Macrophage activation syndrome triggered by methotrexate-related lymphoproliferative disease in a patient with rheumatoid arthritis.

A 56-year-old woman was treated for rheumatoid arthritis for 17 years with methotrexate (MTX). Night sweats, fever and weight loss made her visit our hospital. Although levofloxacin failed to resolve her fever, she was suspected of having sepsis because of pancytopenia, elevated procalcitonin and a nodular lesion in the lung. After urgent hospitalization, she was diagnosed finally with the methotrexate-related lymphoproliferative disorder (MTX-LPD) associated with macrophage activation syndrome (MAS). Her general condition was improved with MTX withdrawal and 5-day high-dose glucocorticoid administration. Thus, even when the patient was critically ill with MAS, no cytotoxic agents were required to control MTX-LPD.

Leucine-rich alpha-2 glycoprotein as a potential biomarker for large vessel vasculitides.

Objectives: Serum levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been used as useful biomarkers for reflecting the activity of large vessel vasculitides (LVV). However, a novel biomarker that could have a complementary role to these markers is still required. In this retrospective observational study, we investigated whether leucine-rich α-2 glycoprotein (LRG), a known biomarker in several inflammatory diseases, could be a novel biomarker for LVVs. Methods: 49 eligible patients with Takayasu arteritis (TAK) or giant cell arteritis (GCA) whose serum was preserved in our laboratory were enrolled. The concentrations of LRG were measured with an enzyme-linked immunosorbent assay. The clinical course was reviewed retrospectively from their medical records. The disease activity was determined according to the current consensus definition. Results: The serum LRG levels were higher in patients with active disease than those in remission, and decreased after the treatments. While LRG levels were positively correlated with both CRP and erythrocyte sedimentation rate, LRG exhibited inferior performance as an indicator of disease activity compared to CRP and ESR. Of 35 CRP-negative patients, 11 had positive LRG. Among the 11 patients, two had active disease. Conclusion: This preliminary study indicated that LRG could be a novel biomarker for LVV. Further large studies should be required to promise the significance of LRG in LVV.

Branched chain amino acids in the treatment of polymyositis and dermatomyositis: a phase II/III, multi-centre, randomized controlled trial.

OBJECTIVES: To assess the efficacy and safety of branched chain amino acids (BCAAs) in the treatment of PM/DM prior to official approval of their use in Japan. METHODS: Treatment naïve adults with PM/DM were enrolled in a randomized, double-blind trial to receive either TK-98 (drug name of BCAAs) or placebo in addition to conventional treatment. After 12 weeks, patients with an average manual muscle test (MMT) score <9.5 were enrolled in an open label extension study for a further 12 weeks. The primary endpoint was the change of the MMT score at 12 weeks. The secondary endpoints were the clinical response and the change of functional index (FI). RESULTS: Forty-seven patients were randomized either to the TK-98 (n = 24) or placebo (n = 23) group. The changes of MMT scores at 12 weeks were 0.70 (0.19) [mean (s.e.m.)] and 0.69 (0.18), respectively (P = 0.98). Thirteen patients from the TK-98 group and 12 from the placebo group were enrolled in the extension study. The MMT scores in both groups improved similarly. The increase of the FI scores of the shoulder flexion at 12 weeks was significantly greater in the TK-98 group [27.9 (5.67) vs 12.8 (5.67) for the right shoulder flexion, and 27.0 (5.44) vs 13.4 (5.95) for the left shoulder; P < 0.05]. Frequencies of adverse events up to 12 weeks were similar. CONCLUSION: BCAAs showed no effect on the improvement of the muscle strength evaluated by MMT and the clinical response. However, they were partly effective for improving dynamic repetitive muscle functions. TRIAL REGISTRATION: UMIN-CTR Clinical Trial, https://center6.umin.ac.jp/, UMIN000016233.

Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies.

Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.

Therapeutic Effect of Cyclin-Dependent Kinase 4/6 Inhibitor on Dermal Fibrosis in Murine Models of Systemic Sclerosis.

OBJECTIVE: One of the histologic characteristics of systemic sclerosis (SSc) is an increased number of dermal myofibroblasts, and transforming growth factor ß (TGFß) plays a crucial role in the promotion of myofibroblast differentiation from fibroblasts, leading to dermal fibrosis. This study was undertaken to 1) examine whether inhibition of the cell cycle with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor suppresses the proliferation of fibroblasts and their differentiation into myofibroblasts, and 2) assess the therapeutic effects of a CDK4/6 inhibitor, administered as monotherapy or in combination with a TGFß receptor (TGFßR) inhibitor, on dermal fibrosis in murine models of SSc. METHODS: Fibroblasts obtained from the skin of patients with SSc were cultured in the presence or absence of TGFß. The effects of palbociclib, a CDK4/6 inhibitor, on fibroblast proliferation and TGFß-induced differentiation into myofibroblasts were examined using bromodeoxyuridine uptake assays as well as immunofluorescence and immunoblotting analyses. Murine models of HOCl- and bleomycin-induced dermal fibrosis were used to study the effect of a CDK4/6 inhibitor on dermal fibrosis, with the CDK4/6 inhibitor treatment administered as monotherapy or in combination with galunisertib, a TGFßR inhibitor. RESULTS: Addition of a CDK4/6 inhibitor to the cell cultures suppressed the proliferation of human dermal SSc fibroblasts and their TGFß-induced differentiation into myofibroblasts, without inhibiting canonical and noncanonical TGFß signals. In murine models of dermal fibrosis, treatment of mice with a CDK4/6 inhibitor decreased dermal thickness and collagen content, as well as dermal fibroblast proliferation and the numbers of myofibroblasts. Combination therapy with the CDK4/6 inhibitor and TGFßR inhibitor resulted in additive antifibrotic effects. Mechanistically, the CDK4/6 inhibitor suppressed the expression of cellular communication network 2 and cadherin-11, which are proteins that have important roles in the development and progression of fibrosis. CONCLUSION: Results of this study demonstrate the therapeutic effect of a CDK4/6 inhibitor on dermal fibrosis when administered as monotherapy or in combination with a TGFßR inhibitor. CDK4/6 inhibitors, including palbociclib used in the present study, may represent novel agents for the treatment of SSc, which, if used in combination with a TGFßR inhibitor, might result in increased efficacy.

Chondroprotective effects of CDK4/6 inhibition via enhanced ubiquitin-dependent degradation of JUN in synovial fibroblasts.

OBJECTIVE: Targeting synovial fibroblasts (SF) using a cyclin-dependent kinase (CDK) 4/6 inhibitor (CDKI) could be a potent therapy for RA via inhibition of proliferation and MMP-3 production. This study was designed to elucidate the mechanism of chondroprotective effects on SFs by CDK 4/6 inhibition. METHODS: CDK4/6 activity was inhibited using CDKI treatment or enhanced by adenoviral gene transduction. Chondroprotective effects were evaluated using a collagen-induced arthritis model (CIA). Gene and protein expression were evaluated with quantitative PCR, ELISA and Western blotting. The binding of nuclear extracts to DNA was assessed with an electrophoresis mobility shift assay. RNA-Seq was performed to identify gene sets affected by CDKI treatment. RESULTS: CDKI attenuated cartilage destruction and MMP-3 production in CIA. In RASFs, CDKI impaired the binding of AP-1 components to DNA and inhibited the production of MMP-1 and MMP-3, which contain the AP-1 binding sequence in their promoter. CDK4/6 protected JUN from proteasome-dependent degradation by inhibiting ubiquitination. The RNA-Seq analysis identified CDKI-sensitive inflammatory genes, which were associated with the pathway of RA-associated genes, cytokine-cytokine receptor interaction and IL-17 signalling. Notably, the AP-1 motif was enriched in these genes. CONCLUSION: The mechanism of chondroprotective effects by CDK4/6 inhibition was achieved by the attenuation of AP-1 transcriptional activity via the impaired stability of JUN. Because the pharmacologic inhibition of CDK4/6 has been established as tolerable in cancer treatment, it could also be beneficial in patients with RA due to its chondroprotective and anti-inflammatory effects.

The validity of the claims-based definition of rheumatoid arthritis evaluated in 64 hospitals in Japan.

BACKGROUND: An administrative database covering a whole population such as the national database in Japan may be used to estimate the nationwide prevalence of diseases including rheumatoid arthritis (RA) when a well-validated definition of the disease is available. In Japan, the record linkage between the administrative database and medical charts in hospitals is strictly prohibited. A "hospital-based" validation study is one of few possible validation studies where claims kept inside the study hospital are rearranged into the database structure. METHODS: We selected random samples of 19,734 patients from approximately 1.6 million patients who received medical care between February 2018 and January 2019 in one of the 64 hospitals of the Tokushukai Medical Group. We excluded patients whose observation period was less than 365 days and identified 334 patients who met the definition of "possible cases of RA" whose medical charts were then independently evaluated by two rheumatologists. In a sensitivity analysis, we assessed bias due to misclassifying some patients with RA who did not meet the definition of "possible cases of RA" as a patient with no RA. RESULTS: The kappa coefficient between the two rheumatologists was 0.80. The prevalence of RA in the study population was estimated to be 0.56%. We found that [condition code of RA] and ([any disease-modifying antirheumatic drug] or [oral corticosteroid with no systemic autoimmune diseases (other than RA) and no polymyalgia rheumatica]) had a relatively high sensitivity (approximately 73%) and a high positive predictive value (approximately 80%). In a sensitivity analysis, we found that when some patients with RA who did not meet the definition of "possible cases of RA" were misclassified as a patient with no RA, then this would lead to underestimation of the prevalence of the definition-positive patients and the adjusted prevalence. CONCLUSIONS: We recommend using the claims-based definition of RA (found in the current validation study) to estimate the prevalence of RA in Japan. We also suggest estimating the adjusted prevalence using the quantitative bias analysis method, since the prevalence of the disease in the "hospital-based" validation study is different from that in the administrative database. TRIAL REGISTRATION: The current study is not a clinical trial and hence not subject to trial registration.

[What the New International Classification Criteria for Idiopathic Inflammatory Myopathy Tell Us].

The classification criteria of idiopathic inflammatory myopathy in the past include the criteria defined phenotypically by Peter and Bohan in 1975 and the criteria defined based on pathological findings in affected muscles. New criteria were defined by international researchers, including the author of this article, from multiple fields to be used widely in different fields in 2017. The criteria were approved by rheumatological societies in Europe and the United States, and validated as excellent criteria in a Japanese cohort. It is known that the criteria used for etiological studies and those for diagnosis should be different in their stringency. In the new criteria, the stringency can be set freely by the users to be used for a wide range of purposes.

Response to tocilizumab and work productivity in patients with rheumatoid arthritis: 2-year follow-up of FIRST ACT-SC study.

OBJECTIVES: We evaluated long-term control of rheumatoid arthritis (RA) in Japanese paid workers (PWs) and house workers (HWs) treated with subcutaneous tocilizumab (TCZ-SC) and explored factors affecting response to TCZ-SC regarding work productivity. METHODS: This study collected data from patients with RA in the TCZ-SC +/- conventional synthetic disease-modifying antirheumatic drugs group. Factors affecting the response to tocilizumab regarding work productivity were explored using logistic regression. Differences in quality-adjusted life years (QALYs) between with/without response were analysed by a linear regression. RESULTS: Data were analysed for 357/360 patients. Patients with a ≥ 75% improvement in activity impairment (AI) were considered responders. EuroQol-5 Dimension (EQ-5D), six-item Kessler psychological distress scale score (K6), Health Assessment Questionnaire Disability Index (HAQ-DI), and the patient's disease global health by visual analogue scale were significant contributors to TCZ-SC response based on improvements in AI. Work Functioning Impairment Scale, presenteeism, EQ-5D, K6, and HAQ-DI significantly contributed to the improvement of overall work impairment in PWs. Shorter disease duration also was related to TCZ-SC response based on AI improvements. Responders had significantly larger mean QALYs than non-responders (difference = 0.2614; p < .001). CONCLUSIONS: These real-world clinical data support long-term work productivity control with TCZ-SC for biologic-naïve HWs and PWs with RA.

Long-term efficacy and safety of tocilizumab in refractory Takayasu arteritis: final results of the randomized controlled phase 3 TAKT study.

OBJECTIVE: To investigate the long-term efficacy and safety of the IL-6 receptor antibody tocilizumab in patients with Takayasu arteritis (TAK). METHODS: Patients completing the randomized, double-blind, placebo-controlled period of the TAKT (Takayasu arteritis Treated with Tocilizumab) trial were followed up during open-label extended treatment with weekly s.c. tocilizumab 162 mg for up to 96 weeks or longer, with oral glucocorticoid tapering performed at the investigators' discretion. Endpoints of the extension analysis included steroid-sparing effects of tocilizumab, imaging data, patient-reported outcomes (36-Item Short Form Health Survey) and safety. RESULTS: All 36 patients enrolled in the double-blind period entered the open-label extension; 28 patients received tocilizumab for 96 weeks. The median glucocorticoid dose was 0.223 mg/kg/day at the time of relapse before study entry, 0.131 mg/kg/day (interquartile range 0.099, 0.207) after 48 weeks and 0.105 mg/kg/day (interquartile range 0.039, 0.153) after 96 weeks. Overall, 46.4% of patients reduced their dose to <0.1 mg/kg/day, which was less than half the dose administered at relapse before study entry (mean difference -0.120 mg/kg/day; 95% CI -0.154, -0.087). Imaging evaluations indicated that most patients' disease was improved (17.9%) or stable (67.9%) after 96 weeks compared with baseline. Mean 36-Item Short Form Health Survey physical and mental component summary scores and 7 of 8 domain scores were clinically improved from baseline and maintained over 96 weeks of tocilizumab treatment. No unexpected safety issues were reported. CONCLUSION: These results in patients with Takayasu arteritis provide evidence of a steroid-sparing effect and improvements in well-being during long-term treatment with once-weekly tocilizumab 162 mg, with no new safety concerns. TRIAL REGISTRATION: JAPIC Clinical Trials Information, http://www.clinicaltrials.jp/user/cteSearch_e.jsp, JapicCTI-142616.

The development of quality indicators for systemic lupus erythematosus using electronic health data: A modified RAND appropriateness method.

Objective: Quality indicators (QIs) are tools that standardize evaluations in terms of the minimum acceptable quality of care, presumably contributing for the better management of patients with systemic lupus erythematosus (SLE). This study aimed to develop QIs for SLE using electronic health data.Methods: The modified RAND/UCLA Appropriateness Method was used to develop the QIs. First, a literature review was conducted. Second, the candidate QI items that were available to be evaluated using the electronic health data were extracted. Third, the appropriateness of the items was assessed via rating rounds and panelists' discussions.Results: We found 3621 articles in the initial search. Finally, 34 studies were reviewed, from which 17 potential indicators were extracted as candidate QIs. Twelve indicators were selected as the final QI set through the process of appropriateness. The median appropriateness of these 12 indicators was at least 7.5, and all of them were without disagreement. The QI included assessment of disease activity, treatment of SLE, drug toxicity monitoring, treatment of glucocorticoid complications, and assessment of SLE complications.Conclusion: We formulated 12 QIs for the assessment of patients with SLE based on electronic medical data. Our QI set would be a practical tool as a quality measure.

A new in vitro model of polymyositis reveals CD8+ T cell invasion into muscle cells and its cytotoxic role.

OBJECTIVES: The hallmark histopathology of PM is the presence of CD8+ T cells in the non-necrotic muscle cells. The aim of this study was to clarify the pathological significance of CD8+ T cells in muscle cells. METHODS: C2C12 cells were transduced retrovirally with the genes encoding MHC class I (H2Kb) and SIINFEKL peptide derived from ovalbumin (OVA), and then differentiated to myotubes (H2KbOVA-myotubes). H2KbOVA-myotubes were co-cultured with OT-I CD8+ T cells derived from OVA-specific class I restricted T cell receptor transgenic mice as an in vitro model of PM to examine whether the CD8+ T cells invade into the myotubes and if the myotubes with the invasion are more prone to die than those without. Muscle biopsy samples from patients with PM were examined for the presence of CD8+ T cells in muscle cells. The clinical profiles were compared between the patients with and without CD8+ T cells in muscle cells. RESULTS: Analysis of the in vitro model of PM with confocal microscopy demonstrated the invasion of OT-I CD8+ T cells into H2KbOVA-myotubes. Transmission electron microscopic analysis revealed an electron-lucent area between the invaded CD8+ T cell and the cytoplasm of H2KbOVA-myotubes. The myotubes invaded with OT-I CD8+ T cells died earlier than the uninvaded myotubes. The level of serum creatinine kinase was higher in patients with CD8+ T cells in muscle cells than those without these cells. CONCLUSION: CD8+ T cells invade into muscle cells and contribute to muscle injury in PM. Our in vitro model of PM is useful to examine the mechanisms underlying muscle injury induced by CD8+ T cells.

First external validation of sensitivity and specificity of the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria for idiopathic inflammatory myopathies with a Japanese cohort.

OBJECTIVE: To externally validate the performance of the new European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria set for idiopathic inflammatory myopathies (IIM) with a Japanese cohort. METHODS: This study included 420 IIM and 402 non-IIM cases. Probability of having IIM in each patient was calculated using the collected data set. The cut-off probability was set at 55%, as recommended by EULAR/ACR. Patients classified as IIM by the criteria were further subclassified with classification trees. RESULTS: When the probability cut-off was set at 55%, the sensitivity/specificity of the new criteria to diagnose IIM were 89.3%/91.0% in the total cohort, 88.1%/95.1% without muscle biopsy data and 90.4%/65.5% with biopsy data. The cohort included 12 overlap syndrome patients with biopsy data, who were included as non-IIM cases in accordance with traditional Japanese methods. When they were included in the IIM cases, the specificity in patients with biopsy increased to 74.4%. The sensitivity/specificity of the new criteria to diagnose polymyositis/dermatomyositis (PM/DM) plus juvenile and amyopathic DM in the Japanese cohort was 87.4%/92.4%, which were greater than those of the Tanimoto's criteria revised to enable classification of amyopathic DM (ADM) (71.2%/87.8%) and were comparable with those of Bohan & Peter's criteria to diagnose those diseases except for ADM (88.4%/88.3%). CONCLUSIONS: Our study externally validated high specificity of the new criteria for the first time, although with several limitations, including low percentage of child patients. The new criteria have higher sensitivity and/or specificity in classification of PM/DM than the previously reported criteria, demonstrating its usefulness for interethnic patients.

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector.

BACKGROUND: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. METHODS: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. RESULTS: We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. CONCLUSION: This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Higher risk of hospitalized infection, cardiovascular disease, and fracture in patients with rheumatoid arthritis determined using the Japanese health insurance database.

Objective: To evaluate the risk of hospitalized infection (HI), cardiovascular disease (CVD), stroke, and fracture in rheumatoid arthritis (RA) patients compared with non-RA patients using the Japanese health insurance database. Method: Among individuals aged ≥18 years, RA cases were defined to have one RA diagnostic code and receiving ≥1 disease-modifying antirheumatic drug between 2005 and 2013 (n = 6,712). Age-, sex-, calendar year of the observation start-, and observation length-matched non-RA cases were selected at 1:5 (n = 33,560). Hazard ratios (HRs) were calculated using the time-dependent Cox regression analysis. Results: Median age of the patients was 52.0 years. The incidence rates of HI, CVD, and fracture in the RA group were 2.42/100 person-years (PY), 4.94/1,000 PY, and 10.59/1,000 PY. The crude incidence rate ratios (95% CI) (RA vs. non-RA) for HI, CVD, and fracture were 2.47 (2.20-2.77), 1.89 (1.49-2.41), and 3.35 (2.80-4.02). The adjusted HR (95% CI) (RA vs. non-RA) was significantly elevated (HI, 1.74 [1.52-1.99], CVD, 1.38 [1.04-1.85], and fracture, 1.88 (1.54-2.31)]. Conclusion: The relatively young RA population had significantly higher risks of these complications than the non-RA, indicating importance of prevention of them even at young ages in clinical settings.

Tacrolimus successfully used to control refractory eosinophilic granulomatosis with polyangiitis complicated by invasive aspergillosis and chronic hepatitis B.

While several alternatives to cyclophosphamide have been proposed for refractory eosinophilic granulomatosis with polyangiitis (EGPA), therapeutic options are limited in patients with chronic infections. We report a case of refractory EGPA complicated by invasive aspergillosis and chronic hepatitis B. Although multiple immunosuppressants, including cyclophosphamide, were not effective, tacrolimus was used successfully to control disease without exacerbating concomitant infections in the long term. Tacrolimus could be an alternative choice in the treatment of EGPA, especially when aggressive immunosuppression is unfeasible.