G Dessole

Bleomycin-induced scleroderma: Report of a case with a chronic course rather than the typical acute/subacute self-limiting form

Abstract: We report a case of bleomycin-induced scleroderma in a 35-year-old woman treated with chemotherapy for Hodgkin’s disease. Approximately 6 months after the first chemotherapy cycle, the patient developed skin sclerosis in both arms. The lesion showed no signs of spontaneous clinical amelioration and treatment with steroids was unsuccessful. A partial remission of the skin sclerosis was instead obtained by the administration of d-penicillamine. A family history revealed other cases of autoimmune diseases and HLA typing showed the presence of antigens associated with scleroderma. The association between bleomycin therapy and scleroderma is discussed.

Susceptibility to ankylosing spondylitis but not disease outcome is influenced by the level of HLA-B27 expression, which shows moderate variability over time

Objective: Previous reports have highlighted the relevance of HLA-B27 expression in the pathogenesis of ankylosing spondylitis (AS). The aim of the current study was to estimate the level of HLA-B27 expression on the cell surface of ex vivo monocytes and lymphocytes by a quantitative method and to correlate this with AS disease susceptibility, disease clinical indexes, and the occurrence of acute anterior uveitis (AAU). Method: We recruited 32 B27-positive patients with AS and 32 B27-positive healthy normal controls (NCs) for evaluation at different time points. The expression of HLA-B27 molecules was quantified by flow cytometry on ex vivo peripheral blood mononuclear cells (PBMCs). Patients were also evaluated by scores on the Bath AS disease activity (BASDAI), functional (BASFI), and metrology (BASMI) indexes. Results: The expression of HLA-B27 molecules was significantly higher in patients with AS than in B27-matched controls in the case of both monocytes [219K (IQR 174K–308K) vs. 137K (IQR 96K–170K), p < 0.0001] and lymphocytes [82K (IQR 58K–118K) vs. 54K (IQR 44K-61K), p < 0.0001]; AS only vs. AS with AAU: p = 0.744 in monocytes and p = 0.701 in lymphocytes. Comparisons with metrology and functional indexes were also not significant (BASMI: r = 0.05, p = 0.77; BASFI: r = −0.09, p = 0.67). The overexpression of HLA-B27 molecules was stable after 1 week of follow-up. At 3 years follow-up, the variability was moderate and did not correlate with variations in disease activity (BASDAI: r = −0.01, p = 0.92 ns). Conclusions: The level of HLA-B27 expression in PBMCs correlates with the susceptibility to AS but not with the disease outcome, nor with the occurrence of extra-articular manifestations such as AAU.

Killer‐Cell Immunoglobulin‐Like Receptors (KIR) and HLA‐Class I Heavy Chains in Ankylosing Spondylitis

Postmarketing Phase IV

Clinical potential of apremilast in the treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a frequent chronic inflammatory disease characterized by joint and skin involvement, and by typical extra-articular manifestations. Although the pathogenesis of PsA is still under investigation, the available evidence suggests the importance of the patient’s genetic background, microbial or environmental triggers, and an imbalance in the adaptive and acquired immune system, resulting in the production of inflammatory mediators. New therapeutic approaches have been proposed, among them the use of modulators of intracellular signals and gene transcription such as PDE4-inhibiting compounds, which are able to modulate the activity of transcription factors such as CREB and NF-κB and therefore the synthesis of inflammatory mediators, resulting in immunoregulation. This paper summarizes the mechanism of action of apremilast, a PDE4 inhibitor, and the clinical data available on its clinical efficacy and safety profile in the treatment of PsA patients.

Expression analysis of HLA-E and NKG2A and NKG2C receptors points at a role for natural killer function in ankylosing spondylitis

Background Ankylosing spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of human leucocyte antigen (HLA)-B27 alleles. HLA-E molecules are non-classical major histocompatibility complex (MHC) class I molecules that specifically interact with the natural killer receptors NKG2A (inhibitory) and NKG2C (activating), and have been recently proposed to be involved in AS pathogenesis.‘’ Objective To analyse the expression of HLA-E and the CD94/NKG2 pair of receptors in HLA-B27-positive patients with AS and healthy controls (HC) bearing the AS-associated B*2705 and the non-AS-associated B*2709 alleles. Methods The level of surface expression of HLA-E molecules on CD14+ peripheral blood mononuclear cell was evaluated in 21 HLA-B*2705 patients with AS, 12 HLA-B*2705 HC, 12 HLA-B*2709 HC and 6 HLA-B27-negative HC using the monoclonal antibody MEM-E/08 by quantitative cytofluorimetric analysis. The percentage and density of expression of HLA-E ligands NKG2A and NKG2C were also measured on CD3−CD56+ NK cells. Results HLA-E expression in CD14+ cells was significantly higher in patients with AS (587.0, IQR 424–830) compared with B*2705 HC (389, IQR 251.3–440.5; p=0.0007), B*2709 HC (294.5, IQR 209.5–422; p=0.0004) and HLA-B27-negative HC (380, IQR 197.3–515.0; p=0.01). A higher number of NK cells expressing NKG2A compared with NKG2C were found in all cohorts analysed, as well as a higher cell surface density. Conclusion The higher surface level of HLA-E molecules in patients with AS compared with HC, concurrently with a prevalent expression of NKG2A, suggests that the crosstalk between these two molecules might play a role in AS pathogenesis, accounting for the previously reported association between HLA-E and AS.

AB0608 Elevated Circulating Tumor-Associated Antigens in Systemic Sclerosis: Association with Lung Fibrosis

Background Some tumor-associated antigens (TAAs), apart from cancer cells, are expressed on the surface of inflammatory cells. The production of some TAAs may also be increased in some autoimmune diseases including systemic sclerosis (SSc). Objectives To assess serum carcinoembryonic antigen (CEA), CA-15.3, CA 125, CA-19.9 levels in SSc patients, and to identify any possible associations with lung involvement parameters. Methods Eighty-two SSc patients (70 females and 12 males), mean age 64±13 years (range 34-91), disease duration 6±5 years (range 1-27), were consecutively studied. None of the patients ever had any malignancies. Serum TAAs determination were considered and all patients underwent to high-resolution scan (HRCT) and pulmonary function tests (PFTs). CEA, CA 19.9, CA 15.3 and CA125 were determined by electrochemiluminescence immunoassays; the normal upper limit determined by the manufacturer, were as follows: CEA <2.5 ng/ml, CA19.9 <33 U/ml, CA 15.3 <46.5 U/ml, CA125 <21 U/ml. Results CEA was elevated in 28 (32%) of SSc patients, CA 19.9 in 7 patients (9%), CA 15.3 in 28 patients (36%), CA 125 in 6 patients (8%). Lung fibrosis at HRCT significantly associated with CEA (p<0.0003, r=0.4), CA15.3 (p<0.001, r=0.4), and CA125 (p<0.01, r=0.3) while no association was seen for CA 19.9. Forced vital capacity (FVC) significantly associated only with CA 15.3 (p=0.0001), and diffusion lung capacity for carbon monoxide (DLCO) only with CEA (p=0.04). There was an inverse correlation between CA 15.3, FVC and DLCO (r= -0.52 and r= -0.44, respectively). Conclusions The production of some TAAs may be elevated in SSc patients; CEA, CA15.3, and CA125 may have a negative prognostic role, being associated with lung fibrosis as documented by HRCT and PFTs. Further studies on higher proportion of patients could be addressed for identifying a surrogate biomarker, among TAAs, for lung involvement in SSc, to assess extent of the disease and possibly with prognostic significance. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5074

FRI0160 Comparable Amount of Free Heavy Chain and β2M in the Cytoplasm of Ex Vivo Peripheral Blood Mononuclear Cells of B*2705 Ankylosing Spondylitis Patients VS B*2705 and B*2709 Healthy Subjects Does not Support the UPR Theory. Influence of ERAP1 Polymorphisms

Background Ankylosing Spondylitis (AS) is a chronic inflammatory disease of the spine strongly associated with the majority of HLA-B27 alleles, with the exception of B*2709 and B*2706. Genome wide association studies (GWAS) have revealed that besides HLA-B27, other genes are involved in AS pathogenesis, such as ERAP1, an ER aminopeptidase that is implicated in peptide trimming thus influencing B27-peptide-β2microglobulin (β2m) complex stability. Several theories have been proposed to explain the B27 association with AS. Among them, the unfolded protein response (UPR) theory suggests that the tendency of B27 trimeric complex to misfold determines free heavy chain (FHC) accumulation in the endoplasmic reticulum, leading to a stress response and activation of pro-inflammatory pathways. Objectives To our knowledge this is the first ex vivo study investigating the intracellular (ic) level of FHC and β2m in peripheral blood mononuclear cells (PBMC) and the possible influence of ERAP1 allelic variance in HLA-B27 positive AS patients and healthy subjects (HSs) bearing the AS-associated (B*2705) and the non-AS-associated (B*2709) allele. Methods The ic amount of FHC and β2m in CD14+ cells from ex vivo PBMC was evaluated in 12 HLA-B*2705 patients with AS, 12 HLA-B*2705 HSs and 12 HLA-B*2709 HSs by flow cytometry analysis. HC10 (gift of Dr. Chella David) and TU99 clone (BD Biosciences, USA) monoclonal antibodies were used to detect FHC and β2m, respectively, and quantified by comparison with standard beads (antibody binding capacity ABC units, Dako Denmark). Cells were fixed and permeabilized by the Intraprep Permeabilization technique (Beakman Coulter, USA) according to standard procedure. Patients and controls were also genotyped for two ERAP1 SNPs associated with AS (rs27044 C/G and rs30187 C/T). Optimized allelic discrimination assays were purchased from Applied Biosystem (Life Technologies, Italy). Values were expressed as mean ± standard deviation. Differences between AS patients and healthy subjects were analyzed by Mann-Whitney U test. Results FHC expression in AS patients was 37486±30346 compared to B*2705 HSs 35673±16723 and B*2709 HSs 26683±10592 ABC units (p=ns). β2m quantity was also not significantly different in AS patients 174930±90441 compared to B*2705 HSs 156471±123855 and B*2709 HSs 153478±42117 ABC units (p=ns). The majority of AS patients and HSs were heterozygous for both rs27044 (C/G) and rs30187 (C/T) SNPs; the intracellular amount of FHC and β2m in the PBMC of the analysed cohorts appeared not influenced by ERAP1 allelic distribution, as shown in Fig. 1. Figure 1 Conclusions This study shows equal amount of FHC and β2m in the cytoplasm of B*2705 AS patients compared to B*2705 and B*2709 healthy controls, regardless of ERAP1 allelic variance. These data, therefore, do not provide support to the UPR theory in the pathogenesis of AS. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4442

AB0059 Effect of anti-tnf alpha treatment on serum levels of light (tnfsf14), cathepsin k, dkk-1 and sclerostin in ankylosing spondylitis: the osteoclast/osteoblast function balance and its relation to disease activity

Background Ankylosing Spondylitis (AS) is characterized by erosions and new bone formation wich resulting in classical syndesmophytes. These processes appear to be influenced by the fine balance of mediators involved in osteoclast activation, such as LIGHT (TNFSF14) protein and cathepsin K, and inhibition of osteoblastogenesis influenced by DKK-1 and sclerostin, although data available is still controversial. Objectives To investigate in AS the serum levels of mediators involved in bone homeostasis, their relation to disease activity/disease severity and the modifications induced by anti-TNF-α treatment. Methods 63 patients with AS have been consecutively recruited as well as 19 healthy controls (HC). 15 patients underwent anti-TNF-α treatment (Adalimumab), and were evaluated at baseline (w0) and 12 weeks after treatment (w12). Serum levels of DKK-1, sclerostin, cathepsin K and LIGHT were measured by ELISA assay (Biomedica, R&D System) and correlated with clinical scores (BASMI, BASFI e BASDAI), inflammatory markers (ESR and CRP) and disease duration. Data were expressed as mean ± SD. Differences among groups and statistical correlation have been analyzed according to data distribution by means of paired t-test, unpaired t-test with Welch’s correction and Spearman’s test. Results Mediators of osteoclast activation were increased in AS patients compared to HC: LIGHT (115.1 pg/mL ± 74.8 vs 75.8 pg/mL ± 49.2; p=0.009), cathepsin-k (21 pmol/L ± 35.9 vs 9.6 pmol/L ± 5.8; p= 0.03), as well as mediators involved in osteoblast function: DKK-1 (36.4 pmol/L ± 18.8 vs 23.5 pmol/L± 17.6; p= 0.009), sclerostin (36.4 pmol/L ± 21.8 vs 25.1 pmol/L ±9.1; p= 0.001). The increase of these mediators resulted unrelated to disease activity, as evidenced by the lack of correlation of DKK-1, sclerostin, LIGHT and cathepsin-K with clinical scores and with disease duration; only LIGHT showed to correlate with ESR (p=0,01; r=0,4). It is also noteworthy the little modulation induced on these mediators by TNF-α antagonists, regardless the good clinical response according to EULAR criteria: DKK-1, LIGHT and cathepsin-k, w0 vs w12 p= ns, while increased sclerostin levels were observed at w12 compared with baseline (p=0.03). Conclusions AS patients showed an increased bone metabolism, characterized by an increase of both mediators responsible of osteoclast and osteoblast function. This increased bone turn-over appear not linked to the inflammatory process and to disease activity, as perceived by the patient. Furthermore it appear to be independent to TNF-α related mechanisms, as recently suggested by radiological progression in patients with disease in clinical remission following anti-TNF-α treatment. Disclosure of Interest None Declared

SAT0253 HLA-E as ligand for NKG2A/NKG2C in ankylosing spondylitis: Increased expression of HLA-E and prevalence of the inhibitory receptor

Background Ankylosing Spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of HLA-B27 alleles. HLA-E are non classical MHC class I molecules that specifically interact with the natural killer receptors NKG2A (inhibitory) and NKG2C (activating), and have been recently proposed to be involved in AS pathogenesis. The two known HLA-E variants differ only at position 128, with either an arginine or a glycine, and this polymorphism has been demonstrated to be relevant in host immunity thus influencing the protection against HIV and HCV infection. Objectives To analyze the expression of HLA-E and the CD94/NKG2 pair of receptors in HLA-B27 positive AS patients and healthy controls (HC) bearing the AS-associated (B*2705) and the non-AS-associated (B*2709) allele. Methods The level of surface expression of HLA-E molecules on CD14 positive peripheral blood mononuclear cell was evaluated in 21 HLA-B*2705 patients with AS, 12 HLA-B*2705 healthy subjects (HC) and 12 HLA-B*2709 HC by using the monoclonal antibody MEM-E/08 (gift of Dr. Horejsi) in quantitative cytofluorimetric analysis and correlated with the Arg128/Gly128 genotype. Moreover, the HLA-E mRNA was also evaluated by real-time polymerase chain reaction (RT-PCR), GAPDH was used as reference. The percentage and density of expression of HLA-E ligands NKG2A (clone Z199; Beckman Coulter) and NKG2C (clone 134522; R&D) were also measured on CD3-CD56+ NK cells. Values were expressed as median percentage of positive cells (interquartile range) and as cell surface antigen density in antibody binding capacity (ABC) units. The differences between AS patients and HCs were analyzed by one-way ANOVAs with Bonferroni post test and the two-tailed unpaired t-test with Welch’s correction. Results HLA-E expression on CD14 positive cells was significantly higher in AS patients (587.0 IQR 424-830) compared to B*2705 HC (389 IQR 251.3-440.5, p=0.0007) and B*2709 HC (294.5 IQR 209.5-422, p=0.0004). The amount of HLA-E molecules does not correlate with the polymorphism Arg128Gly as also confirmed by mRNA analysis on total PBMC (1.8 IQR 0.7-2.6 in AA bearing subjects, 1.8 IQR 1.0-2.7 in AG bearing subjects, GG not found). An increased number of NK cells expressing the inhibitory receptor NKG2A compared to the activating receptor NKG2C was found in AS patients (45.9% IQR 35.1-52.9 vs 8.5%, IQR 6.3-16.7; p<0.0001) as well as in either B*2705 HC (38.0% IQR 30.0-54.8 vs 8.0%, IQR 4.8-12.9; p<0.0001) or B*2709 HC (45.9% IQR 35.1-52.9 vs 7,5% IQR 6.6-15.9; p<0.001). NKG2A also showed a higher cell surface density compared with the NKG2C receptor reaching statistical significance in AS patients (p=0.02) and B*2705 HCs (p=0.007). Conclusions This study shows a higher expression of HLA-E molecules in AS patients compared to HC and an imbalance of NKG2A/NKG2C ratio with a larger expansion of NK cells expressing the inhibitory receptor which appears also more expressed on the cell surface. The higher surface level of HLA-E molecules in AS patients concurrently with a prevalent expression of NKG2A suggests that the crosstalk between these two molecules might play a role in AS pathogenesis accounting for the previously reported association between HLA-E and AS. Disclosure of Interest None Declared

OP0240 Higher Expression of TNFR1 and IL-1R2 on Cell Surface of B*2705 Ankylosing Spondylitis Patients Vs B*2705 and B*2709 Healthy Subjects. Influence of Erap1 Polymorphism

Background Ankylosing Spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of HLA-B27 alleles, with the exception of B*2706 and B*2709. GWAS studies have revealed that other genes are also involved in AS pathogenesis such as ERAP1 which is able to cleave TNFR1 and IL-1R2 cell surface receptors, supposed to play a pivotal role in AS pathogenesis. Objectives To analyze the cell surface expression of TNFR1 and IL-1R2 receptors and the possible influence of ERAP1 allelic variance. Methods The percentage of CD14 negative peripheral blood mononuclear cells (PBMC) expressing TNFR1 and IL-1R2 receptors on the cell surface was evaluated in 12 HLA-B*2705 patients with AS, 12 HLA-B*2705 healthy subjects (HC) and 12 HLA-B*2709 HC by means of flow cytometry analysis and CD120a and CD121b monoclonal antibodies, respectively. Patients and controls were genotyped for two ERAP1 SNPs associated with AS (rs27044 C/G and rs30187 C/T). Values were expressed as median percentage of positive cells (interquartile range). The differences between AS patients and HC were analyzed by Mann Whitney U-test. Results TNFR1 expression on PBMC was significantly higher in AS patients (35.5 IQR 15.9-60.4) compared to B*2705 HC (18.9 IQR 9.7-25.5, p=0.04) and B*2709 HC (13.1 IQR 9.3-21.5, p=0.02). IL-1R2 was also significantly higher in AS patients (1.5 IQR 0.6-2.5) compared to B*2705 HC (0.2 IQR 0.1-0.8, p=0.01) and B*2709 HC (0.6 IQR 0.1-1.6, p=ns). The majority of AS patients and HC were heterozygous both for rs27044 (C/G) and rs30187 (C/T); the higher numbers of TNFR1 and IL-1R2 positive cells found in AS patients compared to HC were not due to differences in the ERAP1 allelic distribution in patients and controls as shown in the figure below: Image/graph Conclusions This study shows a higher expression of TNFR1 and IL-1R2 on the PBMC surface of AS patients compared to B*2705 and B*2709 regardless of ERAP1 allelic variance, underlining the important role of these two cytokines in the pathogenesis of AS. Disclosure of Interest None Declared