Shuichi Nakayama

Regulation of Glucocorticoid Receptor Transcription and Nuclear Translocation during Single and Repeated Immobilization Stress

We have previously reported reduced glucocorticoid receptor (GR) mRNA levels in the hippocampus and hypothalamic paraventricular nucleus (PVN) during repeated immobilization, which is potentially associated with persistent activation of the hypothalamic-pituitary-adrenocortical axis. We used in situ hybridization and Western blot to examine the transcriptional regulation of the GR gene, GR nuclear translocation, and expression of cytosolic heat shock protein 90 (hsp90), a chaperone protein essential for GR nuclear translocation, in the hippocampus, PVN, and anterior pituitary (AP) during single immobilization (sIMO) and the final immobilization on d 7 after daily IMO for 6 days (rIMO). As with GR mRNA, GR heteronuclear RNA levels decreased in the hippocampus and PVN and increased in the AP during sIMO and rIMO, indicating that the GR mRNA levels in these regions were regulated at the transcriptional level. In both sIMO and rIMO, nuclear GR levels were significantly increased in the hippocampus, medial basal hypothalamus (MBH), and AP. However, GR nuclear translocation was reduced in the hippocampus, unchanged in the MBH, and enhanced in the AP during rIMO, as compared with sIMO. Cytosolic hsp90 expression was unchanged in the hippocampus and MBH, whereas it significantly increased in the AP at 30 min during rIMO but not during sIMO. These results suggest that the site-specific changes in GR nuclear translocation during sIMO vs. rIMO are partially linked to hsp90 responses to immobilization. The reduced nuclear translocation of GR in the hippocampus during rIMO may reflect decreased glucocorticoid-mediated negative feedback on the hypothalamic-pituitary-adrenocortical axis.

Glucocorticoid Receptor-β and Receptor-γ Exert Dominant Negative Effect on Gene Repression But Not on Gene Induction

Glucocorticoid has diverse biological effects through induction or repression of its target genes via glucocorticoid receptor (GR). In addition to the wild-type GR (GR-alpha), a variety of GR variants has been reported, and these are thought to modify glucocorticoid action. Among others, GR-beta is reported be responsible for the glucocorticoid resistance frequently observed in steroid-resistant nephrotic syndrome, rheumatoid arthritis, and hematologic tumors, although the precise molecular mechanism remains unclear. In this study, we examined the function of GR-beta and some GR variants (GR-gamma and GR-Delta313-338) using GR-deficient BE(2)C and T84 cells in vitro. We found that GR-beta, when expressed alone, completely lost the capacity of both trans-activation and trans-repression on GR target genes. Interestingly, however, GR-beta showed a dominant-negative effect on GR-alpha only for its trans-repressive effects on cAMP-mediated and cAMP response element-dependent genes. Furthermore, both GR-beta and GR-gamma had dominant-negative effects on GR-alpha selectively for its trans-repressive effects on nuclear factor-kappaB-mediated and inflammation-related genes. These results suggest that 1) the GR-beta variant by itself has no receptor function, but 2) GR-beta and GR-gamma have properties to exert dominant-negative effects on the GR-alpha-mediated trans-repression, which may be responsible for the steroid resistance frequently observed in chronic inflammatory diseases under glucocorticoid therapy.

Liver X Receptor α Is Involved in the Transcriptional Regulation of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase Gene

The activity of 6-phosphofructo-1-kinase is strictly controlled by fructose-2,6-bisphosphate, the level of which is regulated by another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2). PFK2/FBP2 is a bifunctional enzyme, having kinase and phosphatase activities, and regulates both glycolysis and gluconeogenesis. Here, we examined the hormonal regulation of the PFK2/FBP2 gene in vitro using the reporter assay, the electromobility shift assay (EMSA), and the chromatin immunoprecipitation (ChIP) assay in HuH7 cells and also using the mouse liver in vivo. We found that the transcriptional activity of the PFK2/FBP2 gene was stimulated by insulin and inhibited by cAMP and glucocorticoid. Liver X receptor (LXR) α showed a potent and specific stimulatory effect on PFK2/FBP2 gene transcription. Deletion and mutagenesis analyses identified the LXR response element (LXRE) in the 5′-promoter region of the PFK2/FBP2 gene. Binding of LXRα was confirmed by the EMSA and ChIP assay. Endogenous PFK2/FBP2 mRNA in the mouse liver was increased in the fasting/refeeding state compared with the fasting state. Altogether, PFK2/FBP2 gene transcription is found to be regulated in a way that is more similar to other glycolytic enzyme genes than to gluconeogenic genes. Furthermore, our data strongly suggest that LXRα is one of the key regulators of PFK2/FBP2 gene transcription.

Clinical implications of ultrasonography in monitoring disease activity of relapsing polychondritis

A 78-year-old man presented with a 2-month history of hearing loss, auricular pain and oligoarthralgia. Thickening, deformity and tenderness of the bilateral ear auricles with sparing of the ear lobules (Fig. 1A) were revealed. Ultrasonography of the auricle (Fig. 1B and C) showed low-echoic swollen auricular cartilage (arrows) with increased power Doppler signals (arrowheads). Biopsy specimens showed degeneration of the cartilages, with infiltration of inflammatory cells (Fig. 1D) and increased capillary vascularization (Fig. 1E). Biopsy findings corresponded to the above-mentioned US findings, and thus he was diagnosed with relapsing polychondritis. After treatment with prednisolone combined with MTX, the auricular swelling resolved completely (Fig. 1F), and US findings (Fig. 1G and H) also showed dramatic reductions in swelling of cartilage (arrows) with a decrease in the power Doppler signal (arrowheads). US imaging can be used to differentiate between inflammation, vascular lesions and tumours in the ear pinna [1]. Relapsing polychondritis could be differentiated from the damage of repeated trauma (i.e. rugby) with production of a subperichondrial serous effusion [1]. As in the present case, US imaging of the external ear and auricular cartilage in relapsing polychondritis also facilitates evaluation of auricular lesions and monitoring of disease activity, especially when we consider the treatment response and the timing of drug tapering.

Lupus nephritis with positive myeloperoxidase/proteinase 3-antineutrophil cytoplasmic autoantibody that developed after 17 years of propylthiouracil therapy

Dear Editor, Propylthiouracil (PTU) is a thiouracil-derived drug used to treat hyperthyroidism. A number of adverse effects have been reported in association with this drug, including fever, agranulocytosis, skin rash, and vasculitis. Increasing evidence has recently suggested that PTU can induce autoimmune syndromes with the production of antinuclear antibodies or antineutrophil cytoplasmic autoantibody (ANCA) in patients with Graves’ disease. We herein report a patient who developed lupus nephritis with positive myeloperoxidase (MPO)/proteinase 3 (PR3)-ANCA after taking PTU for 17 years. A 51-year-old man with a history of Graves’ disease, treated with PTU at 50 mg/day for approximately 17 years, presented to another institution with persistent high fever, polyarthralgia, and foot edema and was subsequently referred to our hospital. Upon presentation, he had thrombocytopenia (platelet count, PLT: 3.8 9 10/lL), leukocytopenia (WBC: 2.9 9 10/lL with 47 % neutrophils), microhematuria ([100 red blood cells per highpower field), proteinuria (1.01 g/day, peak value 4.26 g/ day), and an elevated serum creatinine level (1.42 mg/dL, peak value 5.66 mg/dL; Fig. 1). Thyroid function tests were within the normal range. Serological examinations showed a high titer of antinuclear antibodies (1:320, speckled pattern), positive MPO-ANCA (140.0 U/mL), positive PR3-ANCA (19.5 U/mL), positive double-stranded DNA antibody (Ds-DNA IgG Ab: 19.1 IU/mL), positive single-stranded DNA antibody (Ss-DNA IgG Ab: 140.0 IU/mL), and hypocomplementemia (C3: 41.7 mg/dL, C4: 7.1 mg/dL, and CH50: 13.9 U/mL). Renal biopsy showed mesangial proliferative glomerulonephritis, and immunofluorescence revealed positive staining for IgG, IgM, C3, and C1q. The pathological finding of a crescentic lesion indicating ANCA-associated renal vasculitis was not seen. Based on these findings, ANCA-positive lupus nephritis (WHO class II) was diagnosed. PTU treatment was discontinued, and he was effectively treated with plasmapheresis and hemodialysis combined with two pulses of cyclophosphamide and three pulses of methylprednisolone, prednisolone, and intravenous immunoglobulin therapy. After 3 months, his thrombocytopenia, proteinuria, and MPO/PR3-ANCA titer were significantly improved (Fig. 1). Potassium iodide and radioiodine therapy was administered to treat Graves’ disease. At the current time point of 2-years follow-up with low-dose prednisolone, his serum creatinine level is 1.28 mg/dL with no proteinuria and microhematuria. MPO-ANCA is 12.5 IU/mL (normal range is \3.5 IU/ml) without any remarkable findings of vasculitis, and Ds-DNA/Ss-DNA IgG Ab and complement titers are within the normal range. While the renal biopsy showed lupus nephritis, this patient had overlapping clinical and laboratory findings of lupus and vasculitis. In a previous study, ANCA was detected in 37.3 % of patients with idiopathic systemic lupus erythematosus, and patients with this condition are almost always positive for MPO-ANCA [1]. In addition, MPO-ANCA is found in a similar proportion of patients with PTU-induced lupus and patients with vasculitis; however, PR3-ANCA is detected only in patients with PTU-induced vasculitis [2]. In the present case, the regular long-term use of PTU and overlapping clinical findings of lupus and vasculitis with positive PR3-ANCA highly indicated that this patient developed an autoimmune T. Taguchi (&) S. Nakayama S. Fujimoto Y. Terada Department of Endocrinology, Metabolism and Nephrology, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku 783-8505, Japan e-mail: ttagu27k@axel.ocn.ne.jp

THU0302 Clinical Characteristics of Japanese Patients with Reactive Arthritis Induced by Intravesical BCG Therapy for Bladder Cancer: A Retrospective Two-Center Study

Background Intravesical instillation of BCG is used as an effective immunotherapy of bladder cancer. However it may have, as adverse event, a reactive arthritis (ReA) and the incidence are known as about 0.5 to 1%. Objectives To evaluate clinical characteristics and incidence of Japanese patients with ReA induced by intravesical BCG therapy (iBCG) for bladder cancer. Methods The clinical symptoms, laboratory, and imaging findings of Japanese patients who received iBCG (n=405 (134 and 271 in Kochi University Hospital (KUH) and Kurashiki Medical Center (KMC), respectively)) for bladder cancer from March 1997 to October 2012 were retrospectively assessed. Especially, the patients with ReA and conjunctivitis/uveitis were examined. All data are presented as mean ± SD. Results Of the patients received iBCG (age 71±10 and 69±11; male/female 95/39 and 215/56 in KUH and KMC, respectively), 40 (30%) and 35 (13%), 41 (31%) and 48 (18%), and 59 (44%) and 73 (27%) in KUH and KMC presented fever, hematuria and painful urination, respectively. ReA was revealed in 3/134 (2.2%) and 3/271 (1.1%), uveitis in 3/134 (2.2%) and 1/271 (0.4%), and conjunctivitis in 12/134 (8.9%) and 14/271 (5.2%) in KUH and KMC, respectively. As the total evaluation, ReA was revealed in 6/405 (1.5%). Moreover, most patients with ReA also had hepatic dysfuntion. All ReA were developed after 3-times of iBCG. Clinical, ultrasound and FDG-PET/CT findings of ReA induced by iBCG showed asymmetric polyarthritis/polyenthesitis pattern in shoulder, sternoclavicular joints, spinous process, sacroiliac joints, ischial tuberosity, hip, knee and ankle. Laboratory examinations showed high CRP (>10mg/dl) and 33% HLA-B27 positivity (in 1 of 3 cases). All ReA patients were improved by treatments with prednisolone and isoniazid. Conclusions The incidence of ReA induced by iBCG in Japanese population was 1.5% and it might be almost equal or more than the incidence, 0.5 to 1%, in the Western countries from previous reports. Positive HLA-B27 was revealed in 33% of ReA patients as background in our study, suggesting lower frequency than 51 to 55% in the Western countries. Therefore, besides HLA phenotype, a cross reaction between a mycobacterium epitope and an antigen of joint cartilage might be suggested in the pathogenesis of ReA induced by iBCG in Japan. Acknowledgements No funding support Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4230

AB0571 The study of the clinical characteristics between late-onset and early-onset spondyloarthritis

Background Spondyloarthritis (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis (ReA), enteropathic arthritis, undifferentiated spondyloarthritis (uSpA), and synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome, often involves the entheses. The differences between early-onset and late-onset SpA are poorly understood. Objectives To evaluate comparatively clinical characteristics, including positron emission tomography/computed tomography (PET/CT) using 18F-fluorodeoxyglucose (FDG) findings, of patients with between both early-onset and late-onset SpA in our hospital. Methods We detected SpA group that developed in less than 45 years old as early-onset SpA (EOSpA), and in more than 65 years old as late-onset SpA (LOSpA). The clinical symptoms, laboratory findings and the results of PET/CT scans of twenty patients with EOSpA and twenty LOSpA were comparatively examined. Results LOSpA had fever, body weight loss and severe systemic symptoms. Three of LOSpA (3/20) developed PMR-like symptoms. LOSpA showed significantly higher inflammatory reaction than EOSpA. Images of PET/CT scans revealed widespread FDG accumulations at the entheses in LOSpA patients, in comparison with EOSpA. The maximum SUVs were statistically higher in LOSpA patients compared EOSpA patients at the entheses (P < 0.05). Furthermore, three of LOSpA (3/20) had peripheral edema in lower extremities, and it pathologically showed panniculitis. There were no significant differences in HLA typing between EOSpA and LOSpA. Conclusions LOSpA revealed severe clinical symptoms, inflammatory reactions, and widespread and strong enthesitis and arthritis compared with EOSpA. It is important to differentiate SpA, RS3PE syndrome and PMR in cases with peripheral edema, myalgia and pantalgia. We must investigate further cases of LOSpA in order to elucidate its pathophysiology and mechanisms of development. Disclosure of Interest None Declared