Masahiro Ayano

The Cytotoxic Effects of Certolizumab Pegol and Golimumab Mediated by Transmembrane Tumor Necrosis Factor α

Background:Anti–tumor necrosis factor &agr; (anti-TNF-&agr;) agents have been successfully applied for the treatment of rheumatoid arthritis, Crohns disease, and other chronic inflammatory diseases. Not only the neutralization of soluble TNF-&agr; but also the effect on transmembrane TNF-&agr; is important mechanisms of action of anti-TNF-&agr; agents. This study investigated the cytotoxic effects of new anti-TNF-&agr; agents, certolizumab pegol and golimumab, which are mediated by transmembrane TNF-&agr;. Methods:Transmembrane TNF-&agr;–expressing Jurkat T cells that did not express TNF receptors were used. The binding ability of each anti-TNF-&agr; agent to transmembrane TNF-&agr;, antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and the apoptotic effect were examined. Results:Certolizumab pegol and golimumab bound to transmembrane TNF-&agr;. Golimumab induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, which was comparable to infliximab and adalimumab. However, certolizumab pegol did not induce antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. Certolizumab pegol directly induced nonapoptotic cell death in transmembrane TNF-&agr;–expressing cells. Golimumab induced a weaker apoptotic effect than infliximab and adalimumab. Conclusions:The cytotoxic effects of anti-TNF-&agr; agents on TNF-&agr;–expressing cells are considered to be associated with the clinical effect of these agents on granulomatous diseases. The direct cytotoxic effect of certolizumab pegol on TNF-&agr;–producing cells may contribute to its clinical efficacy in Crohns disease. Golimumab may be less effective for granulomatous diseases.

Increased CD226 Expression on CD8+ T Cells Is Associated with Upregulated Cytokine Production and Endothelial Cell Injury in Patients with Systemic Sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage and fibrosis of the skin and internal organs. Because activated and oligoclonally expanded CD8+ T cells can be detected in peripheral blood and lungs of SSc patients, effector memory CD8+ T cells may play a critical role for organ involvement in SSc; however, the pathogenic functions of effector memory CD8+ T cells remain incompletely understood. In this study, we performed DNA microarray analysis of the sort-purified effector memory CD8+ T cells from SSc patients and healthy controls, and showed that the expression of genes related to immune response and cell adhesion, including CD226 (also known as DNAX accessory molecule-1 [DNAM-1]), was significantly altered. Moreover, detailed analysis of CD226 revealed that CD226highCD8+ T cells were increased in SSc patients (mean, 50.7%) compared with healthy controls (32.9%) and were appreciably associated with the severity of skin sclerosis and interstitial lung disease. Furthermore, CD226+CD8+ T cells produced higher amount of various cytokines than CD226− ones, and CD226highCD8+ T cells from SSc patients showed upregulated IL-13 production and positive correlation with the cytotoxic capacity of CD8+ T cells against HUVECs. Finally, the neutralization of CD226 in CD8+ T cells impaired costimulation, cytokine productions, and cytolysis against HUVECs. These findings indicate that upregulated CD226 expression on CD8+ T cells reflects disease severity and is involved in SSc pathogenesis via the production of various cytokines, including profibrotic IL-13 and endothelial cell injury, and that CD226 may be a useful target in the treatment of SSc.

Genome-wide Association Study of Idiopathic Osteonecrosis of the Femoral Head

Idiopathic osteonecrosis of the femoral head (IONFH) is an ischemic disorder that causes bone necrosis of the femoral head, resulting in hip joint dysfunction. IONFH is a polygenic disease and steroid and alcohol have already known to increase its risk; however, the mechanism of IONFH remains to be elucidated. We performed a genome-wide association study using ~60,000 subjects and found two novel loci on chromosome 20q12 and 12q24. Big data analyses identified LINC01370 as a candidate susceptibility gene in the 20q12 locus. Stratified analysis by IONFH risk factors suggested that the 12q24 locus was associated with IONFH through drinking capacity. Our findings would shed new light on pathophysiology of IONFH.

Effects of tumor necrosis factor inhibitors and tocilizumab on the glycosylated hemoglobin levels in patients with rheumatoid arthritis; an observational study

Rheumatoid arthritis (RA) and diabetes mellitus (DM) are associated with inflammation. We tried to investigate the influence of tumor necrosis factor inhibitors (TNFi) and tocilizumab (TCZ) on the glucose metabolism of RA patients. RA patients in whom treatment with TNFi or TCZ was initiated from 2008 to 2015 were studied based on their medical records. We analyzed patients whose glycosylated hemoglobin (HbA1c) levels were measured both before and 3 months after the initiation of these biologic agents. The association between HbA1c reduction and the treatment was evaluated. From 971 cases treated with these biologic agents, 221 cases whose medical records of HbA1c were available, were included (TNFi, n = 154; TCZ, n = 67). Both the TNFi and TCZ groups had significantly lower HbA1c values at 1 month and 3 months after the initiation of treatment (TNFi, p<0.001; TCZ, p<0.001). Although the pretreatment HbA1c values did not differ (TNFi, 6.2%; TCZ, 6.2%; p = 0.532), the 3-month treatment HbA1c values were lower (TNFi, 6.1%; TCZ, 5.8%; p = 0.010) and the changes in HbA1c (ΔHbA1c) were greater (TNFi, 0.1%; TCZ, 0.4%; p<0.001) in the TCZ group. The reduction of HbA1c—defined by the achievement of a ΔHbA1c of ≥0.5%—was associated with baseline diagnosis of diabetes mellitus, baseline diabetes treatment, hospitalization, medical change during the observation period, and TCZ. In the multivariate logistic regression analysis, TCZ was associated with the reduction of HbA1c in comparison to TNFi (adjusted OR = 5.59, 95% CI = 2.56–12.2; p<0.001). The HbA1c levels in RA patients were significantly lower after the initiation of TNFi or TCZ. Our study suggests that TCZ decreases the HbA1c levels in RA patients to a greater extent than TNFi.

A Japanese case of TNF receptor-associated periodic syndrome (TRAPS) successfully treated by canakinumab

Abstract Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is one of the representative autoinflammatory diseases characterised by prolonged periodic fever caused by a genetic mutation of the type 1 TNF receptor (TNFR1). Although its molecular pathophysiology mechanism has not been completely elucidated, therapeutic strategies have been developed based on the recent pathological findings, such as the intracellular stress due to deposition of misfolded mutant TNFR1. The effectiveness of the IL-1β inhibitor for TRAPS has been reported, and canakinumab was approved on December 2016 in Japan. We report a case of TRAPS who became secondarily resistant to predonisolone and etanercept, an anti-TNF agent, after eleven years of treatment. Subsequently, canakinumab, a IL-1β inhibitor, was introduced, resulting in prompt improvement of inflammatory symptoms. This is the first case report of Japanese TRAPS patient successfully treated by canakinumab.

Long-term efficacy of infliximab treatment and the predictors of treatment outcomes in patients with refractory uveitis associated with Behçet’s disease

OBJECTIVE To assess the long-term efficacy and safety of infliximab (IFX) treatment for refractory uveitis associated with Behçets disease (BD) and to identify predictors of long-term IFX therapy outcomes. METHODS We retrospectively studied 44 consecutive BD patients with uveitis who were started on IFX therapy and analyzed the efficacy and safety of IFX and the treatment continuation rate. To determine predictors of IFX responsiveness, we analyzed the clinical characteristics of the patients who received regular maintenance therapy and those who required treatment intensification. The serum cytokine levels prior to IFX were measured through the Bio-Plex human cytokine assays. RESULTS IFX significantly reduced the frequency of ocular attacks and improved the visual acuity of patients with BD-related uveitis. However, approximately half of the patients required dose escalations, necessitating a shortening of the intervals between IFX infusions due to loss of efficacy during the 5-year treatment. The frequency of ocular attacks was significantly higher in patients with complete BD than in patients with incomplete BD. A multiplex cytokine analysis revealed that patients with BD-related uveitis exhibited increased serum IL-2, IL-6, IL-8, and MCP-1 levels. Moreover, among BD patients, the serum IL-2 and IL-6 levels were particularly high in those who maintained remission and received regular IFX treatments. CONCLUSION We confirmed the long-term efficacy and tolerability of IFX in patients with BD-related uveitis. Our results indicate that complete BD may be less responsive to IFX and that the pretreatment serum cytokine profiles may be useful for predicting the long-term IFX therapy outcomes.

OP0093 Comparison of CD34-Selected and Unmanipulated Autologous Hematopoietic Stem Cell Transplantation for Systemic Sclerosis: Four-Year Follow-Up Results

Background The superior efficacy of autologous hematopoietic stem cell transplantation (auto-HSCT) versus intravenous pulses cyclophosphamide was demonstrated in a randomized phase II trial in 19 severe systemic sclerosis (SSc) patients and a large randomized phase III trial in 156 patients. However, the usefulness of CD34-selection in auto-HSCT for SSc has not been investigated. Objectives The aim of this study is to compare the safety and the efficacy of CD34-selected auto-HSCT with those of unmanipulated auto-HSCT for severe SSc during a four-year follow-up period. Methods Nineteen patients (4 male and 15 female) with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSC more than 2×106CD34+ cells/kg by apheresis, CD34+ cells were immunologically selected in 11 patients. All of the patients were treated with high-dose CY (200 mg/kg) and received CD34-selected (n=11; group A) or unmanipulated (n=8; a group B) auto-HSCT. Immune reconstitution was evaluated serially by analyzing lymphocyte subpopulations for 48 months after HSCT. Results The age of the patients was 52.3±7.5 years in group A and 55.1±5.5 years in group B. The modified Rodnans skin score was 21.5±14.0 in group A and 22.9±9.0 in group B. Numbers of the infused CD34+ and CD3+ cells were 4.7±2.6×106 and 0.011±0.012×106/kg, respectively, in group A and 7.6±10.7×106 and 45.6±28.1×106/kg, respectively, in group B. The time to engraftment of neutrophil and platelet was not different between two groups. There was no treatment-related mortality in both groups. As toxicity, 2, 5 and 6 out of 11 patients had adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia, respectively, in the group A, on the other hand, no patients had adenoviral hemorrhagic cystitis, herpes zoster and only 2 out of 8 patients had cytomegaloviral antigenemia, respectively, in group B. All of the patients in both groups had marked improvement of skin sclerosis within 3 months and the improvement was sustained for 48 months after auto-HSCT. Surprisingly, the reduction of the skin scores was significantly greater at 1 and 12-48 months after auto-HSCT in group A than in group B. About the effect on interstitial pneumonia, %VC increased continuously until 48 months after auto-HSCT in group A, on the other hand, it once increased but decreased after 12 months in group B. The recovery of lymphocyte subpopulations after auto-HSCT was not significantly different between two groups. Conclusions CD34-selected auto-HSCT had greater effects but more susceptiblity to viral infections than unmanipulated auto-HSCT. References Tsukamoto H, Nagafuji K, Horiuchi T, et al. A Phase I-II Trial of Autologous Peripheral Blood Stem Cell Transplantation in the Treatment of Refractory Autoimmune Disease. Ann Rheum Dis. 65: 508-14, 2006. Tsukamoto H, Nagafuji K, Horiuchi T, et al. Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells. Rheumatology (Oxford) 50: 944-52, 2011. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3166

AB0201 Cell Surface Expression of DNAM-1 (CD226) on CD8+ T Cells is Increased in Patients with Systemic Sclerosis

Background DNAM-1 (CD226) is well known as a genetic risk factor of many autoimmune diseases such as systemic sclerosis (SSc). More recently, Avouac et al. have reported that mice deficient for DNAM-1 are protected from bleomycin-induced dermal fibrosis. In human, however, the roles of DNAM-1 in SSc are still unknown. Objectives To evaluate cell surface expression of DNAM-1 on peripheral blood lymphocytes from SSc patients and to identify the roles of DNAM-1 in the pathogenesis of SSc. Methods The expression of DNAM-1 was analyzed by flow cytometry (FCM) on surface of CD4+ T cells, CD8+ T cells and CD19+ B cells from 41 SSc patients (median age, 59 years; female, 37) and 23 healthy controls (HCs). We evaluated the relationship between DNAM-1 expression level on CD8+ T cells and clinical manifestations of SSc. Intracellular and extracellular cytokine production in CD8+ T cells with or without DNAM-1 was measured by FCM (intracellular staining and multiplex bead array, respectively) after stimulation with PMA/Ionomycin. The cytotoxic capacity of CD8+ T cells against human umbilical vein endothelial cells (HUVECs) was assessed in the presence or absence of anti-DNAM-1 neutralizing antibody in SSc patients and HCs. Results The proportion of DNAM-1high CD8+ T cells was increased in SSc patients (median, 52.5%) compared to HCs (29.7%, P=0.0002). We found no significant difference between SSc patients and HCs in CD4+ T cells and CD19+ B cells. The expression of DNAM-1 on CD8+ T cells was higher in patients with diffuse cutaneous subtype than those with limited cutaneous subtype (P=0.0009) and was higher in those with interstitial pneumonia than those without (P=0.0016). The percentage of DNAM-1high CD8+ T cells was significantly correlated with modified Rodnan skin thickness score (r =0.4295, P=0.0071). The production of IL-13 was significantly increased in DNAM-1 positive CD8+ T cells compared with DNAM-1 negative ones and the neutralization of DNAM-1 impaired the IL-13 production from CD8+ T cells. The cytotoxic ability of CD8+ T cells was significantly elevated in SSc patients and positively correlated with the percentage of DNAM-1high CD8+ T cells. The cytotoxicity against HUVECs was partially inhibited in the presence of anti-DNAM-1 neutralizing antibody. Conclusions These findings indicate that DNAM-1high CD8+ T cells are involved in the pathogenesis of SSc via the production of profibrotic IL-13 and endothelial cell injury. References Avouac J, Elhai M, Tomcik M, et al. Critical role of the adhesion receptor DNAX accessory molecule-1 (DNAM-1) in the development of inflammation-driven dermal fibrosis in a mouse model of systemic sclerosis. Ann Rheum Dis. 2013;72:1089-98. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3217

THU0063 Serum Progranulin Levels are Elevated in Dermatomyositis Patients with Acute Interstitial Lung Disease, Reflecting Severity and Prognosis

Background Recently, it has been reported that granulin (GRN) and/or progranulin (PGRN), precursor of GRN, is a soluble cofactor for TLR9 signaling [1]. We reported that serum PGRN levels are associated with SLE global activity and PGRN may have a role in the pathogenesis via increased cytokine production [2]. TLR9 is also involved in the pathological condition of dermatomyositis (DM). Moreover, DM is occasionally complicated with interstitial lung disease (ILD). However, the roles of PGRN and GRN in DM are still unknown. Objectives To determine if serum PGRN levels are elevated in DM patients, in particular, complicated with ILD and are associated with severity and prognosis. Methods The serum levels of PGRN were measured by ELISA in patients with DM (n=50; acute/subacute interstitial pneumonia (A/SIP), defined as a rapidly progressive ILD within 3 months from the onset of symptoms: n=13, chronic interstitial pneumonia (CIP): n=20, without ILD: n=17), polymyositis (PM, n=21) and normal healthy controls (NHCs, n=60). We assessed the correlation between the serum PGRN levels and the activity indexes of ILD. The sera from some of the patients were reevaluated after the disease was ameliorated by treatment (n=6). Moreover, we calculated the cumulative survival rate for 6 months in DM patients with ILD, which is classified two groups, serum PGRN levels > or =200 ng/ml and < 200 ng/ml. Results The serum PGRN levels were significantly higher in the DM patients (median: 100 ng/ml) than in the PM patients (60.4 ng/ml, P=0.0028) and NHCs (48.3 ng/ml, P<0.0001). Of the total DM patients, the levels were significantly higher in DM with A/SIP than that in DM with CIP (P<0.0001) or without ILD (P=0.0002). Proportion of clinically amyopathic DM is higher in DM with A/SIP than in DM with CIP or without ILD (76.9%, 40%, and 5.9%, respectively). The serum PGRN levels correlated significantly with serum ferritin (rs=0.71, P=0.0001), LDH (rs=0.59, P=0.0003), and CRP (rs=0.57, P=0.0005) levels. They significantly decreased following successful treatment of DM (P=0.0313). Moreover, the cumulative survival rate for 6 months was significantly lower in the group with serum PGRN levels > or =200 ng/ml (60%) than that in the group with serum PGRN levels < 200 ng/ml (P=0.001). Conclusions These findings indicate that PGRN is associated with severity and prognosis of DM with ILD. PGRN may play a role in the pathogenesis of DM by affecting the TLR9 signaling and could be a useful biomarker. References Park B, Buti L, Lee S, et al. Granulin is a soluble cofactor for toll-like receptor 9 signaling. Immunity 2011;34:505-513. Tanaka A, Tsukamoto H, Mitoma H, et al. Serum progranulin levels are elevated in patients with systemic lupus erythematosus, reflecting disease activity. Arthritis Res Ther 2012;14(6):R244. Disclosure of Interest None Declared

THU0238 Long-term follow-up of autologous hematopoietic stem cell transplantation for severe systemic sclerosis

Background Recent phase II study demonstrated that autologous hematopoietic stem cell transplantation (auto-HSCT) is more effective than conventional intravenous cyclophosphamide in the treatment of severe systemic sclerosis (SSc). However, the long-term results of CD34-selectiod or unmanipulated auto-HSCT for SSc has not been investigated. Objectives The aim of this study is to investigate the long-term results of auto-HSCT for severe SSc and to compare efficacy of CD34-selected auto-HSCT with that of unmanipulated auto-HSCT. Methods Nineteen patients (4 male and 15 female) with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSCs more than 2×106CD34+cells/kg by apheresis, CD34+ cells were immunologically selected in 11 patients. All of the patients were treated with high-dose CY (200 mg/kg) and received CD34-selected (n=11; a group with CD34-selection) or unmanipulated (n=8; a group without CD34-selection) auto-HSCT. Immune reconstitution was evaluated serially by analyzing lymphocyte subpopulations for 36 months after HSCT. Results There was no treatment-related mortality. As toxicity, there were a variety of post-transplant infections such as adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia. Sclerosis of the skin was markedly improved in all of the patients within 6 months and the improvement was sustained for 60 months after auto-HSCT. Vital capacity was significantly increased at 48 months after HSCT and KL-6, a marker for interstitial pneumonia (IP), was significantly decreased in patients with IP during 12-60 months after HSCT. A titer of anti-Scl-70 was significantly decreased during 1-60 months after HSCT. Progression-free and overall 5-year survivals were 68 and 95%, respectively. Interestingly, the reduction of the skin scores was significantly greater and viral infections were more frequent in the patients with CD34-selection than those without. Effect of auto-HSCT on interstitial pneumonia, and the recovery of lymphocyte subpopulations after auto-HSCT were not significantly different between two groups. Conclusions Auto-HSCT had long-term effects on skin sclerosis and IP, resulting in improved prognosis in patients with severe SSc. CD34-selected auto-HSCT had greater effect on skin sclerosis than unmanipulated auto-HSCT. References Tsukamoto H, Nagafuji K, Horiuchi T, et al. A Phase I-II Trial of Autologous Peripheral Blood Stem Cell Transplantation in the Treatment of Refractory Autoimmune Disease. Ann Rheum Dis. 65: 508-14, 2006. Tsukamoto H, Nagafuji K, Horiuchi T, et al. Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells. Rheumatology 2011; 50: 944-52. Disclosure of Interest None Declared