Beata Nowak

Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis

INTRODUCTION Despite the fact that therapy with TNF-α inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. METHODS Five SNPs within the TNF-α and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-α inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. RESULTS At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P=0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P=0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. CONCLUSIONS Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment.

The influence of bexarotene, a selective agonist of the retinoid receptor X (RXR), and tazarotene, a selective agonist of the retinoid acid receptor (RAR), on bone metabolism in rats

PURPOSE The purpose of this study was to investigate the influence of selective agonists of the retinoid receptor X (RXR) and the retinoid acid receptor (RAR) on bone metabolism in rats. METHODS Thirty six male Wistar rats were divided into three groups: receiving bexarotene, or tazarotene, or to control group. Serum biochemical markers of bone turnover (osteocalcin - OC, tartrate resistant acid phosphatase 5 - TRACP5b and osteoprotegerin - OPG) and mechanical properties of bones were analyzed. RESULTS There was a significant decrease in the femur index value in groups receiving tazarotene and bexarotene on Day 14 (8% and 20% respectively, p=0.0039). On Day 28, 14 days after discontinuation of tazarotene and bexarotene, the difference in femur indexes was still significant (4% for T1-6 and B1-6, p=0.0270). In the bexarotene group an increase in mean plasma osteocalcin level and mean plasma TRACP5b level was detected. In the tazarotene group the mean osteocalcin level remained unchanged and the mean plasma TRACP5b level decreased. An increased yield stress was detected in groups receiving retinoids comparing to controls after 14 days of tazarotene and bexarotene administration. CONCLUSION Although bexarotene and tazarotene administration caused decrease in the femur index, mechanisms responsible for that effect seem to be different. Our results suggest that bexaroten increases bone turnover. On the contrary, tazaroten seems to have inhibitory effect on bone turnover. A counter influence of selective RAR and RXR agonists on the bone turnover might be the reason for inconsistency in results from published research concerning the influence of retinoids on bone metabolism.

Effects of long-term administration of pantoprazole on bone mineral density in young male rats

BACKGROUND Epidemiological studies suggest that long-term administration of proton pump inhibitors (PPIs) may decrease bone mineral density (BMD) and increase the risk of osteoporotic fractures. The aim of the study was to assess the influence of pantoprazole on bone metabolism in growing rats. METHODS The experiment was carried out on twenty-four young male Wistar rats divided into two groups receiving either pantoprazole at the dose of 3mg/kg or vehicle for 12 weeks. Femoral bone mineral density (BMD) and bone histomorphometry were assessed. Serum total calcium, inorganic phosphate and markers of bone turnover were measured. RESULTS In pantoprazole-treated rats a decreased BMD was detected (0.2618±0.0133g/cm(2)vs. 0.2715±0.0073g/cm(2), p<0.05). Bone histomorphometry revealed a decrease in growth plate thickness (G.Pl.RTh.) (161.0±27.8μm vs. 195.0±20.8, p<0.05) in pantoprazole-treated animals. Serum total calcium level and osteocalcin concentrations were decreased in the pantoprazole-treated group (9.62±0.55mg/dl vs. 10.15±0.38mg/dl, p<0.05 and 242.7±44.4pg/ml vs. 342.5±123.3pg/ml, p<0.05, respectively). CONCLUSION We observed that PPIs might have a negative impact on bone formation in growing rats mainly due to their inhibitory effects on the gastric proton pump, with probable deterioration of calcium absorption and decrease in growth plate thickness.

OP0071 Tnf-Alpha, TNF Receptor, HLA-E and NKG2A Gene Polymorphisms and Response to Anti-TNF-Alpha Treatement in Rheumatoid Arthritis

Background Despite the fact that therapy with TNF-alpha inhibitors constitutes a breakthrough in rheumatoid arthritis (RA) management, no improvement is still achieved in approximately 30% of cases. Objectives The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor, and HLA-E and NKG2A receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. Methods For these purpose 280 RA patients who had been treated with TNF-alpha inhibitors for at least 6 months or they stopped therapy because of adverse events were investigated and genotyped for 9 SNPs within the TNFA promoter (rs1800629 G>A; rs361525 G>A; rs1799724 C>T); TNF receptors (TNFR1A: rs767455 G>A; TNFR1B: rs1061622; T>G) while 89 patients were studied for the HLA-E (rs1264457 C>T; HLA-E*01:01, HLA-E*01:03, A>G) and NKG2A (rs7301582 C>T; rs2734440 A>G) genes using LightSNiP typing or Custom TaqMan® SNP Genotyping Assays. Results Among polymorphisms located within TNF-alpha and receptors genes only the TNFR1A (rs767455, G>A) and one of the TNFA (rs1799724, C>T) promoter polymorphisms were found to be associated with response to anti-TNF therapy. Significantly more patients with the homozygous TNFR1A AA genotype achieved a good EULAR response at 3 months compared to patients carrying the G allele (p=0.011). At week 24 DAS28 was significantly lower in patients homozygous for the TNFA T variant (DAS28 – 2.05) compared to the C allele carriers (p=0.045). As for HLA-E and NKG2A genes, after 3 months of anti-TNF treatment the significantly worse (EULAR DAS28) response was observed in patients carrying the HLA-E C allele (20/45 vs. 9/28, p=0.030), HLA-E*01:03/01:03 genotype (8/10 vs. 30/73, p=0.038), NKG2A-(rs7301582)-CC genotype (28/51 vs. 10/33, p=0.043) or NKG2A-(rs2734440)-AA genotype (15/26 vs. 15/50, p=0.026). At week 12 low disease activity or remission was not observed in any of the patients with the HLA-E CC genotype (p=0.09). Also treatment failure (inefficiency or loss of effectiveness of therapy) was more frequently observed in the HLA-E CC homozygous patients (5/8 vs. 12/61, p=0.018) as well as in those with the NKG2A-(rs2734440)-AA genotype (15/37 vs. 3/33, p=0.005). Conclusions These results imply that the polymorphisms within genes coding for TNF-alpha and its TNFR1 receptor as well as HLA-E and NKG2A affect the response to anti-TNF therapy in patients with RA. Acknowledgements Supported by the UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 grants from the National Science Center. Disclosure of Interest J. Swierkot Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, M. Iwaszko Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Gebura Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, B. Nowak Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, L. Korman Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Kolossa: None declared, S. Jeka: None declared, P. Wiland: None declared, K. Bogunia-Kubik Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 DOI 10.1136/annrheumdis-2014-eular.5416

Effect of long-term administration of ranitidine, a histamine H2 receptor antagonist, on bone metabolism in young growing rats

BACKGROUND Histamine regulates function of osteoclasts and osteoblasts, however data regarding the influence of histamine H2 receptors antagonists on bone tissue are ambiguous. Factors that influence growing skeleton may have an important impact on the peak bone mass and future risk of fractures. The aim of our study was the assessment of influence of ranitidine, on growing bones. METHODS The experiment was carried out on young male Wistar rats divided into two groups receiving either ranitidine (10mg/kg ip) or vehicle. RESULTS A significant decrease in femoral BMD in ranitidine-treated rats (R) compared to vehicle-treated ones (C) was detected (0.262±0.009g/cm2vs. 0.271 ±0.007g/cm2, p<0.05). In group R we observed elevated serum C-terminated telopeptide of type I collagen (CTX) level with concomitantly lowered serum osteocalcin (OC) concentration comparing to control group (151.2±27.2pg/ml vs. 101.5±55.6, p<0.05 and 229.1±50.0pg/ml vs. 292.0±52.9, p<0.05, respectively). Serum concentration of inorganic phosphorus was lower in group R than in group C (134±13mmol/L vs. 157±28mmol/L, p<0.05). CONCLUSIONS Long-term administration of ranitidine increases bone resorption and decreases bone formation in growing rats leading to decrease in BMD.BACKGROUND Histamine regulates function of osteoclasts and osteoblasts, however data regarding the influence of histamine H2 receptors antagonists on bone tissue are ambiguous. Factors that influence growing skeleton may have an important impact on the peak bone mass and future risk of fractures. The aim of our study was the assessment of influence of ranitidine, on growing bones. METHODS The experiment was carried out on young male Wistar rats divided into two groups receiving either ranitidine (10 mg/kg ip) or vehicle. RESULTS A significant decrease in femoral BMD in ranitidine-treated rats (R) compared to vehicle-treated ones (C) was detected (0.262 ± 0.009 g/cm2vs. 0.271 ±0.007 g/cm2, p < 0.05). In group R we observed elevated serum C-terminated telopeptide of type I collagen (CTX) level with concomitantly lowered serum osteocalcin (OC) concentration comparing to control group (151.2 ± 27.2 pg/ml vs. 101.5 ± 55.6, p < 0.05 and 229.1 ± 50.0 pg/ml vs. 292.0 ± 52.9, p < 0.05, respectively). Serum concentration of inorganic phosphorus was lower in group R than in group C (134 ± 13 mmol/L vs. 157 ± 28 mmol/L, p < 0.05). CONCLUSIONS Long-term administration of ranitidine increases bone resorption and decreases bone formation in growing rats leading to decrease in BMD.

THU0404 Vitamin D level in patients with early arthritis. A preliminary report

Background Vitamin D, because of its pleiotropic effect, is involved in many aspects of pathophysiology including calcium-phosphorus metabolism, cellular growth and differentiation and immune functions. Epidemiological studies suggest that deficiency of vitamin D may be a risk factor of many conditions including autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus [1,2]. There are reports showing an inverse relationship between vitamin D concentrations and measures of disease activity in early inflammatory arthritis [3]. But some other data are conflicting [4]. Objectives To estimate the vitamin D status in patients with early arthritis and to find relationship with some relevant features of disease activity. To examine associations between 25(OH)D level and current treatment. Methods The study participants were patients with the diagnosis of early arthritis (lasting <2years) hospitalized in the Dept. of Rheumatology University Hospital, Wroclaw, Poland. 25(OH)D was measured in serum samples using commercial ELISA kit, all the necessary data concerning disease activity were also collected. Functional status was derived from HAQ questionnaire. Patients were diagnosed as having vitamin D deficiency when serum level of 25(OH)D were under 30 ng/ml. Results A total of 39 patients (31 women, 8 men,), mean age 45.2±16.8 years were included in the study. In analyzed group mean disease duration was 6.3±7.3 months. Estimated disease activity according to DAS28 had a mean value 4.8±1.4. Mean serum level of 25(OH)D was 18.1±9.4 ng/ml. Only 2 out of 39 patients had 25(OH)D level over 30 ng/ml, the rest had under 12 ng/ml (severe deficiency). There was no correlation between vitamin D level and parameters of disease activity (ESR, C reactive protein, DAS28, morphologic parameters of the peripheral blood) or functional status. There was also no differences in 25(OH)D serum concentrations among women and men. Analyzing the influence of the treatment on 25(OH)D levels, the levels was significantly higher in patients treated with sulphasalazin, compared to those not treated with any of the disease modifying antirheumatic drugs. The use of corticosteroids (mean dose of prednisone in the study group was 10.4±8.6 mg per 24h) did not influence the 25(OH)D serum level significantly. Conclusions The prevalence of vitamin D deficiency among patients with early arthritis is very high. The appropriate treatment regiments seem to influence the serum 25(OH)D levels. In contrast to earlier reports with recent-onset inflammatory arthritis, in our study, there were no associations between 25(OH)D levels and disease activity. References Fletcher JM, Basdeo SAet al. Therapeutic use of vitamin d and its analogues in autoimmunity.Recent Pat Inflamm Allergy Drug Discov.2012 Jan 1;6(1):22-34. Cutolo M, Pizzorni C, Sulli A. Vitamin D endocrine system involvement in autoimmune rheumatic diseases.Autoimmun Rev.2011 Dec;11(2):84-7. Patel S, et al. Association between serum vitamin D metabolite levels and disease activity in patients with early inflammatory polyarthritis. Arthritis Rheum. 2007; 56(7):2143–9. Craig SM, Yu F, Curtis JR, Alarcόn GS et al. Vitamin D status and its associations with disease activity and severity in African Americans with recent-onset rheumatoid arthritis.J Rheumatol.2010 Feb;37(2):275-81. Disclosure of Interest None Declared

AB0412 Plasma levels of angiogenic factors – association with clinical manifestations in systemic lupus erythematosus.

Background The data concerning systemic lupus erythematosus (SLE) and angiogenesis are relatively limited. Most of them are focused on the activity of the vascular endothelial growth factor (VEGF) as the key regulator of the angiogenesis. However other cytokines involved in the new vessels formation are also studied e.g. angiopoietin-2, endothelin-1, E-selectin. In SLE recent studies indicate the relation of different angiogenic factors with disease activity, skin manifestations, renal involvement or abnormal capillaroscopic findings [1,2,3]. Objectives To evaluate the serum concentrations of angiogenic factors and the relationships among them. To asses relations of their levels with disease activity and organ involvement in SLE patients. Methods Serum levels of VEGF, fibroblast growth factor 2(FGF-2), angiopoietn-1 (Ang-1), angiopoietin-2 (Ang-2) and endostatin were assessed by ELISA in a group of 22 patients with SLE and 25 healthy controls. Clinical examination and basic laboratory tests to asses disease activity were performed. Results A total of 22 SLE patients (20 women, 2 men,) with mean age 45.3±13.4 years were enrolled in the study. Mean disease duration was 9.2±6.0 months. The levels of VEGF (49.4 vs 39.3), FGF-2 (256.5 vs 93.9), Ang-2 (6.3 vs 3.4) and endostatin (172.0 vs 126.0) were significantly higher in SLE patients than in healthy populatin. Only Ang-1 concentrations did not differ between SLE patient and healthy control. There was no significant correlation among angiogenic factors except strong positive correlation between Ang-1 and VEGF (R=0.63). Non of the angiogenic cytokines correlated with laboratory markers of disease activity (ESR, C3, C4, complete blood count). Higher level of endostatin in patients with renal involvement (proteinuria, microscopic haematuria) due to SLE, then in those with normal renal function (201.4±66.9 vs 155.2±37.6) was noted. Ang-2 levels were significantly higher in SLE patients with skin involvement (excluding skin changes due to vasculitis) (10.8±10.5 vs 4.9±3.0). Conclusions Our results confirm earlier facts indicating the involvement of angiogenesis in the SLE pathogenesis. We showed significant difference in angiogenic activity of the sera from SLE patients and healthy population. Our data are in some fields similar with earlier, suggesting that Ang-2 may be a useful marker of disease activity in SLE. However in present study we did not show the relation between Ang-2 and proteinuria or components of the complement system. The presented data confirm the necessity of further study on the role of angiogenic factors in the pathogenesis of SLE. References El-Banawy HS, Gaber EW, Maharem DA, Matrawy KA. J Nephrol. 2012 Jul-Aug;25(4):541-50. Salama MK, Taha FM, Safwat M et al. Immunol Invest. 2012;41(8):864-75. MoneibHA, SalemSA, Aly DG et al. J Dermatol. 2012 Jan;39(1):52-7. Disclosure of Interest M. Madej Grant/research support from: Wroclaw Medica University, ST557, B. Nowak: None Declared, A. Luczak: None Declared, L. Korman: None Declared, P. Wiland: None Declared

FRI0053 Analysis of associations between tumor necrosis factor-alpha (TNFA -308 g>a, tnfa -238 g>a, tnfa -857 c>t) and tnf receptors (TNFRA 36 a>g, tnfrb 676 t>g) gene polymorphisms status and responsiveness to tnf-a blockers in rheumatoid arthritis.

Background Despite the fact that therapy with TNF-α inhibitors constitutes a breakthrough in rheumatoid arthritis (RA) management, no improvement is still achieved in approximately 30% of casess. There is ongoing search for biochemical and clinical markers that would allow prediction of a good response to therapy with biologicals, including TNF-α inhibitors. Besides clinical factors, genetic predisposition, may be also helpful in that prediction. Objectives The aim of the study was to evaluated whether single nucleotide polymorphisms (SNPs) within the tumour necrosis factor (TNF-α) [TNFA -308 G>A (rs1800629), TNFA -238 G>A (rs3615525), TNFA -857 C>T (rs1799724)], and TNF receptors [TNFR1 36 A>G (rs767455), TNFR2 676 T>G (rs1061622)] encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. Methods Genetic polymorphism was determined in 274 RA patients who had been treated with TNF-α inhibitors (adalimumab, etanercept, infliksimab, certolizumab) for at least 3 ? 6 ? months or they stop therapy because of adverse events. Outcomes were parameters of efficacy of TNF treatment and adverse events. DNA was extracted from peripheral blood taken on EDTA using Maxwell 16 Blood DNA Purification Kit (Promega Corp., Madison, WI, USA) following the recommendation of the manufacturer. Gene polymorphisms were analyzed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays designed by TIB MOLBIOL GmbH, Berlin, Germany. The efficacy of therapy was evaluated according to EULAR response criteria at 3 month. Association between the relative change in DAS28 and SNP genotypes was tested by linear regression. In addition, logistic regression was applied to compare genotypes in non-responders versus, moderate and good-responders. Results Clinical data of 274 Caucasians patients with RA treated with TNF-α inhibitors were analyzed. EULAR moderate was achieved in 69% of patients, while good EULAR response in 23% of patients at 3 months. The TNFRA GG genotype was present in 56 patients (20%), TNFRA AA in 74 patients (27%), and 144 patients (53%) were heterozygous. More patients with TNFRA AA genotype achieved a EULAR good response at 3 months compared to patients carrying the G allele (with TNFRA GG and TNFRA AG genotypes) 34%/20%/19%, respectively; p=0.011). Presence of the other polymorphism separately were not significantly associated with EULAR response at week 12. Conclusions The TNFRA gene polymorphism was found to affect the responsiveness to TNF-α blockers in rheumatoid arthritis. The presence of the TNFRA AA genotype was associated with better response to treatment. Polymorphisms located within the TNFA promoter (-308 G>A, -238 G>A, -857 C>T) showed no association with TNF-α inhibitor related efficacy. Disclosure of Interest J. Swierkot Grant/research support from: National Centre of Science - 2011/01/B/NZ5/05367, K. Bogunia-Kubik Grant/research support from: National Centre of Science - 2011/01/B/NZ5/05367, B. Nowak Grant/research support from: National Centre of Science - 2011/01/B/NZ5/05367, K. Białowąs: None Declared, L. Korman: None Declared, K. Gębura: None Declared, K. Kolossa: None Declared, S. Jeka: None Declared, P. Wiland: None Declared