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Nitric oxide (NO), originally found as endothelium-derived relaxing factor (EDRF), is a free radical synthesized by NO synthases (NOS). Two isoforms exist in NOS,i.e. constitutive NOS (cNOS) and inducible NOS (iNOS). Inflammatory cytokines such as interleukin-1, interferon-γ, tumor necrosis factor-α induce iNOS expression in various cells including macrophages. Enhanced NO production is observed in arthritic conditions both in rodent models and human. The onset of arthritis in rodent models is significantly inhibited by the NOS inhibitor,N G-monomethyl-l-arginine. These data suggest a possible involvement of NO in the induction and/or maintenance of rheumatoid arthritis.

We recently found that retinoic acids (RAs) exert anticoagulant effects by upregulating thrombomodulin (TM) and downregulating tissue factor (TF ) expression in acute promyelocytic leukemia (APL) cells and monoblastic leukemia cells. Two classes of nuclear RA receptors, termed retinoic acid receptors (RARs) and retinoid X receptors, have been identified. Each receptor class consists of three subtypes. In the present study, we have used several synthetic retinoids to determine which receptor subtypes are involved in the regulation of TM and TF expression in NB4 APL cells, U937 monoblastic leukemia cells, and human umbilical vein endothelial cells (HUVECs). Am80, which has no binding affinity for RARγ, and Ch55, which does not bind to cytoplasmic retinoic acid binding protein (CRABP), upregulated TM and downregulated TF in NB4 and U937 cells, similar to all-trans RA (ATRA). A specific RARα antagonist, Ro41-5253, significantly suppressed the upregulation of TM by ATRA and Am80 in NB4 cells, U937 cells, and HUVECs. In contrast, only with preincubation with both RARα and RARβ antagonists was downregulation of TF by retinoids suppressed in NB4 cells. These findings indicate that the mechanism of transactivation and transrepression functions of RARs are distinct and also elucidate the major role of RARα in TM upregulation by retinoids in leukemic cells and HUVECs and the cooperation of RARα and RARβ in TF downregulation by retinoids. They also indicate that binding to CRABP is not required for the anticoagulant effect of retinoids and that synthetic retinoids will prove very useful in controlling distinct targets, the TM and TF genes, at the level of transcription, and will permit the development of retinoids with a new type of anticoagulant effect.

The receptors for erythropoietin (Epo) and interleukin-3 (IL-3) both induce the ligand-dependent activation of the Jak2 tyrosine kinase. Activated Jak2 then phosphorylates these receptors and thereby recruits various signaling molecules containing the Src homology (SH)-2 domain, including Stat5, to the tyrosine phosphorylated receptors. In the present study, we demonstrate that Epo stimulation induces unidirectional cross-phosphorylation of the IL-3 receptor β subunit (βIL3) on tyrosines and its rapid and transient association with Stat5 in murine IL-3–dependent cell lines engineered to express the Epo receptor (EpoR). Using cell lines expressing various EpoR mutants, it was demonstrated that the Epo-induced tyrosine phosphorylation of βIL3 is dependent on the membrane-proximal EpoR cytoplasmic region involved in the activation of Jak2, but not on the extracellular and transmembrane regions or on the carboxy-terminal 145 amino acid region containing all the intracellular tyrosine residues. It was also shown that IL-3 induces rapid and dose-dependent association of Jak2 with βIL3. However, Epo failed to induce any detectable association of βIL3 with Jak2 or the EpoR. The present study also demonstrates that in IL-3–stimulated cells, an ovine Stat5 mutant harboring a substitution of Tyr694 to Phe, which abolishes the tyrosine phosphorylation required for activation, fails to dimerize with endogenous Stat5, shows sustained binding with tyrosine-phosphorylated βIL3, and inhibits the tyrosine phosphorylation of endogenous Stat5. These results suggest that βIL3 may have Stat5 docking sites, similar to those found in the EpoR, that facilitate the activation of Stat5 by Jak2 and raise the possibility that Epo may cross-activate or transmodulate the IL-3 receptor signaling pathways.

Nucleobindin (Nuc) was originally identified as a 55 kDa protein that enhanced anti-DNA antibody production in cultures of autoimmune MRL/ lpr mouse spleen cells. cDNA cloning and the production of recombinant protein (rNuc) have revealed that rNuc binds to DNA and Ca2+ by means of leucine zipper and EF-hand motif structures. In vitro and in vivo effects of rNuc to induce autoantibodies including anti-dsDNA antibody in normal mice have been demonstrated; however, whether Nuc is involved in the state of autoimmunity occurred in MRL/ lpr, C3H/ gld and male BXSB mice has not been elucidated. Here we report that the expression of Nuc mRNA is upregulated with age in the lymphatic organs and is positively correlated to the serum levels of Nuc in these lupus-prone strains of mice. Upon immunization with KLH in Freund's complete adjuvant, normal BALB/c mice also showed an elevated level of Nuc in their serum and elevated Nuc mRNA in their lymphatic organs. In addition, in vitro stimulation of BALB/c splenocytes with Con A or LPS remarkably enhanced Nuc mRNA expression. These results suggest that upregulation of Nuc mRNA in lymphatic organs and serum Nuc of lupus-prone mice is related to spontaneous activation of immunocompetent cells; the present data are also consistent with our previous hypothesis on the role of Nuc in the induction or enhancement of autoimmunity in lupus models of mice.

Studies of transcriptional activation by interferons and various cytokines have led to the identification of a family of proteins that serve as signal transducers and activators of transcription (STAT). STAT4 is phosphorylated following interleukin (IL)-12 stimulation and is required for IL-12 signal transduction. By immunoprecipitation and PCR amplification, a specific consensus sequence for DNA binding of the STAT4 complex was determined. The binding sequence of the STAT4 complex, (T/A)TTCC(C/G)GGAA(T/A), proved to be palindromic and similar to the IFN-gamma activated site (GAS)-like sequence. The first (T/A) and last (T/A) sites of the consensus sequence were critical for the binding affinity of the STAT4 complex.

Enlarged lymph nodes of mice with lpr mutation consist predominantly of CD4- CD8- (double-negative: DN) T cells. Among them, TCRBV8S3 (V beta 8.3) T cells are overrepresented as compared to those in single-positive (SP) T cells. To address the question of whether the expansion of oligoclonal T cells is responsible for the increase in TCRBV8S3 cells, we examined the TCRBJ gene repertoires of BV8S3 DN and SP T cells from multiple MRL lpr/lpr mice. The BJ repertoires of BV3 (V beta 3), BV8S1 (V beta 8.1) and BV8S2 (V beta 8.2) were studied for comparison with those of BV8S3 T cells. The employed method, which was based on a PCR-ELISA technique, was newly developed and allowed us to make a precise quantitation of TCRBJ gene usage of the multiple lymphocyte samples. The results showed that there were no biases of the BJ gene usage by BV8S3 DN T cells as well as other BV T cells. Furthermore, the BJ gene usage of CD4 and CD8 BV8S3 T cells was conserved by the DN T cells. It is suggested that the BV8S3 DN T cells were not expanded by specific antigens. The expansion may result from aberrant regulation specific to the BV8S3-expressing T cells.

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6−/−RM). Lymphogenesis in primary lymphoid tissues of Bcl6−/−RM is normal, and Bcl6−/−RM produced control levels of primary IgG1 antibodies specific to T cell–dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.

The feasibility of transcatheter laser ablation of the canine left ventricle (LV) was tested using a newly developed cardioscope. In 17 anesthetized dogs, a combined laser-endoscope catheter, consisting of an endoscope encased in a 7-French flexible catheter with an inflatable and transparent balloon at the distal end, was introduced into the LV via the carotid artery. A 1064-nm neodymium-yttrium-aluminum-garnet (Nd:YAG) laser was delivered by laser optic fiber, which was introduced through the transport channel and positioned inside the saline-filled balloon. In 16 of 17 dogs, the endocardial surface of the LV was clearly observed. Laser energy totaling 500-5,000 J was applied sequentially in 13 dogs and laser irradiation was completed in all but 2 of the dogs. The excised hearts revealed well-demarcated oval-shaped lesions 2.5-9.5 mm deep in 7 of 11 dogs. Histologic sections revealed coagulation necrosis surrounded by a rim of contraction band necrosis. Thus, transballoon laser photocoagulation of the beating LV is feasible. The newly combined laser-endoscope catheter, which is still in its preliminary stages and needs to be improved to increase the success rate of photocoagulation, appears to be a promising alternative modality for catheter ablative therapy for ventricular tachycardia. (Jpn Circ J 1997; 61: 695 - 703)

Objectives. To evaluate the long-term efficacy and safety of certolizumab pegol (CZP) treatment and to assess the efficacy of two CZP maintenance dosing schedules in Japanese rheumatoid arthritis (RA) patients who could not receive methotrexate (MTX). Methods. HIKARI double-blind (DB) patients were entered into an open-label extension (OLE) study. Patients withdrawn at 16 weeks due to lack of efficacy and DB completers without a 24-week American College of Rheumatology (ACR)20 response received CZP 200 mg every 2 weeks (Q2W). DB completers with 24-week ACR20 responses were randomized to CZP 200 mg Q2W or CZP 400 mg every 4 weeks. Results. The ACR20/ACR50/ACR70 response rates of DB completers (n = 98) were 82.7%/56.1%/34.7% at OLE entry, and 83.7%/65.3%/48.0% at 52 weeks, respectively. Other clinical, functional, and radiographic outcomes were sustained during long-term administration of CZP, even without MTX. No new unexpected adverse events were observed during long-term CZP treatment. The efficacy and safety of CZP treatment were similar between the two dosing schedules. Conclusions. Long-term CZP administration is efficacious and safe for RA patients. No obvious differences in clinical efficacy and safety were observed between the two dosing schedules. The choice between two maintenace regimens adds flexibility in administration schedules for RA patients and physicians.

Reactivation of hepatitis B virus (HBV) and de novo HBV hepatitis in patients with rheumatic diseases given intensive and long-term immunosuppressive therapy with or without biological disease-modifying antirheumatic drugs is of great concern, especially in regions where the virus is endemic, including Japan. To ascertain a better benefit–risk balance for immunosuppressive therapy for patients with rheumatic diseases, the Japan College of Rheumatology developed this proposal. All patients with rheumatic diseases commencing immunosuppressive therapy should be screened for hepatitis B surface antigen (HBsAg); those who are negative for HBsAg should be screened for hepatitis B core antibody (HBcAb) and hepatitis B surface antibody (HBsAb) as well. HBV carriers and serum HBV DNA positive patients with resolved infection should receive nucleoside analog as soon as possible, prior to commencing immunosuppressive therapy. For serum HBV DNA negative patients with resolved infection, careful monthly monitoring using serum levels of aspartate and alanine aminotransferases and HBV DNA is recommended during and at least 12 months after withdrawal of immunosuppressive therapy. If serum HBV DNA becomes positive, patients should receive nucleoside analog treatment as soon as possible, while ongoing immunosuppressive therapy should be continued to avoid severe or fulminant hepatitis development. To facilitate proper management of patients with HBV infection, collaboration between rheumatologists and hepatologists is strongly encouraged.

A post-marketing surveillance (PMS) program was implemented to assess the safety and effectiveness of tacrolimus (TAC) in Japanese rheumatoid arthritis (RA) patients and to identify risk factors related to adverse drug reactions (ADRs).Patients were registered centrally and monitored for all adverse events (AEs) for 24 weeks. Effectiveness was evaluated using the Disease Activity Score 28-CRP (DAS28-CRP).Data from 3,172 patients (mean age 62.2 years) were evaluated in the safety analysis. Of the safety population, 78.5 %were female and 25.9 % were in Steinbrocker's functional class 3 or 4. TAC was prescribed as monotherapy in 52.5 % and the most common concomitant disease modifying antirheumatic drug (DMARD) was methotrexate, used in 28.9 % of the patients. The incidence of AEs, serious AEs (SAEs), ADRs and serious ADRs were 41.2, 6.4, 36.0, and 4.9 %, respectively. The most frequent serious ADR category was infections and infestations. Age ≥ 65 years, concurrent renal dysfunction, and concurrent diabetes mellitus were identified as significant risk factors for ADR. Based on EULAR response criteria, 65.4 % of the patients showed moderate or good response.The results demonstrate that TAC is well tolerated by Japanese patients with active RA, including those receiving concomitant methotrexate, in the real world.

Introduction of methotrexate (MTX) and biologic agents caused a paradigm shift in the treatment of rheumatoid arthritis(RA). Development of new classification criteria for RA was established in 2010, which makes early diagnosis of RA possible. Treating to target (T2T) by setting treatment goal avoids irreversible joint damage and disability in RA. Remission, thus became a realistic goal with early diagnosis and intervention. Consequently, not only clinical but also structural and functional remission can be achieved with the current treatment. However, risk managements with the use of these agents are also of importance. In addition, there remains unmet needs in RA treatment.

To the Editor: Fibroblast-like synoviocytes (FLS) proliferate in the synovial tissue of patients with rheumatoid arthritis (RA), and contribute to chronic inflammation and the destruction of articular cartilage due to the production of a variety of cytokines, chemokines, and matrix metalloproteinases (MMP)1. The expression of CC chemokine ligand 18 (CCL18) was shown to be increased in the RA synovium2,3; however, the pathogenic role of CCL18 remains unclear. In this study, we investigated the expression of a recently identified receptor for CCL18, phosphatidylinositol transfer membrane-associated phosphatidylinositol transfer protein 3 (PITPNM3)4 in the RA synovium, and the stimulatory effects of CCL18 on FLS. Synovial tissue samples were obtained from patients with RA (n = 4) and osteoarthritis (OA) (n = 4) undergoing joint replacement surgery. RA patients were a median 65 years old (range 48–85 yrs), with median disease duration 9 years (range 2.5–30 yrs) and median C-reactive protein level 1.23 mg/dl (range 0.68–2.85). All RA patients were positive for rheumatoid factor and anticitrullinated protein antibodies. All subjects provided informed consent. The experimental protocol was approved in advance by the Ethics Committee of Tokyo Medical and Dental University. CCL18-positive cells were observed in the synovial lining, sublining, and perivascular regions of the RA synovium (Figure 1A). CCL18 expression was minimal in the OA synovium (Figure 1C). Western blotting analysis showed that CCL18 expression was significantly higher in the RA synovium than in the OA synovium (Figure 1E, 1F). Double-immunofluorescence staining revealed that most CD68-positive macrophages expressed CCL18 (Figures 1G–1I), and von Willebrand factor (vWF)-positive vascular endothelial cells were also … Address correspondence to Dr. T. Nanki, Department of Medicine and Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: nanki.rheu{at}tmd.ac.jp

Hypocomplementaemia is frequently observed in IgG4-related diseases, however the clinical significance is unclear. We describe herein the clinical courses of 4 patients with IgG4-related disease with hypocomplementaemia. Our cases showed autoimmune pancreatitis, retroperitoneal fibrosis, Mikulicz's disease, interstitial lung disease, lymphadenopathy and mesenteric fibrosis around the aorta. A decrease in serum complement preceded deterioration of the disease and clinical improvement was observed in accordance with normalisation of serum complement. These clinical courses suggest that serum complement is a biomarker of the disease activity.

A 37-year-old male with systemic lupus erythematosus (SLE) presented with high fever, subcutaneous indurations, anemia, thrombocytopenia, elevated liver enzymes and hyperferritinemia. Skin biopsy revealed hemophagocytic histiocytes in the adipose tissues. The patient was diagnosed with SLE with cytophagic histiocytic panniculitis (CHP). Treatment with high-dose glucocorticoids and cyclosporine A induced remission of SLE and CHP. CHP is generally a systemic disorder affecting subcutaneous adipose tissues with a high mortality rate. However, based on the present and previously reported cases, we believe that intensive immunosuppression can ameliorate CHP that occurs as a skin manifestation of SLE.

We analyzed the expression of the BCL6 gene in mouse tissues by in situ hybridization. The expression was strong in the upper layer but undetectable in the basal layer of epidermis from adult mice. When human keratinocytes were cultured with a high concentration of calcium ion, these cells stopped their proliferation and differentiated to their terminal stage. In these keratinocytes, BCL6 expression was induced after stimulation and progressively up-regulated. The kinetics was very similar to that of a cyclin dependent kinase inhibitor p21sdil/cip1/WAF1 in these cells. These results suggest that BCL6 plays a role in keratinocytes at terminal differentiation stage.

Although both ceramide and interleukin-1β converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both antiFas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.

The amino acids encoded at the junctions of T cell receptor (TCR) V and J genes directly interact with MHC bound peptides. However, the regulation of the human TCRBJ gene repertoire has been difficult to analyze, because of the potentially complex number of BJ gene rearrangements. To overcome this problem, we developed a PCR-ELISA method to study BJ gene expression, and compared peripheral T lymphocytes from 12 pairs of monozygotic twins, including 6 rheumatoid arthritis (RA) discordant pairs, and 5 normals. Analyses of the TCRBV5, 13 and 17 gene families, which have been reported to be increased in RA patients, showed: (a) the three TCRBV transcripts have common features of BJ gene usage; (b) TCR transcripts from each TCRBV family display a distinctive BJ gene profile, which is displayed better by CD4+ than CD8+ lymphocytes; (c) the BJ gene repertoires of monozygotic twins are more similar than those of unrelated individuals; and (d) the inflammation of RA does not induce specific changes in the genetically determined pattern of BJ expression. These results indicate that the frequency of expression particular TCRBV-TCRBJ recombinants in human lymphocytes is controlled genetically, and is maintained despite the presence of a chronic inflammatory disease.

To clarify the nature of the accumulated lung T cells of pulmonary sarcoidosis, we studied bronchoalveolar and peripheral blood (PB) lymphocytes from Japanese patients for expressed T-cell receptor beta-chain variable (TCR V beta) gene usage. Quantitative polymerase chain reaction technique revealed that the V beta repertoires of bronchoalveolar and PB T cells were heterogeneous. However, the bronchoalveolar lymphocytes from five out of 10 patients showed overrepresentation of T cells, with a few TCR V beta families, which varied among individuals, possibly reflecting a difference in the human leukocyte antigen (HLA) backgrounds. Nucleotide sequence analysis of third complementarity determining region (CDR3) segments in overrepresented V beta families demonstrated limited diversity of the transcripts. Two HLA-DR 12(5)+ individuals with overrepresented V beta 6 gene transcripts in the lungs shared a common amino acid motif within their CDR3. By contrast, non-overrepresented TCR V beta transcripts did not show clonal expansion, and the oligoclonal clones in the lung were not found in PB of the corresponding patient. These results showed that, at least in subsets of Japanese patients with pulmonary sarcoidosis, a limited number of T-cell clones are accumulated in the lung in response to a specific antigen. They may mediate an immune response to shape pathology of the disease.

Objective. To determine the involvement of human thioredoxi/adult T cell leukemia–derived factor (TRX/ADF) in Sjogren's syndrome (SS) and the correlation with Epstein-Barr virus (EBV). Methods. Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and the presence of EBV, using 6 normal tissues and 23 surgical specimens. The kinetics of expression of TRX/ADF induced by EBV was examined in vitro with peripheral blood B cells from EBV-seronegative donors. Results. Marked expression of TRX/ADF was found in the infiltrating B cells and the epithelial cells of salivary gland tissues from patients with SS (11 of 12 cases), but not in those from patients with other salivary gland inflammatory conditions (0 of 11 cases) or those of normal individuals (0 of 6 cases). In immunohistologic analyses, a striking topographic correlation between TRX/ADF and EBV was found. The coexistence of TRX/ADF messenger RNA and EBV DNA was detected by polymerase chain reaction (r = 0.75, P < 0.01). Peripheral blood B cells from EBV-seronegative donors showed de novo synthesis of TRX/ADF following in vitro infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRX/ADF was not detected in non-EBV-infected cells. Tumors in SCID mice reconstituted with mononuclear cells of salivary glands from SS patients, which were composed of human B cells carrying EBV DNA, were positive for TRX/ADF. Conclusion. These findings suggest that TRX/ADF expression closely reflects the intracellular event of EBV reactivation in SS. This is also the first report to show the ectopic in vivo expression of TRX/ADF in human autoimmune disease.

デルタ波の極性と頻拍中の逆行性P波の極性の不一致より複数副伝導路の存在が推定され, アブレーションによりその存在が証明された1例を報告する.症例は房室回帰性頻拍 (1種類) を合併した顕性WPW症候群の男性である.体表心電図ではII, III, aVF誘導にて陰性デルタ波を認め, 副伝導路 (AP) は (左室) 後壁側に存在すると推定された.頻拍中の逆行性P波はII, III, aVF誘導で陽性であった.同症例に対して電気生理学的検査およびカテーテルアブレーションを施行した.頻拍中の最早期興奮部位は左室前側であり, 同部位での2回の通電により頻拍は誘発不能となり, 逆伝導は房室結節経由となったが, 陰性デルタ波は不変であった.切断された左室前側壁のAP以外に, 後壁寄りに川頁伝導のみ可能な2本目のAPの存在することが推定された.最短房室伝導時間を示す左後中隔部位での1回の通電によりデルタ波は消失し, QRS波は正常化した.体表心電図における陰性デルタ波と頻拍時の陽性P波の所見は2本の副伝導路の可能性を示唆しているものと推察された.

Objective. To investigate the expressions of costimulatory molecules CD86 (B7-2, B70) and CD28 by cells obtained from the synovial tissues (ST) and synovial fluids (SF) of patients with rheumatoid arthritis (RA). Methods. Monoclonal antibodies (MAb) against CD86 and CD28 were used for immunochemical study of synovia from 18 RA patients, 4 osteoarthritis (OA) patients, and 4 normal subjects. These MAb were also used for flow cytometry of isolated ST cells from 8 RA and 5 OA patients and of SF mononuclear cells from 5 RA and 5 OA patients. Results. Immunohistochemical examination revealed that CD86+ cells occurred in 11 of the 18 RA synovia, but in none of the 4 OA or 4 normal synovia. Most of the positive cells had macrophage-like morphology, and surrounded lymphoid aggregates. Most cells within lymphoid aggregates were stained positively for CD28. Flow cytometry showed that CD86+ cells comprised 2.9–33.4% (average 14.3%) of the total ST cells and 2.1–14.9% (average 6.1%) of the total SF mononuclear cells from RA patients. Approximately 40% of the CD86+ cells expressed CD14. A majority (mean 72%, range 57–89%) of the T cells in ST and SF expressed CD28. RA synovia expressed more CD86 molecules than did OA synovia (mean frequency of positive cells 14.3% versus 2.8%; mean fluorescence intensity 104.6 versus 40.9). Conclusion. This study has shown that the expression of the CD86 molecule was up-regulated in RA synovia. The CD86+ cells appeared macrophage-like and surrounded CD28+ cells in lymphoid aggregates. The simultaneous presence and close localization of CD86+ and CD28+ cells in the RA synovial compartment suggests that their interaction potentially contributes to the sustained immune activation of RA.

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.

To clarify the natural course of synovitis after anterior cru ciate ligament injury, we took synovial tissues at various periods after injury and analyzed them immunohistologi cally. Twelve patients with isolated anterior cruciate liga ment injuries participated in the study. Inflammation in tensity was graded semiquantitatively based on the following parameters: synovial lining cell depth, synovial hyperplasia, vascular hyperplasia, lymphocyte infiltration, and the expression of cell adhesion molecules. The ex pression of fibrogenic cytokines and the degree of fibrosis were also investigated to elucidate the mechanisms of ar throfibrosis. The results demonstrated that synovitis in the infrapatellar fat pad subsides within 3 months after injury with the progression of fibrosis. Fibrogenic cytokines, platelet-derived growth factor and transforming growth factor-β, were detected at fibrotic sites, suggesting their involvement in arthrofibrosis. An increased incidence of arthrofibrosis recently has been recognized to occur when anterior cruciate ligament reconstruction is performed within several weeks after injury. We speculate that when anterior cruciate ligament reconstruction is performed dur ing the stage of acute synovitis, the operation may further promote the synovitis, accelerating the arthrofibrotic re action. Waiting to perform the anterior cruciate ligament reconstruction until the synovitis resolves may possibly decrease the motion problems seen postoperatively.

Among antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), granulomatosis with polyangiitis (GPA) and proteinase 3-ANCA-positive AAV (PR3-AAV) are prevalent in European populations, while microscopic polyangiitis (MPA) and myeloperoxidase-ANCA-positive AAV (MPO-AAV) are predominant in the Japanese. We previously demonstrated association of DRB1*09:01-DQB1*03:03 haplotype, a haplotype common in East Asians but rare in the European populations, with MPA/MPO-AAV, suggesting that a population difference in HLA-class II plays a role in the epidemiology of this disease. To gain further insights, we increased the sample size and performed an extended association study of DRB1 and DPB1 with AAV subsets in 468 Japanese patients with AAV classified according to the European Medicines Agency algorithm (MPA: 285, GPA: 92, eosinophilic GPA [EGPA]: 56, unclassifiable: 35) and 596 healthy controls. Among these patients, 377 were positive for MPO-ANCA and 62 for PR3-ANCA. The significance level was set at α = 3.3x10-4 by applying Bonferroni correction. The association of DRB1*09:01 with MPO-AAV was confirmed (allele model, P = 2.1x10-4, odds ratio [OR] = 1.57). Protective association of DRB1*13:02 was detected against MPO-AAV (allele model, P = 2.3x10-5, OR = 0.42) and MPA (dominant model, P = 2.7x10-4, OR = 0.43). A trend toward increased frequency of DPB1*04:01, the risk allele for GPA in European populations, was observed among Japanese patients with PR3-AAV when conditioned on DRB1*13:02 (Padjusted = 0.0021, ORadjusted = 3.48). In contrast, the frequency of DPB1*04:01 was decreased among Japanese patients with MPO-AAV, and this effect lost significance when conditioned on DRB1*13:02 (Padjusted = 0.16), suggesting that DRB1*13:02 or other allele(s) in linkage disequilibrium may be responsible for the protection. The differential association of DPB1*04:01 with PR3-AAV and MPO-AAV and difference in DPB1*04:01 allele frequencies between populations supported the hypothesis that the HLA-class II population difference may account in part for these epidemiologic characteristics. Furthermore, taken together with our previous observations, the haplotype carrying DRB1*13:02 was suggested to be a shared protective factor against multiple autoimmune diseases.

Recent investigations have revealed the involvement of cytokines in the pathogenesis of psoriasis. This study examined the amount of inflammatory cytokines--interleukin-1 (IL-1), interleukin-6 (IL-6) and granulocyte macrophage colony-stimulating factor (GM-CSF)--released into the supernatants of organ cultures of involved and uninvolved skin from psoriatic patients and normal skin from healthy individuals. Bioassays were employed to detect the activities of IL-1 and IL-6. Enzyme-linked immunosorbent assay (ELISA) methods were used to quantitate immunoreactive IL-1 alpha, IL-1 beta, IL-6 and GM-CSF. The activity of IL-1 in uninvolved psoriatic skin was found to be increased relative to that in involved and normal skin, while immunoreactive IL-1 beta was found only in involved skin. A neutralization experiment showed that bioactive IL-1 was mostly attributable to IL-1 alpha. Uninvolved psoriatic skin also secreted higher amounts of both bioactive and immunoreactive IL-6 compared with involved skin. Immunoreactive GM-CSF was detected in uninvolved skin only. These cytokines detected in uninvolved skin may have been released from epidermal or mesenchymal cells, since uninvolved skin contained fewer inflammatory infiltrates. Our results offer additional evidence that increased amounts of inflammatory cytokines in uninvolved skin may provide a preliminary condition and play important roles in the initial events in the evolution of psoriatic lesions.

To estimate the prevalence of Sjogren's syndrome (SS) in Japan, and to describe the clinicoepidemiological features, a nationwide epidemiological survey was conducted in 1994. The study consisted of two questionnaires distributed to the relevant departments of hospitals throughout Japan. The first questionnaire inquired the number of the patients treated in 1993, and the second one detailed clinico-epidemiological information of the patients reported. Following major epidemiological findings emerged from the study : (a) The total number of patients treated for SS in 1993, in Japan, was estimated as 17, 000 (95% confidence interval 15, 000-20, 000). The estimated crude prevalence rates were 1.9 and 25.6 per 100, 000 population in males and females, respectively. (b) The ratio of female to male patients was 13.7. The peak of age distribution of SS patients reported was in their fifties. The highest prevalence rate was observed among females with their sixth decade ; being 62.2 per 100, 000 population. J Epidemiol. 1995; 5 : 125-129.

A 44-year old woman with eosinophilic granulomatosis with polyangiitis (EGPA) developed sequential paralysis of different cranial nerves despite treatments including methylpredonisolone pulse therapy, intravenous immunoglobulins (IVIG), and cyclophosphamide. Infusions of rituximab ameliorated her neurological symptoms and serological inflammatory findings. Rituximab, a specific B cell-targeting therapy, might offer an alternative for refractory EGPA with possible advantages of cost and ease of use compared to IVIG, which also targets (at least in part) B lymphocytes and immunoglobulin production.

Aseptic abscess (AA) is characterized by accumulation of neutrophils without evidence of infection, no response to antibiotics, and rapid response to corticosteroids. We report a case of multiple abscesses in the subcutaneous tissues and joints, and severe mitral valve regurgitation. Although AA did not respond to antibiotic therapy, it improved dramatically with corticosteroid treatment. However, repeated valvuloplasty was required for the mitral valve regurgitation. The mitral valve tissue showed neutrophil infiltration without any bacterial invasion. This is the first case of AA to show involvement of cardiac valves, indicating the importance of systematic examination for patients with AA and cardiac valve involvement.

Induction of cyclin-dependent kinase (CDK) inhibitor gene p16INK4a into the synovial tissues suppresses rheumatoid arthritis in animal models. In vitro studies have shown that the cell-cycle inhibitor p16INK4a also exerts anti-inflammatory effects on rheumatoid synovial fibroblasts (RSF) in CDK activity-dependent and -independent manners. The present study was conducted to discern how p16INK4a modulates macrophages, which are the major source of inflammatory cytokines in inflamed synovial tissues. We found that p16INK4a suppresses LPS-induced production of IL-6 but not of TNF-α from macrophages. This inhibition did not depend on CDK4/6 activity and was not observed in RSF. p16INK4a gene transfer accelerated LPS-triggered IL-1R–associated kinase 1 (IRAK1) degradation in macrophages but not in RSF. The degradation inhibited the AP-1 pathway without affecting the NF-κB pathway. Treatment with a proteosome inhibitor prevented the acceleration of IRAK1 degradation and downregulation of the AP-1 pathway. THP-1 macrophages with forced IRAK1 expression were resistant to the p16INK4a-induced IL-6 suppression. Senescent macrophages with physiological expression of p16INK4a upregulated IL-6 production when p16INK4a was targeted by specific small interfering RNA. These findings indicate that p16INK4a promotes ubiquitin-dependent IRAK1 degradation, impairs AP-1 activation, and suppresses IL-6 production. Thus, p16INK4a senescence gene upregulation inhibits inflammatory cytokine production in macrophages in a different way than in RSF.

Objectives The aim of this phase 3, double-blind study was to compare the radiographic and clinical effects of etanercept (ETN) versus methotrexate (MTX) over 52 weeks in Japanese subjects with active rheumatoid arthritis. Methods The study population comprised 550 subjects with inadequate response to C1 disease-modifying antirheumatic drugs who were randomized to treatment groups of ETN 25 mg twice weekly (BIW; n = 182), ETN 10 mg BIW (n = 192), or MTX (B8.0 mg/week; n = 176). Results Of the 550 subjects initially enrolled in the three treatment groups, 21.6 % discontinued the study; a significantly higher proportion of those who withdrew from the study due to lack of efficacy were in the MTX (21.6 %) group compared with the ETN 25 mg (3.3 %) and ETN 10 mg (6.8 %) groups (P \ 0.001). Mean change from baseline in the modified total Sharp score at week 52 (primary endpoint) was significantly lower in the ETN 25 mg [3.33; standard error (SE), 0.73] and ETN 10 mg (5.19; SE 0.93) groups than in the MTX group (9.82; SE 1.16; P \ 0.0001 vs. either ETN group). Compared with subjects receiving MTX, significantly higher percentages of subjects treated with ETN 25 and 10 mg achieved American College of Rheumatology (ACR) ACR20 and ACR50 response rates at all time points (P \ 0.01). ETN was well-tolerated, with no unexpected safety findings. Conclusions ETN 25 mg BIW and ETN 10 mg BIW slowed radiographic progression and improved clinical outcomes more effectively than MTX in this Japanese population.

To investigate the clinical characteristics and risk factors of Pneumocystis jirovecii pneumonia (PCP) in rheumatoid arthritis (RA) patients treated with adalimumab.We conducted a multicenter, retrospective, case-control study to compare RA patients treated with adalimumab with and without PCP. Data from 17 RA patients who were diagnosed with PCP and from 89 RA patients who did not develop PCP during adalimumab treatment were collected.For the PCP patients, the median age was 68 years old, with a median RA disease duration of eight years. The median length of time from the first adalimumab injection to the development of PCP was 12 weeks. At the onset of PCP, the median dosages of prednisolone and methotrexate were 5.0 mg/day and 8.0 mg/week, respectively. The patients with PCP were significantly older (p0.05) and had more structural changes (p0.05) than the patients without PCP. Computed tomography of the chest revealed ground-glass opacity without interlobular septal boundaries in the majority of the patients with PCP. Three PCP patients died.PCP may occur early in the course of adalimumab therapy in patients with RA. Careful monitoring, early diagnosis, and proper management are mandatory to secure a good prognosis for these patients.

Many clinical studies have reported the excellent clinical efficacy of infliximab (IFX), an antitumour necrosis factor α (anti-TNFα) monoclonal antibody, in the treatment of rheumatoid arthritis (RA).1 IFX is also reported to induce a rapid and marked reduction in circulating interleukin 6 (IL-6) levels, suggesting that its efficacy may result from the suppression of IL-6 as well as TNF.2,–,5 In the RISING Study (NCT00691028),6 7 we observed patients who showed no response to IFX therapy, despite maintaining a serum IFX level higher than the threshold level for clinical response. Here, we examined data on clinical response to better understand the mechanism of action of IFX. In this study, patients with methotrexate-refractory RA treated with 3 mg/kg of IFX at weeks 0, 2 and 6 were randomly assigned to receive 3, 6 or 10 mg/kg of IFX every 8 weeks from week 14 to 46 in combination …

Objective Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS). Methods Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody–positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay. Results Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide. Conclusion Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.

To investigate associations between continuous treatments with tumor necrosis factor (TNF) antagonists and risk for developing serious infections (SIs) over 3 years in Japanese patients with rheumatoid arthritis (RA) enrolled in the Registry of Japanese RA Patients for Long-Term Safety (REAL) database.We analyzed 727 RA patients who had started either infliximab or etanercept (the anti-TNF group; 1,480.1 patient-years [PY]) and 571 RA patients who had started conventional nonbiologic disease-modifying antirheumatic drugs (the unexposed group; 1,104.1 PY) at the time of enrollment in the REAL. We assessed the occurrence of SIs within a 3-year observation period, including the period after switching to other TNF antagonists, and all SIs, unlimited to the first one in each patient as reported in other studies, to evaluate the real safety of TNF antagonists in daily practice.The incidence rate of SIs per 100 PY was 5.54 (95% confidence interval [95% CI] 4.44-6.84) in the anti-TNF group and 2.72 (95% CI 1.87-3.83) in the unexposed group. Poisson regression analysis revealed that the relative risk (RR) of continuous use of TNF antagonists for SIs after adjusting for baseline and time-dependent covariates was significantly elevated both overall (1.97, 95% CI 1.25-3.19) and for the first year (2.40, 95% CI 1.20-5.03), but not for the second and third years combined (1.38, 95% CI 0.80-2.43). The adjusted RR for SIs of etanercept compared to infliximab was not significantly elevated.Continuous anti-TNF therapy was significantly associated with increased risks for developing SIs during, but not after, the first year.

The objective of this study was to assess the response to abatacept at doses of 2 mg/kg and 10 mg/kg compared to placebo in patients with active rheumatoid arthritis (RA) with an inadequate clinical response to methotrexate (MTX). In this multicenter, placebo-controlled, double-blind, parallel-group, dose–response study, 195 Japanese patients with active RA with an inadequate response to MTX were randomized 1:1:1 to receive 10 mg/kg or 2 mg/kg abatacept plus MTX, or placebo plus MTX, for 24 weeks. Abatacept demonstrated a dose–response relationship when given at 2 and 10 mg/kg. Based on the American College of Rheumatology criteria (20, 50, and 70 %), the responses to 10 mg/kg abatacept were significantly greater than those to placebo at week 24 (p

We investigated associations between treatment with methotrexate (MTX) or biological disease-modifying antirheumatic drugs (DMARDs) and elevation of serum Krebs von den Lungen-6 (KL-6) levels in Japanese patients with rheumatoid arthritis (RA).Using a standardized form, data were collected retrospectively from medical records and analyzed descriptively.Of a total of 198 RA patients with KL-6 serum levels measured at initiation of treatment (month 0) and two or more times by month 12, 27 (17.9 %) of 151 RA patients treated with biological DMARDs, including infliximab, etanercept, adalimumab, and tocilizumab (the biological DMARDs group), and 5 (10.6 %) of 47 patients treated without biological DMARDs but with MTX (MTX group), met criterion B (max. KL-6 ≥500 U/ml and1.5-fold from baseline) by 12 months. The majority of patients (n = 28) meeting criterion B had no apparent interstitial lung disease or malignancy. Of these 28 patients, 21 had serum KL-6 levels available after reaching their maximum level, and 13 (61.9 %) of the 21 then met criterion R [decrease to less than 500 U/ml or to less than (baseline + 0.5 × (maximum - baseline))] by month 12.Serum KL-6 levels may increase during treatment with MTX or these biological DMARDs without significant clinical events.

Objective To investigate associations between continuous treatments with tumor necrosis factor (TNF) antagonists and risk for developing serious infections (SIs) over 3 years in Japanese patients with rheumatoid arthritis (RA) enrolled in the Registry of Japanese RA Patients for Long-Term Safety (REAL) database. Methods We analyzed 727 RA patients who had started either infliximab or etanercept (the anti-TNF group; 1,480.1 patient-years [PY]) and 571 RA patients who had started conventional nonbiologic disease-modifying antirheumatic drugs (the unexposed group; 1,104.1 PY) at the time of enrollment in the REAL. We assessed the occurrence of SIs within a 3-year observation period, including the period after switching to other TNF antagonists, and all SIs, unlimited to the first one in each patient as reported in other studies, to evaluate the real safety of TNF antagonists in daily practice. Results The incidence rate of SIs per 100 PY was 5.54 (95% confidence interval [95% CI] 4.44–6.84) in the anti-TNF group and 2.72 (95% CI 1.87–3.83) in the unexposed group. Poisson regression analysis revealed that the relative risk (RR) of continuous use of TNF antagonists for SIs after adjusting for baseline and time-dependent covariates was significantly elevated both overall (1.97, 95% CI 1.25–3.19) and for the first year (2.40, 95% CI 1.20–5.03), but not for the second and third years combined (1.38, 95% CI 0.80–2.43). The adjusted RR for SIs of etanercept compared to infliximab was not significantly elevated. Conclusion Continuous anti-TNF therapy was significantly associated with increased risks for developing SIs during, but not after, the first year.

We investigated the effects of inflammatory cytokines and periodontopathic bacteria on expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1, and E-selectin (endothelial leukocyte adhesion molecule 1) in cultured human gingival fibroblasts (HGF). Cell surface ICAM-1 was upregulated on HGF under transcriptional control by exposure not only to interleukin-1 beta, tumor necrosis factor alpha, and gamma interferon but also to sonic extracts prepared from Porphyromonas gingivalis and Prevotella intermedia (nigrescens) and lipopolysaccharides from Escherichia coli. However, these stimuli induced only minimal expression of vascular cell adhesion molecule 1 and E-selectin on HGF. Binding assays using HGF and Molt 4, the human T-cell leukemia cell line, showed induced ICAM-1 to be functional, and the increased binding was blocked by a combination of monoclonal antibodies against ICAM-1 and leukocyte function-associated antigen 1. Furthermore, gingival tissues from adult periodontitis patients showed increased mRNA expression of ICAM-1 compared with that in tissues from normal healthy donors. In immunohistological analysis, we also observed in vivo that the expression of ICAM-1 on fibroblasts in adult periodontitis tissues was greater than that in normal gingiva. Thus, the overexpression of ICAM-1 on gingival fibroblasts induced by cytokines and periodontopathic bacteria is speculated to be deeply involved in the accumulation and retention of leukocyte function-associated antigen 1-bearing leukocytes in adult periodontitis lesions.

We examined the possible involvement of Epstein-Barr virus (EBV) in the pathogenesis of Sjogren's syndrome (SS) using B cell lines (BCLs) spontaneously established from the peripheral blood. These BCLs were positive for EBNA and produced a large amount of infecting EBV in culture, a feature unique to SS-BCLs. Polymerase chain reaction analysis revealed that EBV with a B95-8-like U2 region was dominant in SS-BCLs. The nucleotide sequences of the U2 region of SS-EBV obtained so far have shown high homology to those of B95-8 EBV. However, there was a substantial amount of deletion and substitution of nucleotides within the U2 region of SS-EBV when compared with B95-8 EBV. This might enable SS-BCLs to escape immune recognition by cytotoxic T cells specific to EBNA-2. In addition, SS-EBV contains the sequence that spans the B95-8-deleted region. Furthermore, transfer of SS-BCLs to SCID mice induced monoclonal lymphoproliferative disorders that resembled those arising in SS. These data further support the motion that reactivation of EBV is deeply involved in polyclonal B cell activation and the development of B cell malignancies in SS.

This study was designed to clarify the important association between eosinophilia-myalgia syndrome (EMS) and the L-tryptophan contaminant, "Peak E." To determine the functional activation of eosinophils induced by Peak E, eosinophil cationic protein (ECP) release was examined. Peak E augumented the release of ECP from peripheral blood normodense eosinophils by degranulation. Proliferative analysis using the human eosinophilic leukemia cell line EoL-3 showed prominent cellular replication in the presence of Peak E. Moreover, Peak E upregulated interleukin 5 (IL-5) receptor levels on normodense eosinophils. Of particular interest, Peak E-stimulated human splenic T cells produced bioactive and immunoreactive IL-5. Marked induction of IL-5 mRNA in Peak E-stimulated T cells was also shown by reverse-transcriptase polymerase chain reaction (RT-PCR). In contrast, L-tryptophan without the contaminant showed none of these effects. Thus, these data suggest that Peak E might be involved in the pathogenesis of EMS through bimodal mechanism including IL-5 generation by T cells and potentiation of eosinophil functional activation.

Cloning report of complete coding region of HLA-DRB1*1405 has never appeared in the literature. We have isolated and sequenced it from a B cell line with a DRB1*1405/1502 genotype. The sequence information of this gene and previously published DRB1 genes for the other DR serotypes facilitated us to find that the cytoplasmic domain sequences of DRB1 genes fall into three groups. It is known that antigen presenting cells are differentially activated when products of different MHC class II genes are triggered. Functional analyses would discern if these allelic differences in the cytoplasmic domain are of functional significance.

Introduction Polymyositis (PM) and dermatomyositis (DM) are chronic inflammatory muscle diseases, in which chemokines are thought to contribute to inflammatory cell migration into muscle. In this study, we retrospectively analyzed the expressions of CX3CL1/fractalkine and its corresponding receptor, CX3CR1, in muscle and lung with interstitial lung disease (ILD) of PM patients and DM patients, and determined the correlation between serum soluble CX3CL1 level and disease activity. Methods Expressions of CX3CL1 and CX3CR1 in muscle and lung tissue were analyzed by immunohistochemistry. Serum CX3CL1 concentrations were measured by ELISA. For evaluation of patients' disease activity, serum creatinine kinase, manual muscle testing, and the alveolar-arterial oxygen pressure difference were used independently. Results CX3CL1 was expressed on infiltrated mononuclear cells and endothelial cells in muscle affected by PM and DM and in lung with ILD, whereas CX3CR1 was expressed on some CD4+ T cells, a majority of CD8+ T cells, and most macrophages in muscle, and on infiltrated mononuclear cells in the lung. Serum soluble CX3CL1 was significantly higher in PM patients and DM patients than in healthy controls. The CX3CL1 level was correlated with serum creatinine kinase and manual muscle testing score. In patients with PM and DM with ILD, serum CX3CL1 was also correlated with alveolar-arterial oxygen pressure difference. Furthermore, CX3CL1 was significantly decreased after conventional treatment. Conclusions The interaction between CX3CL1 and CX3CR1 might contribute to the inflammatory cell infiltration into affected muscle and lung with ILD in PM patients and DM patients. Serum CX3CL1 level could be a surrogate marker of disease activity.

To the Editor: We read with interest the recent article by Katchamart, et al 1 about Canadian recommendations for the use of methotrexate (MTX) in patients with rheumatoid arthritis (RA). The authors conclude that trimethoprim-sulfamethoxazole (TMP-SMX) should be avoided in RA patients treated with MTX. This recommendation was based on several case reports and a retrospective case-control study in which concomitant use of TMP-SMX and MTX was associated with blood dyscrasias1. In their cases, therapeutic doses of TMP-SMX were prescribed to treat urinary tract infection or other infectious diseases, but chemoprophylactic doses of TMP-SMX for Pneumocystis pneumonia (PCP) were not included. We believe that these 2 clinical indications for treatment with TMP-SMX should be considered separately. In fact, the contraindication for TMP-SMX use for … Address correspondence to Prof. M. Harigai, Department of Pharmacovigilance, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan. E-mail: mharigai.mpha{at}tmd.ac.jp