Published papers

B cell-activating factor of the tumor necrosis factor (TNF) family, or BAFF, is mainly produced in monocytes and dendritic cells, and indispensable for proliferation, differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF), which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). In this study, we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand, the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line, Loucy (American Type Tissue Collection CRL-2629), in response to several stimuli, while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38, and that shedding of BAFF was catalyzed by a membrane-bound protease, furin. © 2006 Oxford University Press.

Receptors for platelet-derived growth factor (PDGF) are abundantly expressed on synovial fibroblast-like (SFL) cells from patients with rheumatoid arthritis (RA), and stimulation with PDGF enhances both the anchorage-dependent and -independent growth of RA-SFL cells. To elucidate the molecular mechanisms responsible for the excessive growth of RA-SFL cells and to seek a novel molecular-targeting therapy for RA, we examined the expression of adapter proteins and the effect of the specific inhibition of PDGF receptor activation by imatinib mesylate. Cultured SFL cells were used in the present study after 2-5 passages. The anchorage-dependent and -independent growth patterns of the SFL cells were evaluated using a tetrazolium-based assay and colony formation in 0.3% agar, respectively. Adapter proteins Gab1 and Gab2 were expressed in RA-SFL cells, and both proteins were rapidly (< 1 min) tyrosine-phosphorylated after the stimulation of RA-SFL cells with 10 ng/ml of PDGF and, to a lesser extent, after stimulation with 100 ng/ml of epidermal growth factor (EGF). The inhibition of PDGF receptor tyrosine kinase activation by 1 μM or less of imatinib mesylate specifically suppressed the PDGF-dependent, but not EGF-dependent, tyrosine phosphorylation of various proteins. Moreover, imatinib mesylate abolished both the anchorage-dependent and -independent proliferation of RA-SFL cells induced by PDGF stimulation. These results suggest that Gab adapter proteins are expressed and likely to be involved in the growth signalling of rheumatoid synovial cells and that imatinib mesylate, a key drug in the treatment of chronic myeloid leukaemia, may also be effective for the treatment of RA. © 2006 British Society for Immunology.

Japanese rheumatologists consider bucillamine (Buc) to be a useful disease-modifying antirheumatic drug (DMARD) and often give Buc to patients with rheumatoid arthritis (RA) prior to administering methotrexate (MTX). However, no large studies on the efficacy and safety of Buc in RA patients have been published in English to date. We therefore investigated the clinical course of RA patients treated with Buc and compared the results with those for patients treated with MTX to evaluate and confirm the place of Buc in therapeutic strategies for RA in Japan. Our results suggested that Buc should be given to patients with moderately active RA either before or after the administration of MTX because its efficacy can be judged within 3 months and because serious adverse events are rare. Issues like the ability of Buc to prevent joint destruction and its efficacy and safety when combined with agents like etanercept require future study. © Japan College of Rheumatology 2006.

We have reported that the TCRζ mRNA with alternatively spliced 3′ UTR (ζ mRNA/as-3′-untranslated region (UTR)) and ζ mRNA lacking exon 7 (ζ mRNA/exon 7-) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both ζ and TCR/CD3 complexes. To determine whether these T cells expressing decreased ζ exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type ζ cDNA, ζ cDNA/as-3′ UTR, and ζ cDNA/exon 7- into murine T cell hybridoma MA5.8 cells which lack ζ expression to construct the MA5.8 mutants WT, AS3′ UTR, and EX7-, respectively. FACS analyses demonstrated reduced cell surface expression of ζ and TCR/CD3 complexes on the AS3′ UTR mutant and the EX7- mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in both the AS3′ UTR mutant and the EX7- mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the ζ and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus. Copyright © 2006 by The American Association of Immunologists, Inc.

Biological agents targeting a specific molecule have extraordinarily fine specificity and powerful functional capabilities. By the introduction of biological therapy, management of rheumatoid arthritis has undergone a revolution and a paradigm shift. In this review, I will summarize the role in the pathogenesis of rheumatoid arthritis of the molecules targeted by biological agents. Providing evidence obtained in clinical trials and investigator-initiated clinical studies in Japan, the effectiveness and safety of biological therapy in rheumatoid arthritis are discussed. Finally, studies aiming at a personalized strategy with biological agents are listed and the future perspectives toward tailor-made medicine in the field of rheumatology are discussed. © 2011 by The Keio Journal of Medicine.

Tocilizumab, a humanized monoclonal antibody to the interleukin 6 (IL-6) receptor, was approved for use as rheumatoid arthritis (RA) therapy in Japan in 2008, but its efficacy and tolerability in daily practice has not yet been reported. We report the results of a multicenter retrospective study on the efficacy and safety of tocilizumab involving all patients (n = 229) who were started on tocilizumab therapy at three rheumatology institutes in Japan from April 2008 through to March 2009. Tocilizumab was infused every 4 weeks at a dose of 8 mg/kg according to the drug labeling. Among the 229 patients, 55% concomitantly received methotrexate (MTX) and 63% had previously received anti-tumor necrosis factor (TNF) therapy. Average disease activity score (DAS) 28 of all 229 patients significantly decreased from 5.70 to 3.25 after 24 weeks of therapy. A European League Against Rheumatism (EULAR) good response and DAS28 remission was achieved in 57.4 and 40.7% of the patients, respectively, at 24 weeks. White blood cell counts significantly decreased and liver enzymes and total cholesterol slightly but significantly increased; however, liver enzyme levels did not increase in patients without MTX. Tocilizumab was discontinued in 47 cases (20.5%) due to lack of efficacy (5.2%), adverse events (11.4%), and other reasons (3.9%). The overall retention rate at 24 weeks was 79.5%. Based on these results, we conclude that tocilizumab therapy in daily rheumatology practice appears to be highly efficacious and well tolerated among active RA patients, including the anti-TNF therapy-refractory population. Tocilizumab infusion is therefore applicable not only as an alternative approach for anti-TNF therapy-resistant patients, but also as primary biologic therapy for active RA patients. © Japan College of Rheumatology 2010.

Objective: Infusion reaction is a major adverse event in patients with rheumatoid arthritis (RA) treated with infliximab. The possible factors including Fcγ receptor (FcγR) polymorphism associated with the development of infusion reactions in patients with RA receiving infliximab were prospectively examined. Methods: 96 patients with RA were enrolled and scheduled to receive infliximab at a dose of 3 mg/kg at weeks 0, 2 and 6 and every 8 weeks thereafter. Genetic polymorphisms for FcγR were examined in FCGR3A 176F/V and FCGR3B NA1/2 alleles by allele-specific PCR analysis. Results: An infusion reaction was observed in 17 patients (18%) during 52 weeks of treatment with infliximab. The FCGR3B NA1/NA1 genotype was found in 75% of the patients with infusion reactions and in only 37% of those without (p=0.01), whereas the FCGR3A 176F/V genotype was equally distributed in the patients with or without infusion reactions. Glucocorticoids were used in 53% of the patients who developed an infusion reaction and in 80% of those without an infusion reaction (p=0.02). A multivariable logistic regression model showed that the FCGR3B NA1/NA1 genotype and use of glucocorticoids at baseline could be used as independent predictive factors for infusion reactions (OR 6.1 (95% CI 1.9 to 24.3) and OR 0.26 (95% CI 0.08 to 0.84), respectively). The presence of anti-infliximab antibody during infliximab treatment was also associated with infusion reactions. Conclusion: FCGR3B NA1/NA1 genotype, use of glucocorticoids and the presence of anti-infliximab antibody accounted for nearly all patients with RA who developed infusion reactions.

Our aim was to assess the ability of tocilizumab monotherapy to reduce progressive structural joint damage in rheumatoid arthritis patients at high risk of progression. This study was a subanalysis from a prospective 1-year, multicenter, X-ray-reader-blinded, randomized controlled trial of tocilizumab [Study of Active Controlled Monotherapy Used for Rheumatoid Arthritis, an IL-6 Inhibitor (SAMURAI) trial]. All patients were categorized into two or three groups according to four independent predictive markers for progressive joint damage [urinary C-terminal crosslinking telopeptide (uCTX-II), urinary pyridinoline/deoxypyridinoline (uPYD/DPD) ratio, body mass index (BMI), and joint-space narrowing (JSN) score at baseline]. One-year progression of joint destruction was assessed in high-risk versus low-risk groups receiving tocilizumab monotherapy and compared with patients receiving conventional disease-modifying antirheumatic drugs (DMARDs) (n = 157 and 145, respectively). In patients at high risk of progression of erosion as estimated by high uCTX-II, uPYD/DPD, or low BMI, and at high risk of progression of JSN as estimated by low BMI or high JSN score, the 52-week changes in radiological erosion and JSN, respectively, were significantly less in patients treated with tocilizumab monotherapy compared with those receiving DMARDs for each type of risk factor. In patients at low risk, those receiving tocilizumab also progressed less than those on DMARDs, although the difference did not reach statistical significance. Tocilizumab monotherapy is more effective in reducing radiological progression in patients presenting with risk factors for rapid progression than in low-risk patients. Patients at high risk for progression may benefit more from tocilizumab treatment. © 2010 Japan College of Rheumatology.

Both the number and functional capacity of T-regulatory (Treg) cells are known to be decreased in various autoimmune diseases. FOXP3, an essential transcription factor for Treg cells, has three isoforms in humans, wild, and exon 2- and exon 2-exon 7-lacking, although their role in autoimmunity is not clearly understood. Here, we investigated the messenger RNA (mRNA) expression of the major wild and exon-2 isoforms in peripheral mononuclear cells by quantitative PCR methods in 56 subjects, consisting of 23 rheumatoid arthritis (RA) and 25 systemic lupus erythematosus (SLE) patients, and 8 healthy controls (HCs). Although mRNA expression of the two isoforms did not directly correlate with clinical disease activity, relative expression of both was significantly lower in SLE and RA patients than in HCs. Furthermore, we found a significant statistical correlation between the two isoforms, suggesting that they are similarly regulated. Decreased expression of these isoforms in RA and SLE may reflect Treg cell abnormalities in these autoimmune diseases. Copyright © by BIOLIFE, s.a.s.

A 74-year-old woman developed fever, numbness of legs and glomerulonephritis. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase (MPO-ANCA) were positive in her serum, and she presented with acute renal failure. She was also simultaneously diagnosed as having both gastric and duodenal cancers. Complete resection of both cancers and renal biopsy was performed. Some glomeruli showed cellular crescentic changes, while submucosal necrotizing vasculitis of small vessels was noted adjacent to the gastric cancer. A diagnosis of microscopic polyangitis was made. After the operation, the patient's fever, renal failure and microscopic hematuria improved and obvious reductions in her serum soluble receptors of interleukin 2 values and MPO-ANCA titer were observed without any further treatment. However, the patient's proteinuria, cylinduria, and elevated C-reactive protein persisted; these findings eventually resolved after treatment with 30 mg of prednisolone daily. An immunohistochemical analysis showed that CD8 T lymphocytes had infiltrated both the carcinomas and the renal lesions. Our case suggests that CD8 T cells induced as part of an immune response against carcinoma may play a pathologic role in ANCA-positive paraneoplastic syndrome. © 2009 Springer-Verlag.

Accumulating evidence suggests that dysfunction of T cells underlies the pathogenesis of systemic lupus erythematosus (SLE). We revealed that SLE T cells produced an abnormally excessive amount of IFN- in vitro upon stimulation through TCR, and the expression level of TCR zeta was significantly reduced. The production of IFN- by SLE T cells was negatively correlated with the expression level of TCR zeta. This correlation was abolished when the cells were stimulated with TPA and ionomycin, which bypass TCR and introduce signals directly into the cells, but the production of IFN- by SLE T cells remained abnormally elevated. Taken together, these data suggest that regulatory mechanisms not only for the expression of TCR zeta but also for the production of IFN- were impaired in SLE T cells. These impairments may be responsible for the aberrant responses of SLE T cells and partly involved in the development of SLE. © 2010 Keiko Yoshimoto et al.

Objectives: We conducted a two-year prospective study to identify possible factors associated with normalisation of physical function by infliximab treatment in 125 patients with rheumatoid arthritis (RA). Methods RA patients who had been scheduled to receive infliximab at 3 mg/kg were registered and prospectively examined for disease activity, joint damage, and physical function for 102 weeks using the Disease Activity Score of 28 Joints (DAS-28) using C-reactive protein, van der Heijde-modified Sharp score (vdH-Sharp score) of hand and foot x-ray, and Health Assessment Questionnaire Disability Index (HAQ-DI). Normal physical function and clinical remission were defined as a HAQ-DI of 0.5 or less, and DAS28 (CRP) <2.6, respectively. Results: Forty-two of 125 patients (42%) achieved normal physical function at 102 weeks. The percentage of normal physical function at 102 weeks was significantly higher in the patients achieving clinical remission at 102 weeks (60%) than in those without (16%). In the patients with clinical remission at 102 weeks, less structural damage at baseline was correlated with a higher rate of normal physical function, suggesting the critical importance of joint destruction prior to infliximab therapy, in addition to clinical response. Logistic regression analysis further identified HAQ-DI, serum MMP-3 level, vdH-Sharp score, and methotrexate (MTX) dose as baseline factors contributing to normal physical function with 2-year infliximab treatment. Conclusion: Treatment with anti-TNF biologics in combination with MTX may achieve the normalisation of physical function in patients with established RA. Critical factors contributing to the normalisation of function were tight control of disease activity and less joint damage. © Copyright Clinical and Experimental Rheumatology 2010.

The superiority of the combination therapy of methotrexate (MTX) and anti-tumor necrosis factor (TNF) biological agents over anti-TNF monotherapy in MTX-naïve patients with rheumatoid arthritis (RA) has been demonstrated. We investigated the efficacy and safety of continuation versus discontinuation of MTX at the commencement of etanercept (ETN) in patients with active RA despite MTX therapy. In total, 151 patients with active RA despite treatment with MTX were randomized to either ETN 25 mg twice a week and MTX 6-8 mg/week (the E + M group) or ETN alone (the E group). Co-primary endpoints included the European League Against Rheumatism (EULAR) good response rate and the American College of Rheumatology (ACR) 50 response rate at week 24. Demographic and clinical features between groups at baseline were similar. The EULAR good response rates were significantly higher in the E + M group (52%) than in the E group (33%) at week 24 (p = 0.0001). Although the ACR50 response rate, one of the co-primary endpoints, and the ACR70 response rate at week 24 were not significantly greater in the E + M group (64 and 38%, respectively) than in the E group (48 and 26%, respectively), the ACR20 response rate was significantly greater in the E + M group (90%) than in the E group (64%; p = 0.0002). Safety profiles were similar for the groups. Thus, MTX should be continued at the commencement of ETN therapy, even in RA patients who show an inappropriate response to MTX. © 2010 Japan College of Rheumatology.

We conducted a 1-year prospective study to clarify differences between the Health Assessment Questionnaire disability index (HAQ-DI) and the modified HAQ (mHAQ) score among rheumatoid arthritis (RA) patients treated with infliximab. A total of 87 patients were scheduled to receive infliximab infusion at a dose of 3 mg/kg at weeks 0, 2, and 6, and every 8 weeks thereafter for 54 weeks; all patients received a full examination at each infusion appointment. The 28-joint disease activity score (DAS28) and functional capability of each patient was assessed at each visit, using the HAQ-DI and the mHAQ score. A strong correlation was observed between the HAQ-DI and the mHAQ score at baseline (r = 0.892). Over the course of the treatment, the mean mHAQ score changed similarly to the HAQ-DI, but the mean HAQ-DI was significantly higher than the mean mHAQ score at each time-point (for the HAQ-DI vs. mHAQ score, baseline: 1.5 +/- 0.7 vs. 0.9 +/- 0.6, p < 0.0001; 6 weeks: 1.1 +/- 0.7 vs. 0.6 +/- 0.5, p < 0.0001; 30 weeks: 1.0 +/- 0.7 vs. 0.6 +/- 0.5, p < 0.0001; 54 weeks: 0.9 +/- 0.7 vs. 0.6 +/- 0.6, p = 0.0006). In the categories of "eating", "reaching", and "other activities", the scores for several items excluded from the mHAQ score were significantly higher than those included in the mHAQ score over the year-long study period. We identified items contributing to significant differences between the HAQ-DI and the mHAQ score among RA patients treated with infliximab.