclerosis pathogenesis by ros and profibrotic genes induction

Background and objectives Systemic sclerosis (SSc) is a chronic multisystem autoimmune disease characterised by skin and internal organs fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play a role in SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction, which contribute to fibrosis progression and chronic inflammation. The exact factors driving TIMP-1, IL-8 and ROS secretion are still unknown. The aim of this study was to investigate the expression pattern of profibrotic IL-8, TIMP-1, AP1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist (ssRNA) stimulation. Materials and methods The expression of Fra2, IL-8 and TIMP-1 and anti-oxidant superoxide dismutase 1 (SOD1) was measured by qRT-PCR in stimulated and unstimulated HC (n = 14) and SSc (n = 17) monocytes. Generation of ROS was determined using luciferase based assay. Fra2 DNA-binding activity was measured in AP-1 transcription assay in monocytic U937 cell line following epigenetic and TLR8 modifications. The level of anti-fibrotic miRNA-5196, which is predicted to bind and inhibit 3’UTR of Fra-2 gene, was also determined in HC and SSc monocytes. Results Combination of DZNep+TLR8 enhanced Fra2 (2-fold, p = 0.02), TIMP-1 (2-fold) and IL-8 (7.87-fold, p < 0.001) expression in SSc monocytes. Fra2 DNA-binding activity was 1.5-fold increased upon stimulation. Secreted level of TIMP-1 was 1.46-fold higher in SSc monocytes compared to unstimulated cells. Generated ROS was 2.21-fold (p = 0.0395) higher following DZNep+TLR8 stimulation in monocytic U937 cells. In contrast, miRNA-5196 expression was 2.13-fold decreased in SSc monocytes upon DZNep+TLR8 stimulation. Also the level of SOD1 was decreased in HC and SSc monocytes following stimulation, 2.16-fold (p = 0.025) and 1.56-fold, respectively. Conclusions These data suggest that DZNep and TLR8 agonist are able to enhance pro-fibrotic TIMP-1, IL-8 and oxidative stress generation. As opposed by the decrease of anti-fibrotic miRNA-5196 and anti-oxidant SOD1 expression in SSc monocytes, which might be used as a potential modulators of fibrogenesis in SSc. Supported by Homing Plus grant/2013–8/4 from Foundation for Polish Science and UMO-2015/16/S/NZ6/00041 from National Science Centre, Poland.

A3.1 Epigenetic and TLR8-dependent regulation of profibrotic genes expression in monocytes in systemic sclerosis and its implication on fibroblasts trans-differentiation

Background and objectives To investigate whether epigenetic changes induced by 3’deazaneplanocin - DZNep and TLR signalling pathway can modulate monocytes to produce tissue-inhibitor of metalloproteinase-1 (TIMP-1) via Fra2 (Fos-related antigen 2) activity, a novel downstream mediator promoting fibrogenesis. Methods AP-1 and TIMP-1 expression following TLR8 treatment was measured by qRT-PCR in monocytes from Systemic sclerosis (SSc) patients (n = 13) and healthy controls (HC) (n = 13). TIMP-1 promoter activity was measured in U397 monocytic cell line using luciferase reporter assay. The effect of DZNep treatment on inhibition of tri-methylation of lysine 27 on histone 3 was analysed by Western Blot. Expression of TIMP-1 and Fra2 was determined in response to DZNep. The functional effect of TLR8 and DZNep-treated HC monocytes was studied on dermal fibroblasts’ trans-differentiation. Results Increased Fra2 and TIMP-1 expression was correlated in SSc monocytes (p = 0.021), but not in HC monocytes upon TLR8 stimulation. In contrast, the expression of anti-fibrotic Fra1 was significantly (p = 0.037) reduced in SSc monocytes compared to HC. Inhibiting AP-1 activity reduced TIMP-1 production in TLR8 stimulated HC and SSc monocytes. Also, TLR8 stimulation induced significant (p = 0.015) TIMP-1 promoter activity in monocytic U937 cells. Combination of DZNep plus TLR8 enhanced Fra2 (4.1 times) and TIMP-1 (4.7 times) expression in HC monocytes. However, the reverse effect on Fra2 and TIMP-1 expression was observed in SSc monocytes following stimulation. Finally, DZNep plus TLR8-treated HC monocytes induced strong production of collagen and a-SMA in dermal fibroblasts reflecting their trans-differentiation, which is a key event in the pathogenesis of SSc. Conclusions These data demonstrate that histone modification induces by DZNep has an opposite effect of Fra2-mediated TIMP-1 production on HC versus SSc monocytes. Therefore, DZNep cloud be used as a selective regulator of downstream mediators, which orchestrates SSc development.