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Background/Aims: The aim of this study was to investigate long-term post-discontinuation outcomes in patients with rheumatoid arthritis (RA) who had been treated with tumor necrosis factor-α inhibitors (TNF-αi) which was then discontinued.Methods: Sixty Korean patients with RA who participated in a 5-year GO-BEFORE and GO-FORWARD extension trials were included in this retrospective study. Golimumab was deliberately discontinued after the extension study (baseline). Patients were then followed by their rheumatologists. We reviewed their medical records for 2 years (max 28 months) following golimumab discontinuation. Patients were divided into a maintained benefit (MB) group and a loss-of-benefit (LB) group based on treatment pattern after golimumab discontinuation. The LB group included patients whose conventional disease-modifying antirheumatic drug(s) were stepped-up or added/switched (SC) and those who restarted biologic therapy (RB).Results: The mean age of patients at baseline was 56.5 years and 55 (91.7%) were females. At the end of follow-up, 23 (38.3%) patients remained in the MB group. In the LB group, 75.7% and 24.3% were assigned into SC and RB subgroups, respectively. Fifty percent of patients lost MB after 23.3 months. Demographics and clinical variables at baseline were comparable between MB and LB groups except for age, C-reactive protein level, and corticosteroid use. Restarting biologic therapy was associated with swollen joint count (adjusted hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.01 to 3.55) and disease duration (adjusted HR, 1.12; 95% CI, 1.02 to 1.23) at baseline.Conclusions: Treatment strategies after discontinuing TNF-αi are needed to better maintain disease control and quality of life of patients with RA.

Abnormal regulation of Wnt/β-catenin signaling followed by increased levels of the β-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit β-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the β-catenin mRNA. We designed a strategy to identify the cleavage sites in β-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave β-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of β-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited β-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to β-catenin significantly reduced β-catenin–dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against β-catenin might have potential as a therapeutic intervention to treat colon cancer. Mol Cancer Ther; 9(6); 1894–902. ©2010 AACR.

// Hyun-Mi Kwon 1, * , Eun-Jin Kang 1, * , Keunsoo Kang 2 , Sung-Dae Kim 1 , Kwangmo Yang 1 and Joo Mi Yi 1 1 Research Center, Dongnam Institute of Radiological & Medical Sciences (DIRAMS), Busan 46033, South Korea 2 Department of Microbiology, Dankook Universty, Cheonan 31116, South Korea * These authors have contributed equally to this work Correspondence to: Joo Mi Yi, email: jmyi@dirams.re.kr Keywords: DNA methylation inhibitor, radiation, pancreatic cancer, cancer stem cell Received: June 29, 2017 Accepted: August 27, 2017 Published: October 06, 2017 ABSTRACT Pancreatic cancer is associated with a high mortality rate, owing to de novo and acquired drug resistance, thereby leading to highly invasive and metastatic pancreatic cancer cells. Therefore, targeting pancreatic cancer stem cells (CSCs) may be a novel therapeutic strategy for the treatment of pancreatic cancer. Here, we combined a DNA methylation inhibitor (5-aza-2’-deoxycytidine; 5-aza-dC) and ionizing radiation (IR) to improve anti-cancer effects by inhibiting growth and proliferation and promoting apoptosis of pancreatic cancer cells in vitro and in vivo . Importantly, the combinatorial effect of 5-aza-dC with IR on sphere-forming pancreatic cancer cells was preferentially targeted toward CSCs through the downregulation of regulatory factors of self-renewal and CSC surface markers. We next performed the RNA sequencing to understand the underlying cellular mechanisms of the combined treatment with IR and 5-aza-dC in pancreatic cancer cells. Global transcriptome profiling indicated that the expression of the Oct4-centered transcriptional network of genes was significantly downregulated in cells with combination treatment. Our data suggested that combination treatment with DNA methylation inhibitor and IR may be a novel therapeutic strategy for pancreatic cancer. Overall, these findings support the use of epigenetic therapy in combination with radiotherapy to improve therapeutic efficacy by targeting and eradicating pancreatic CSCs.

// Joo Mi Yi 1 , Eun-Jin Kang 1 , Hyun-Mi Kwon 1 , Jin-Han Bae 1 , Keunsoo Kang 2 , Nita Ahuja 3 , Kwangmo Yang 1 1 Research Center, Dongnam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Republic of Korea 2 Department of Microbiology, Dankook University, Cheonan, Korea, Republic of Korea 3 Department of Surgery, Oncology, and Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Joo Mi Yi, email: jmyi@dirams.re.kr Kwangmo Yang, email: kmyang@dirams.re.kr Keywords: microRNA, miR-1247, hypermethylation, tumor suppressor, pancreatic cancer Received: October 25, 2016 Accepted: February 13, 2017 Published: February 24, 2017 ABSTRACT Altered expression of microRNAs has been strongly implicated in human cancers, and growing evidence is emerging that a number of miRNAs are downregulated in cancer associated with CpG island hypermethylation. Although pancreatic cancer is one of the most malignant human cancers, the roles of miRNAs underlying the tumorigenesis of pancreatic cancer are still poorly understood. In the present study, we explored the molecular functional role of microRNA-1247 as tumor suppressor associated with epigenetic alteration in pancreatic cancer. CpG islands methylation of miR-1247 is frequently observed in various pancreatic cancer cell lines and in primary pancreatic tumors, but not in normal pancreatic tissue. Ectopic expression of miR-1247 in five pancreatic cancer cell lines results in suppressing of cell growth, proliferation, migration, and invasion in vitro and tumorigenicity of pancreatic cancer cells in vivo . Interestingly, we found one putative target gene of miR-1247, regulator of chromosome condensation 2 ( RCC2 ), harbored miR-1247 target sequences in the 3′ UTR of its mRNA. In functional studies in vitro to understand the interaction between miR-1247 and RCC2 , decreasing of RCC2 gene expression by miR-1247 was observed by immunoblotting and immunohistochemistry at both mRNA and protein levels. Moreover, luciferase reporter assay confirmed that RCC2 was a direct target of miR-1247. Taken together, our data suggest that CpG island hypermethylation of miR-1247 is responsible for its downregulation in pancreatic cancer, and ectopic expression of miR-1247 functions as a potential tumor suppressor targeting RCC2 in pancreatic cancer cells.

Objectives HLA genes are a major genetic risk factor for myositis and myositis specific antibodies (MSAs), exhibiting unique HLA backgrounds for myositis in different ethnic groups. This is the first large scale Korean study to genotype the HLA-DRB1 and -DPB1 alleles and to examine their association with myositis and MSAs. Methods HLA-DRB1 and HLA-DPB1 alleles and MSAs were examined in 179 patients with dermatomyositis (DM, n = 129) or polymyositis (PM, n = 50) and healthy controls (n = 800 for HLA-DRB1, n = 548 for HLA-DPB1). Associations between individual HLA alleles and myositis/MSA were examined. Bonferroni correction was applied for multiple testing comparing patients and controls. Results A total of 33 HLA-DRB1 and 24 HLA-DPB1 alleles were genotyped in patients and controls. MSAs were found in 67.0% of patients. Anti-MDA5 (26.8%) and anti-aminoacyl-tRNA synthetase antibodies (15.6%) were most common, followed by anti-Mi2 (9.5%) and anti-TIF1γ antibodies (8.9%). HLA-DRB1*12:02 and HLA-DRB1*14:03 were associated with DM and PM, respectively. HLA-DRB1*12:02 was associated with anti-MDA5, HLA-DRB1*08:03 with anti-ARS, HLA-DRB1*14:03 with anti-SRP, and HLA-DRB1*07:01 with anti-Mi2 antibodies. Although HLA-DRB1*13:01 was associated with anti-TIF1γ antibodies, the frequency of HLA-DRB1*13:01 was rare. HLA-DPB1*02:01 was negatively associated with myositis and PM while HLA-DPB1*17:01 was associated with anti-Mi2 positive DM. Conclusions Unique immunogenetic background was observed for Korean patients with myositis. Novel myositis susceptibility alleles, HLA-DRB1*12:02 and HLA-DRB1*14:03, were identified, together with MSA-associated HLA alleles unique to Korean patients with myositis.

Interstitial lung disease (ILD) is the most common cause of death in dermatomyositis (DM). Cyclosporine A (CsA) has shown to be effective in DM-associated ILD (DM-ILD). This study aimed to define the optimal time of CsA administration. A total of 47 patients with DM-ILD, who were treated with CsA at Seoul National University Hospital between January 1998 and June 2013, were enrolled. ILD was diagnosed based on typical chest high-resolution computed tomography (HRCT) findings. Patients with early and delayed CsA treatment were compared in regard to the mortality and ILD progression on HRCT. The early (n = 16) and the delayed treatment group (n = 31) did not differ in regard to baseline clinical characteristics including HRCT scores and pulmonary function. Patients with clinically amyopathic DM (CADM) were more common in the early treatment group. The mortality rate was significantly lower in the early treatment group than in the delayed treatment group (p = 0.009). The survival benefit of early CsA treatment remained significant even after adjusting for age, degree of dyspnea, CADM status, and the year of CsA treatment (hazard ratio 0.057, 95 % confidence interval 0.007-0.472). CsA stabilized disease progression on HRCT in the early treatment group (p = 0.738). Delay in CsA treatment is associated with a worse survival in patients with DM-ILD. Early CsA treatment should be considered at DM-ILD diagnosis especially in patients at a higher risk of developing a rapidly progressive ILD.

OBJECTIVE To develop a decision model to identify SSc-associated interstitial lung disease (ILD) patients who are eligible for watchful waiting at ILD diagnosis. METHODS One hundred and fifty-one SSc-ILD patients who received medical care at Seoul National University Hospital from 1986 to 2013 were enrolled in this retrospective cohort study. ILD was diagnosed by chest CT. Patients with and without immunosuppressive treatment were compared in terms of characteristics at ILD diagnosis to identify distinguishing variables. After multivariate analysis, a decision model for watchful waiting was formulated. Its validity was assessed by comparing the survival of patients whose management in real practice did and did not accord with the management recommended by the model. RESULTS The untreated group had better survival than the immunosuppressive treatment group (P = 0.0316, by log-rank test). The untreated group was less likely to have gastrointestinal involvement (P = 0.008) and pulmonary arterial hypertension (PAH), as determined by echocardiography) (P = 0.015) and more likely to have favourable initial forced vital capacity (P = 0.0004), favourable initial lung diffusion capacity for carbon monoxide (P = 0.0002) and a low CT grade (P < 0.001). The final watchful waiting decision model included lack of PAH and limited ILD extent on CT. Application of the model to the cohort revealed that patients who were eligible for watchful waiting (as determined by the model) and underwent this management strategy had better survival than eligible patients who underwent immunosuppressive treatment (P = 0.048, by log-rank test). CONCLUSION Watchful waiting may be effective for SSc-ILD patients who have minimal pulmonary involvement on CT and lack PAH on echocardiography at baseline.

Background T follicular helper cells (Tfh) are necessary for B-cell maturation and differentiation in the germinal centers (GC). The signaling lymphocyte activation molecule family (SLAMF) receptors on the cell surface mediate T cell:B cell interaction for formation and maintenance of GC. But it remains to be elucidated whether SLAMF is expressed in circulating T follicular helper cells (cTfh). Objectives The aim of this study was to investigate the levels of SLAMF3 and SLAMF6 on cTfh and to analyze their association with disease activity in systemic lupus erythematosus (SLE) patients. Methods Blood samples were collected from 50 SLE patients, 24 Sjogren9s syndrome (SS) patients and 25 age- and sex-matched healthy controls (HCs). In this study, CXCR5, programmed cell death protein 1 (PD-1), and CD4 were used as markers to define cTfh in peripheral blood mononuclear cells (PBMCs). The expression levels of SLMAF3 and SLMAF6 on cTfh were compared to that of circulating B cell subsets by flow cytometry. Clinical features including disease activity and laboratory tests were analyzed according to expression of SLAMF3 and SLAMF6. Results Surface expression of SLAMF6 on CD4 T cells was significantly increased in SLE patients compared to SS patients (mean fluorescence intensity [MFI], mean ± SD: 1291±319 vs 1098±147, p=0.015) and HCs (1291±319 vs 1094±243, p=0.011). But there was no difference in expression of SLAMF3 on CD4 T cells between SLE patients and SS patients or HCs. SLAMF6 expressions on cTfh cells, identified as CD4+CXCR5+PD-1+, were significantly increased in SLE patients than in SS patients (1500±270 vs 1296±140, p=0.001) and HCs (1500±270 vs 1295±161, p=0.001). SLAMF6 expressions on cTfh cells had correlation with systemic lupus erythematosus disease activity index (SLEDAI) (Spearman9s rho=0.477, p=0.001), proteinuria (rho=0.321, p=0.030) and double strand DNA (rho=0.340, p=0.026) in SLE patients. Moreover, expression levels of SLAMF6 on cTfh cells were correlated with those of several B cell subsets including total B cells (CD19+), naive B cells (CD19+CD27-IgD+) and class-switched memory B cells (CD19+CD27+IgD-). But SLAMF6 expressions of B cell subsets were not correlated with disease activity. Conclusions Surface expression of SLAMF6 was increased in cTfh cells in patients of SLE and had correlation with disease activity. References T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity. 2013;17;39(4):770–81. Choi JY, His-enHo J, Pasoto SG, et al. Circulating follicular helper-like T cells in systemic lupus erythematosus: association with disease activity. Arthritis Rheumatol. 2015;67(4):988–99. Zhang X, Lindwall E, Gauthier C, et al. Circulating CXCR5+CD4+helper T cells in systemic lupus erythematosus patients share phenotypic properties with germinal center follicular helper T cells and promote antibody production. Lupus. 2015;24(9):909–17. Disclosure of Interest None declared

Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

Objective To investigate the impact of dose reduction of tumor necrosis factor inhibitor (TNFi) on radiographic progression in ankylosing spondylitis (AS). Methods One hundred and sixty-five patients treated with etanercept or adalimumab were selected from a consecutive single-center observational cohort based on the availability of radiographs at baseline and after two- and/or four-years of follow up. Radiographs were assessed by two blinded readers using the modified Stokes AS Spinal Score (mSASSS). Radiographic progression in patients treated with standard-dose TNFi (standard-dose group, n = 49) was compared with patients whose dosage was tapered during the treatment (tapering group, n = 116) using linear mixed models. Results Baseline characteristics between two groups were comparable except for higher BASDAI (7.1 vs. 6.3, p = 0.003) in the standard-dose group. At two years after the treatment, mean dose quotient (S.D.) of the tapering group was 0.59 (0.17). During follow up, rate of radiographic progression in overall patients was 0.90 mSASSS units/year. Radiographic progression over time between the two groups was similar at the entire group level. However, in the subgroup of patients with baseline syndesmophytes, progression occurred significantly faster in the tapering group after the adjustment for baseline status (1.23 vs. 1.72 mSASSS units/year, p = 0.023). Results were consistent when radiographic progression was assessed by the number of newly developed syndesmophytes (0.52 vs. 0.73/year, p = 0.047). Sensitivity analysis after multiple imputation of missing radiographs also showed similar results. Conclusion A dose tapering strategy of TNFi is associated with more rapid radiographic progression in AS patients who have syndesmophytes at baseline.

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5′-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5′-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5′-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5′-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5′-overhang account for the observed enhanced processivity of DNA unwinding.

Time to unwind: Graphene oxide (GO) enables the quantitative measurement of helicase‐dependent double‐stranded DNA (dsDNA) unwinding activity in real time. GO selectively binds to unwound fluorescent‐dye‐labeled single‐stranded DNA and quenches its fluorescence (see picture). The helicase activity is monitored by following the change in fluorescence.WILEY-VCH

The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.

The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. Here, we demonstrate the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by Gram-positive bacteria and fungi. Our analysis shows that ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions.

The innate immune system is important for the first line of host defence against infectious agents, which have penetrated the mechanical barriers. Mannose-binding lectin (MBL or mannan-binding protein, MBP) is a serum protein that is synthesized in the liver as a part of the acute phase response. MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL is synthesized as a monomer that has a carboxy-terminal carbohydrate recognition domain, a neck region and a collagen region. Low MBL level was reported to be the most frequent immuno-deficiency syndrome. Although extensive studies have yielded detailed information on the structure of MBL, functions of the MBL complex are not fully understood yet. We, here, present cloning process of MBL cDNA from the rat liver and production of truncated recombinant MBL protein using a bacterial expression system in order to produce anti-MBL polyclonal antibody. Anti-MBL polyclonal antibody was raised in a New Zealand rabbit and its affinity was tested against recombinant protein using western blot technique. MBL cDNA, recombinant protein and anti-MBL antibody could be used as great arsenals to dissect cellular biochemistry of MBL.

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

Increasing evidences suggests that various cancer types contain a population of cells that display stem cell properties. These cells are called cancer stem cells (CSCs). They are virtually resistant to radiation, and may contribute to treatment resistance and recurrence. Therefore, therapies specifically targeting CSCs will likely be needed for complete tumor eradication. Exposure of cells to ionizing radiation (IR) induces, not only, activation of multiple signaling pathways that play critical roles in cell fate determination, but also alteration of molecular pathways involved in cell death or survival. Recently, DNA methylation has been established as a critical epigenetic process involved in the regulation of gene expression in cancer cells, suggesting that DNA methylation inhibition may be an effective cancer treatment strategy. We therefore investigated the combination effect of a DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine (5-aza-dC), and radiation treatment in pancreatic cancer. Pancreatic cancer cell lines were initially tested for radiation sensitivity by ionizing radiation (IR) in vitro and were treated with 5-aza-dC. The effects of 5-aza-dC along with irradiation on cell growth, cell cycle distribution, apoptosis, and apoptosis-related gene expression were examined. Combination irradiation treatment with 5-aza-dC significantly decreased growth activity compared with irradiation or 5-aza-dC treatment alone. To understand whether combination treatment can target to pancreatic cancer stem cells, we observed that combination treatment can significantly reduce Oct4, Nanog, Sox2, CD133, and CD44 levels which are key factors for self-renewal pathway or cancer stem cell surface markers in pancreatic cancer cells, respectively. Using transcriptome analysis, OCT4 target genes showed differential expression pattern between irradiation or 5-aza-dC treatment alone and combination treatment. In addition, sphere forming assay supports these data in vitro and in vivo, describing that combination effect of IR and 5-aza-dC can reduce significantly sphere forming ability and proportion of side population (putative CSCs) significantly decreased in combination treated cells compared to control or single treatment. Therefore combined treatment of IR and 5-aza-dC is capable of eliminating pancreatic CSCs. Further investigation of this useful approach may lead to the development of a novel intervention for the cancer therapeutic strategy. Citation Format: Joo Mi Yi, Hyun-Mi Kwon, Eun-Jin Kang, Kwangmo Yang. Combined treatment with radiation and epigenetic regulation improve to targeting cancer stem cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-227. doi:10.1158/1538-7445.AM2017-LB-227

Polyarteritis nodosa (PAN) has a broad spectrum of clinical presentation, since it affects small and medium-sized muscular arteries with microaneurysm formation, aneurysmal rupture with hemorrhage, thrombosis, and, consequently, organ ischemia or infarction. Although skeletal muscle involvement is well documented in patients with PAN, it can mimic more common diseases, and cause confusion and delays in diagnosis. PAN muscular involvement may have limited or early systemic forms with a benign course and excellent clinical response to corticosteroid therapy. Herein, we describe the clinical course and outcome of four unusual cases of PAN manifested by acute onset of pain and pitting edema in both lower extremities; in addition, we reviewed the relevant literature. (J Rheum Dis 2017;24:48-54)

E-mail: wsyeo@konkuk.ac.krReceived June 27, 2011, Accepted July 6, 2011Key Words : Aptamers, Helicase, MALDI-TOF MS, Self-assembled monolayersAptamers are oligonucleotides or peptide molecules thatbind to a specific target molecule with high affinity. Inparticular, nucleic acid aptamers can be selected amongrandomized sequences using the Systematic Evolution ofLigands by Exponential enrichment (SELEX) method, inwhich selections of oligonucleotides that bind to the targetmolecule are iterated.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spätzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 A resolution and were suitable for structure determination. The crystals belonged to space group P2(1). The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease.

Background Although dose tapering of tumour necrosis factor (TNF)-blockers in ankylosing spondylitis (AS) showed a comparable clinical efficacy with favourable safety and cost in some studies, there has been no investigation comparing the radiographic progression of patients with tapering regimen versus patients with standard-dose treatment. Objectives To investigate the impact of dose reduction of TNF-blockers on radiographic progression in the AS over 4 years Methods One hundred sixty five patients treated with etanercept or adalimumab were selected from single-centre, prospective observational cohort based on the availability of cervical and lumbar radiographs at baseline and after 2 and/or 4 years of the treatment. Radiographs were scored using modified Stokes AS spinal score (mSASSS) by two independent readers. Radiographic progression of patients treated with standard-dose (standard-dose group, n=49) was compared with that of patients who tapered the dosage during the TNF-blocker treatment (tapering group, n=116) using linear mixed models. Results Demographic and clinical factors at baseline between two groups were not significantly different except for higher initial BASDAI (7.1 vs. 6.3, p=0.003) in the standard-dose group. Baseline mSASSS (SD) of the standard-dose group and the tapering group were 17.3 (17.7) and 11.0 (16.0), respectively (p=0.059). At 2 years after the treatment, mean dose quotient (SD) of the tapering group was 0.59 (0.17) and time to first dose reduction (SD) was 36.6 (28.4) weeks. During the observation period, the rate of radiographic progression in the overall patients was 0.90 mSASSS unit/year. Rate of progression was significantly higher in patients with longer disease duration (≥10 years), elderly patients (≥40 years old), smokers, or patients with a hip involvement at baseline. Radiographic progression over 4 years between two treatment strategies was similar after the adjustment for baseline status. However, in the subgroup of patients who have syndesmophytes at baseline, it occurred significantly faster in the tapering group (1.30 mSASSS unit/year vs. 1.82 mSASSS unit/year, p=0.008) (figure). This result was concordant when radiographic progression was assessed by the development of new syndesmophytes (0.52/year vs. 0.73/year, p=0.047). Conclusions Dose tapering strategy of TNF-blockers is associated with more rapid radiographic progression in AS patients who have syndesmophytes at baseline. References Zavada J, et al. A tailored approach to reduce dose of anti-TNF drugs may be equally effective, but substantially less costly than standard dosing in patients with ankylosing spondylitis over 1 year: a propensity score-matched cohort study. Ann Rheum Dis 2016;75:96–102. Disclosure of Interest None declared

Background Interstitial lung disease (ILD) is associated with a significant morbidity and mortality in systemic sclerosis (SSc). Many studies have reported the association between human leukocyte antigen (HLA) alleles and increased risk of SSc, but the associated between ILD in SSc and HLA alleles remains unclear. Objectives The aim of this study was to investigate the association of HLA class I and HLA class II with ILD in Korean SSc cohort. Methods A total of 170 SSc patient (105 SSc patients with ILD and 65 SSc patients without ILD) were enrolled at Seoul National University Hospital from 1988 to 2014. Two hundred one healthy participants from Korean Marrow Donor Program (KMDP) were recruited as controls. Demographic information, clinical skin subset (limited vs. diffuse), SSc-specific autoantibodies (anti-topoisomerase I (ATA), anti-centromere (ACA), anti-ribonucleoprotein (RNP)), and internal organ involvement (ILD, pulmonary arterial hypertension, gastrointestinal, renal involvement) of the enrolled patients were characterized. ILD were diagnosed based on the findings of the chest computed tomography. HLA-A, -B, -C,-DQB1,-DRB1, and -DPB1 typing were performed PCR amplification with directly sequencing from extracted genomic DNA. Results HLA-B*52:01, C*12:02, DRB1*15:02, DPB1*09:01, and DPB1*13:01 alleles were more frequent in SSc patients with ILD than healthy controls. However, the frequencies of these genes did not differ between SSc patients with and without ILD (Table). ATA positive SSc was associated with HLA-B*52:01, C*12:02, DRB1*15:02, DPB1*09:01, and DPB1*13:01 alleles compared to ATA negative SSc and DPB1*09:01 showed the highest odds ratio for ATA (OR =23.08). Conclusions SSc patients with ILD have several specific HLA genes compared with healthy controls and the frequencies of these HLA genes were significantly higher in ATA-positive SSc as well. Therefore, ATA positivity and ILD are associated with ceratin HLA class I and II in SSc patients. References Zhou X, Lee JE, Arnett FC, Xiong M, Park MY, Yoo YK, et al. HLA-DPB1 and DPB2 are genetic loci for systemic sclerosis: a genome-wide association study in Koreans with replication in North Americans. Arthritis Rheum 2009;60:3807–14. Disclosure of Interest None declared

Background Adult onset of Still9s disease (AOSD) is a systemic inflammatory disease characterized by fever, arthritis, salmon pink rash and elevated serum inflammatory markers. The classical fever of AOSD is intermittent with a quotidian or double-quotidian pattern. However, a significant subset of AOSD exhibits persistently high fever with little temperature fluctuation. It remains unclear as to whether the certain fever pattern is associated with specific clinical manifestations. Objectives To investigate the association between fever pattern and clinical characteristics in patients with AOSD. Methods A total of 70 patients with AOSD who were treated as inpatient at Seoul National University Hospital from 2004 through 2015 were enrolled. Patients were grouped based on the fever curves on days 2, 3 and 4 using hierarchical clustering. The clinical and laboratory characteristics of the groups were compared. Results 70 patients were divided into 2 groups based on the fever curves. The group 1 with 14 patients had a higher mean temperature (38.1±0.4°C vs. 37.2±0.5°C, p Group 1 tended to be younger (38.4 ± 14.7 years vs. 46.1 ± 17.6 years, p=0.141) (Figure). Fever, arthritis, arthralgia and rash did not differ between them. However, group 1 tended to have more pericardial and lung involvement (42.9% v.s 23.2%, p=0.181). Group 1 had significantly lower platelet count (198,900 ± 68,000/μl vs. 312,900 ± 156,900/μl, p=0.0001), higher LDH (816.6 ± 376.4 IU/L vs. 477.5 ± 327.1 IU/L, p=0.002), higher mean ferritin level (27,004.1 ± 48,891.5 ug/mL vs. 6,852 ± 10,628.2 ug/mL, p=0.072) and d-dimer (9.5 ±7.9 ug/mL vs 3.3 ± 3.4 ug/mL) as compared to group 2, whereas white blood cell count, hemoglobin levels, CRP levels did note differ between the both groups. Group 1 tended to require longer time to a clinical remission (455.5 ± 850 days vs. 9.4 ± 115.7 days, p=0.137). Conclusions The unbiased analysis of fever reveals the presence of at least 2 distinctive fever patterns in AOSD. Spiking fever with higher daily variation was more often associated with higher inflammatory markers and coagulopathy and required longer treatment. Thus, fever pattern might be a prognostic factor in AOSD and AOSD patients with spiking fever might need more intensified treatment. Disclosure of Interest None declared

Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spatzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spatzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.

Abnormal regulation of Wnt/β-catenin signaling followed by increased levels of the β-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit β-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the β-catenin mRNA. We designed a strategy to identify the cleavage sites in β-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave β-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of β-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited β-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to β-catenin significantly reduced β-catenin–dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against β-catenin might have potential as a therapeutic intervention to treat colon cancer. Mol Cancer Ther; 9(6); 1894–902. ©2010 AACR.

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

Background Polymyositis (PM) and dermatomyositis (DM) is known to be associated with certain HLA alleles in several ethnic groups including Caucasians, African-Americans, and the Japanese. Objectives To investigate the profile of HLA class II alleles in Korean patients with PM and DM Methods Sequencing based genotyping for HLA-DRB1 and -DPB1 was performed in 140 Korean patients with DM (n=104) or PM (n=36) and in 207 healthy controls. Associations of each HLA-DR or -DP allele with myositis or clinical subsets of myositis were examined. Results HLA-DRB1*14:54 (odds ratio [95% confidence interval] 1.05 [1.01-1.09]), -DRB1*15:02 (2.44 [1.03-5.81]), -DRB1*16:02 (1.04 [1.00-1.07], and -DPB1*09:01 (2.44 [1.03-5.81]) were found to be susceptibility alleles while HLA-DRB1*14:01 (0.93 [0.90-0.97]) and -DRB1*15:01 (0.38 [0.18-0.79]) to be protective alleles for inflammatory myositis (Table 1). In DM, HLA-DRB1*15:02 (3.14 [1.30-7.62]) and -DPB1*09:01 (2.87 [1.17-7.05]) carriers were more frequent while HLA-DRB1*14:01 (0.93 [0.90-0.97]), -DRB1*15:01 (0.30 [0.12-0.74]), and -DPB1*04:02 (0.44 [0.20-0.94]) carriers were less frequent than controls. In PM, HLA-DRB1*14:03 (6.52 [1.78-23.81]), and -DRB1*14:54 (1.13 [1.00-1.23]) carriers were more frequently observed. When PM and DM were compared, HLA-DRB1*14:03 carriers were more frequently observed in PM (5.43 [1.23-24.02]). When the association between each allele and clinical subsets was examined, patients with interstitial lung disease (ILD) carried HLA-DRβ1*04:03 (6.34 [1.35-29.76]), -DRβ1*07:01 (0.22 [0.08-0.64]), -DRβ1*09:01 (0.16 [0.05-0.60]) -DRβ1*12:02 (2.91 [1.16-8.01]) DRβ1*15:01 (0.10 [0.01-0.80]), -DPβ1*14:01 (8.90 [1.08-73.21]), and -DPβ1*17:01 (0.11 [0.01-0.92]) more frequently than patients without. DM patients with ILD showed higher frequency of HLA-DRβ1*04:05 than DM patients without ILD (4.68 [1.24-17.74]). Other associations included dysphagia with HLA-DRβ1*08:02 (6.73 [1.51-30.00]), -DRβ1*14:54 (5.19 [1.09-24.57]), and -DPβ1*03:01 (5.19 [1.09-24.57]), arthralgia with HLA-DRβ1*03:01 (4.79 [1.21-18.98]) and -DPβ1*02:01 (2.30 [1.10-4.82]), and mechanic9s hand with HLA-DRB1*08:03 (3.25 [1.12-9.45]) and -DRB1*12:02 (5.04 [1.66-15.29]). Regarding skin rashes, heliotrope rash was associated with HLA-DRβ1*09:01 (2.98 [1.05-8.46]) and -DPβ1*05:01 (0.41 [0.18-0.87]), Gottron9s papules with HLA-DRβ1*14:05 (8.77 [1.07-72.17]), -DPβ1*02:01 (2.29 [1.11-4.73]), -DPB1*02:02 (0.086 [0.01-0.67]), and -DPB1*05:01 (0.41 [0.20-0.85]), and shawl sign with HLA-DRB1*14:05 (4.67 [1.15-18.99]) and -DPB1*14:01 (4.67 [1.15-18.99]). Cancer development was associated with HLA-DRB1*12:01 (7.25 [1.65-31.89]) and -DRB1*13:02 (0.81 [0.74-0.88]). No HLA-DRB1 or -DPB1 allele was found to be associated with anti-Jo1 antibody. Conclusions Korean patients with inflammatory myositis showed a unique profile of HLA-DR and -DP alleles compared with other ethnic groups. Alleles associated with disease susceptibility were different between PM and DM. Clinical subsets, particularly ILD, showed different immunogenetic backgrounds. Disclosure of Interest None declared

Background Chemokines are multifunctional mediators that control leukocyte recruitment into the inflammatory sites and enhance immune responses. It remains to be investigated which chemokines are important in Behcet9s disease (BD). Objectives The objective of this study was to investigate serum levels of neutrophil and lymphocyte chemoattractants in BD patients and its association with disease activity and symptoms. Methods We collected sera from patients with BD (n=64) and age-, sex matched healthy controls (n=21). Serum levels of chemokines were assayed for the neutrophil chemoattractants (CXCL1 and CXCL8) and lymphocyte chemoattractants (CXCL9, CXCL10, CXCL12, CXCL13 and CXCL16) by using a multiplex assay. Behcet9s disease current activity form (BDCAF) and symptoms were evaluated at the time of blood collection. Results Serum levels of CXCL8, CXCL10 and CXCL12 were significantly higher in the BD patients than in healthy controls (p Conclusions Serum levels of CXCL8, CXCL10 and CXCL12 were significantly higher in the BD patients than in healthy controls. CXCL10 levels were correlated with number of EN in BD patients. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and internal organs. Although interstitial lung disease (ILD) is a major cause of death in this disease, pulmonary function remains stable in a subset of SSc patients with ILD without any treatment. However, clinical characteristics in this subset of patients are not well characterized. Objectives To investigate clinical characteristics of SSc patients with ILD who do not require additional immunosuppressive treatment for ILD. Methods A total of 151 SSc patients with ILD who received medical care at Seoul National University Hospital between 1978 and 2013 were enrolled in this study. ILD was diagnosed based on chest computed tomography (CT) and the extent of ILD was semi-quantitatively graded into 1-4 (grade 1, 75% of lung involvement). Detailed baseline clinical characteristics were obtained from medical record review. Mortality was ascertained from medical record review and data from the Statistics Korea. After confirming survival benefit of untreated group versus treated group with Kaplan Meier analysis, clinical characteristics of untreated group were defined by comparison with treated group (chi-square test or Student t-test). Sensitivity test was performed by proving survival benefit of patients with the defined characteristics in the untreated group (log-rank test). Results The mean (standard deviation) age at diagnosis of 151 patients was 48.7 (12.9) years and 88.7% were women. Eighty (52.9%) patients did not receive immunosuppressive treatment for ILD, while 71 (47.0%) patients required immunosuppressive treatment, which includes cyclophosphamide (n=46), azathioprine (n=8), glucocorticoids (n=14) and others (n=3). Interestingly, median survival of non-treatment group was significantly better than treatment group during the follow-up of 1300.80 person-years (p=0.029 by log-rank test). Compared with treatment group, non-treatment group had higher baseline predicted % of forced vital capacity (FVC, 79.2±16.8% vs. 68.0±18.2, p Conclusions Watch and wait approach may be applied to patients with SSc-associated ILD with minimal pulmonary infiltrates on CT scan and absence of pulmonary arterial hypertension at ILD diagnosis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3686

Background Interstitial lung disease (ILD) is the leading cause of death in patients with systemic sclerosis (SSc). Treatment with cyclophosphamide (CYC) for 1 year has been shown to preserve pulmonary function. However, long-term efficacy and safety of CYC treatment for more than 1 year remains to be known. Objectives To investigate efficacy and tolerability of long-term CYC treatment in SSc patients with ILD. Methods All of the SSc-associated ILD patients who have been treated with CYC for more than 1 year at Rheumatology clinic of Seoul National University Hospital between 1978 and 2013 were enrolled. The information on clinical characteristics and pulmonary function test results were obtained from medical record review. Chest computed tomography (CT) was reviewed by a radiologist and classified into 4 grades depending on the extent of lung involvement. Mortality data were obtained from medical record or mortality record from Statistics Korea. Results A total of 32 SSc patients with ILD were treated with CYC for more than 1 year with the mean treatment duration of 28.0 months (range: 12.0- 70.5 months). The mean age at diagnosis was 47.5±9.4 years and women were dominant at 84.4%. The mean functional vital capacity (FVC) at baseline was 66.0±16.0% of predicted. At baseline 18 (64.3%) patients had CT grade of 2 or more. After mean (standard deviation) follow up period of 4.1 (3.1) years, FVC remained stable with the mean increase of 2.3 (2.5) % (P=0.365, by paired t test). Furthermore, the degree of pulmonary involvement on CT did not progress. Six patients died during the follow-up; pneumonia (n=1), pulmonary hypertension (n=1), subdural hemorrhage (n=1), heart failure (n=2) and malignancy (n=1). Adverse events during CYC treatment included severe infection (n=4), cytopenia (n=4), hemorrhagic cystitis (n=1) and alopecia (n=3). Conclusions Prolonged treatment with CYC for more than 1 year is associated with stabilization of ILD in the respect of pulmonary function and extent of lung involvement on CT. Long-term maintenance treatment with CYC may be an option to treat severe ILD in SSc patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4302

Background Interstitial lung disease (ILD) is the most common cause of mortality in patients with dermatomyositis (DM). Cyclosporine has been reported to improve clinical outcome in some patients with DM-associated ILD. Objectives To investigate the benefit of early introduction of cyclosporine on the survival of patients with DM-associated ILD. Methods All the patients with DM-associated ILD, who were treated with cyclosporine at Seoul National University Hospital (SNUH) and Seoul National University Bundang Hospital (SNUBH) between 1990 and 2013, were enrolled. Enrolled patients were diagnosed with DM according to Bohan-Peter9s classification criteria (n=28) or with clinically amyopathic dermatomyositis (CADM) according to Sontheimer9s classification criteria (n=19). ILD was diagnosed based on the typical findings on computed tomography (CT) and relevant respiratory symptoms or signs. Kaplan-Meier survival analysis was applied to compare overall mortality rates between patients who took cyclosporine as the initial treatment and those as the delayed treatment. Cox regression analysis was used to estimate the benefit of early treatment with cyclosporine after adjusting confounding characteristics. Results Among the 47 patients who were diagnosed with DM-associated ILD, 16 patients (34.04%) received cyclosprorine initially after diagnosis and 31 patients (65.96%) received cyclosporine after the trial of other immunosuppressants such as corticosteroids, cyclophosphamide or azathioprine. The mean time of follow-up was 43.57 person-years in initial treatment group and 76.23 person-years in delayed treatment group. The mortality rate was significantly lower in the initial treatment group than the delayed treatment group (0.02 person-years vs 0.18 person-years, p=0.0092 by log-rank test). The two groups did not differ in regard to age, sex, duration of disease, presence of autoantibodies, degree of dyspnea at initial visit, initial requirement of oxygen supplement, functional vital capacity (FVC) and diffusion capacity (DLCO) on pulmonary function test (PFT) and the presence of malignancy. Only the proportion of patients with CADM was higher in the initial treatment group than in the delayed treatment group (62.53% vs 29.03%, p=0.027). Survival benefit in the initial cyclosporine treatment remained significant even after adjusting for the CADM status (HR 0.075, 95% CI 0.009 - 0.603, p=0.015 by Cox-regression analysis). Conclusions This study showed significant survival benefit of initial cyclosporine treatment in patients with DM-associated ILD. A randomized controlled study is warranted to clearly demonstrate the therapeutic benefit of early use of cyclosporine in this potentially fatal disease. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3718

Background Interstitial lung disease (ILD) is a common cause of death in patients with dermatomyositis (DM). Especially, patients with clinically amyopathic DM (CADM) have a worse outcome due to ILD which is often refractory to corticosteroids. Lung involvement as an additional target of inflammation increases the total inflammatory burden in patients with DM. Objectives To investigate whether erythrocyte sedimentation rate (ESR) elevation is associated with ILD and a worse prognosis in patients with DM. Methods All patients with DM including CADM receiving clinical care in our institution from January 2004 through June 2013 were enrolled. Patients who had shortness of breath or abnormal finding on chest radiograph underwent a high resolution computed tomography (HRCT) and pulmonary function test (PFT). ILD was diagnosed when HRCT showed typical findings compatible with ILD. Results A total of 114 DM patients were enrolled in this study. The mean age of patients at diagnosis was 49.4±12.3 years. 69.3% were female. The mean follow-up duration was 32.3±29.3 months. ILD was present in 53 (46.5%) patients. Among 28 patients with CADM, 14 (50%) had ILD. The levels of ESR were significantly higher in patients with ILD than those without ILD (49.9±23.3 vs. 34.4±24.1 mm/hr, p=0.001). The difference was more profound between the CADM patients with ILD and those without ILD (56.3±23.8 vs. 25.4±22.9 mm/hr, p=0.002). The ESR levels were inversely correlated with the forced vital capacity (r=−0.238, p=0.036) and diffusing capacity of carbon monoxide (r=−0.334, p=0.004). Further, the patents with baseline ESR ≥30mm/hr had a worse survival as compared to those with ESR Conclusions The ESR elevation at the time of DM diagnosis is associated with pulmonary impairment and a poor outcome in patients with DM. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3276

Relapsing polychondritis is an episodic, systemic inflammation of the cartilage with unknown autoimmune etiology. It leads to the destruction of cartilaginous structures of the ear, eye, nose, respiratory tract, joints, skin, and heart valve, and its presented symptoms are diverse. It can be improved mainly by corticosteroid or immunosuppressive agents. Recently, the use of biologic agents (TNF inhibitors, rituximab, tocilizumab et al) was reported from abroad. However, there is no reported case of relapsing polychondritis, which is treated by biologic agents in Korea. We report this first case of refractory relapsing polychondritis, which was improved with a treatment of Infliximab.

Correspondence to Eun Young Lee, M.D., Ph.D. Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea Tel: +82-2-2072-0852, Fax: +82-2-762-9662, E-mail: elee@snu.ac.kr Copyrightc 2013 The Korean Association of Internal Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 피부근염에 동반된 혈전혈소판감소자색반병에서 리툭시맙 치료 1예

Objective. To assess the efficacy and safety of rituximab (RTX) on disease activity and muscle strength in patients with inflammatory myopathies refractory to conventional therapy. Methods. Four inflammatory myopathy patients who had been refractory to glucocorticoids, one or more immunosuppressive therapies and intravenous immunoglobulin were treated on an open-label basis. Each patient received two 500 mg doses of RTX 2 weeks apart in one cycle. In one patient who did not respond after the first cycle of RTX, the infusion schedule was modified by the physician. We measured muscle enzyme including CPK, LDH and assessed muscle strength individually to evaluate RTX response. Additionally anti-CD19 antibody was measured. Results. Three patients responded to the first cycle of RTX treatment with improvements in muscle enzyme and muscle strength, and then maintained physical function over the duration of several infusion cycles. In one patient, muscle enzyme did not decrease after the first cycle of RTX, and a high dose glucocorticoid was given. After modifying the treatment schedule with monthly RTX infusion, his muscle enzyme level and muscle strength improved. Anti-CD19 antibody decreased after RTX generally, but responses were variable. Herpes zoster infection occurred in two patients. Conclusion. Rituximab may be a therapeutic choice in refractory inflammatory myopathy. However a further trial is needed to confirm the efficacy and prove the safety.

Peripheral nervous system dysfunction is a rare complication in Henoch-Schonlein purpura, but it tends to recover spontaneously without treatment. A 78-year-old man who had ankylosing spondylitis presented with Henoch-Schonlein purpura associated with progressive sensorimotor polyneuropathy. He was diagnosed with chronic inflammatory demyelinating polyneuropathy, which did not improve despite intravenous immunoglobulin therapy. We describe a case of Henoch-Schonlein purpura, accompanied by chronic inflammatory demyelinating polyneuropathy in a patient with ankylosing spondylitis. (Korean J Med 2012;83:538-542)

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by beta-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation.