Modulation of methotrexate-induced intestinal mucosal injury by dietary factors.

Methotrexate (MTX)-induced intestinal mucosal injury in animals has been studied to understand how MTX can cause gastrointestinal disorders, but the pathogenesis of gastrointestinal disorders is still uncertain. We have attempted to reveal how dietary factors influence intestinal toxicity due to MTX. Mice were fed normal chow (NC) or a high-fat high-sucrose diet (HFHSD) before oral administration of MTX. While MTX significantly decreased the survival rates of mice fed HFHSD, the intestinal epithelial injury was detected. MTX excretion in the feces of mice fed HFHSD was reduced. Change of diets between NC and HFHSD influences the survival. The survival rates of the mice fed a high-sucrose diet or control diet were higher than those fed HFHSD. Higher survival rates were observed in mice fed a high-fat high-sucrose diet modified (HFHSD-M) in which casein was replaced by soybean-derived proteins. The survival rates of mice treated with vancomycin were lower than those administered neomycin. Microbiome and metabolome analyses on feces suggest a similarity of the intestinal environments of mice fed NC and HFHSD-M. HFHSD may modify MTX-induced toxicity in intestinal epithelia on account of an altered MTX distribution as a result of change in the intestinal environment.

The role of common protective alleles HLA-DRB1*13 among systemic autoimmune diseases.

Associations between human leukocyte antigen (HLA) and susceptibility to systemic autoimmune diseases have been reported. The predisposing alleles are variable among ethnic groups and/or diseases. On the other hand, some HLA alleles are associated with resistance to systemic autoimmune diseases, including systemic sclerosis, systemic lupus erythematosus and rheumatoid arthritis. Interestingly, DRB1*13 alleles are the protective alleles shared by multiple autoimmune diseases. DRB1*13:01 allele is protective in European populations and DRB1*13:02 in Japanese. Because alleles in multiple HLA loci are in strong linkage disequilibrium, it is difficult to determine which of the protective alleles is functionally responsible for the protective effects. Thus far, association studies suggested that DRB1*13:02 represents at least one of the causally associated protective factors against multiple systemic autoimmune diseases in the Japanese population. The protective effect of DRB1*13 alleles appears to overcome the predisposing effect of the susceptible alleles in heterozygous individuals of DRB1*13 and the susceptible allele. A gene dosage effect was observed in the associations of DRB1*13:02 with the protection from systemic autoimmune diseases; thus homozygous individuals are more effectively protected from the systemic autoimmune diseases than heterozygotes. DRB1*13:02 also confers protection against organ-specific autoimmune diseases and some infectious diseases. Several hypotheses can be proposed for the molecular mechanisms of the protection conferred by DRB1*13, some of which can explain the dominant effect of DRB1*13 molecules over the susceptible alleles, but the actual protective function of DRB1*13 requires further study. Understanding of the protective mechanisms of DRB1*13 may lead to the identification of targets for the curative treatment of systemic autoimmune diseases.

Association of IRF5 polymorphism with MPO-ANCA-positive vasculitis in a Japanese population.

Interferon regulatory factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT4) are shared susceptibility genes for various autoimmune diseases. In this study, we investigated whether these genes also contribute to susceptibility to anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in a Japanese population. A case-control study was carried out on IRF5 rs10954213 and STAT4 rs7574865 in 232 Japanese myeloperoxidase (MPO)-ANCA-positive AAV patients, including 177 microscopic polyangiitis and 710 healthy controls. IRF5 rs10954213G was significantly increased in MPO-ANCA-positive AAV (additive model, P=0.023, odds ratio=1.27, 95% confidence interval=1.03-1.57). The risk allele was previously shown to be associated with lower mRNA level of IRF5. On the other hand, significant association of STAT4 rs7574865T with AAV was not detected. These observations suggested that IRF5 may contribute to susceptibility to MPO-ANCA-positive AAV in a Japanese population.

A novel HLA-DQB1*04 allele, DQB1*04:10, identified in a Japanese individual.

In this paper, we characterize a novel human leukocyte antigen (HLA) DQB1*04:10 allele.

Association of a single nucleotide polymorphism in the SH2D1A intronic region with systemic lupus erythematosus.

SH2D1A, also known as signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), is an adaptor protein. Recently, it was reported that SAP deficient mice were protected from systemic lupus erythematosus (SLE). In this study, we postulated SH2D1A gene to be a candidate susceptibility gene for SLE and analyzed its association with SLE. A case-control association study was conducted on 5 tag single nucleotide polymorphisms (SNPs) in SH2D1A region in 506 Japanese female SLE patients and 330 healthy female controls. The luciferase assay was performed to determine the functional role of the SNP associated with SLE. One SNP in the intron 2, rs2049995, showed association with SLE (p=0.0110, odds ratio (OR) 1.97, 95% confidence interval (CI) 1.16-3.34, under the dominant model). The association of rs2049995 seemed to be stronger in the subset with the age of onset less than 20 years (p=0.0067, OR 2.65, 95% CI 1.28-5.46). Functional evaluation of rs2049995 showed that reporter gene activity was increased 1.9-fold for the susceptible allele compared with the resistant allele. An intronic SNP of SH2D1A is associated with SLE.

Identification of a novel HLA allele, HLA-DQB1*06:51, in a Japanese rheumatoid arthritis patient.

A novel HLA allele, HLA-DQB1*06:51, was identified in a Japanese rheumatoid arthritis patient.

Association of PHRF1-IRF7 region polymorphism with clinical manifestations of systemic lupus erythematosus in a Japanese population.

Interferon regulatory factor 7 (IRF7) has an essential role in the production of type I interferon. Although recent studies detected association of a single nucleotide polymorphism (SNP) rs4963128 in PHD and ring finger domains 1 (PHRF1)/KIAA1542, located closely to IRF7, and IRF7 rs1131665 (glutamine (Gln) 412 arginine (Arg)) with systemic lupus erythematosus (SLE), causal variants have not been established. In this study, we resequenced exons and introns of IRF7 to screen for all common polymorphisms, and examined whether they were associated with SLE in 416 Japanese patients with SLE and 505 healthy controls. We also tested whether the association of PHRF1 rs4963128 with SLE was replicated in a Japanese population. None of the IRF7 polymorphisms was associated with SLE. PHRF1 rs4963128T was not significantly associated with occurrence of SLE either; however, this allele was significantly increased in SLE with anti-Sm antibodies (6.8%) as compared with healthy controls (3.1%, P = 0.014, odds ratio [OR] 2.31) and SLE without anti-Sm antibodies (3.3%, P =0.041, OR 2.12). This allele was also increased in SLE with renal disorder (5.1%) as compared with those without renal disorder (2.4%, P = 0.047, OR 2.17). These results confirmed recently reported association of PHRF1 rs4963128T with anti-Sm antibody positive SLE in African-American populations, and supported the role of PHRF1-IRF7 region in the genetics of SLE.

Changes of human menisci in osteoarthritic knee joints.

OBJECTIVE: To investigate the changes of knee menisci in osteoarthritis (OA) in human. METHODS: OA and control menisci were obtained from 42 end-stage OA knees with medial involvement and 28 non-arthritic knees of age-matched donors, respectively. The change of menisci in OA was evaluated by histology, and gene expression of major matrix components and anabolic factors was analyzed in the anterior horn segments by quantitative PCR (qPCR). In those regions of menisci, the rate of collagen neo-synthesis was evaluated by [(3)H]proline incorporation, and the change of matrix was investigated by ultrastructural observation and biomechanical measurement. RESULTS: In OA menisci, the change in histology was rather moderate in the anterior horn segments. However, despite the modest change in histology, the expression of type I, II, III procollagens was dramatically increased in those regions. The expression of insulin-like growth factor 1 (IGF-1) was markedly enhanced in OA menisci, which was considered to be responsible, at least partly, for the increase in procollagen gene expression. Interestingly, in spite of marked increase in procollagen gene expression, incorporation of [(3)H]proline increased only modestly in OA menisci, and impaired collagen synthesis was suggested. This finding was consistent with the results of ultrastructural observation and biomechanical measurement, which indicated that the change of meniscal matrix was modest in the macroscopically preserved areas of OA menisci. CONCLUSION: Although the expression of major matrix components was markedly enhanced, matrix synthesis was enhanced only modestly, and the changes of matrix in human OA menisci were rather modest in the non-degenerated areas.

Neutrophil CD64 expression in the diagnosis of local musculoskeletal infection and the impact of antibiotics.

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics. These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection.

Methotrexate-related lymphomatoid granulomatosis: a case report of spontaneous regression of large tumours in multiple organs after cessation of methotrexate therapy in rheumatoid arthritis.

We describe a 54-year-old female patient with rheumatoid arthritis (RA) and Sjögren's syndrome (SS) who presented with right chest pain and a large mass visible in the upper right field of a chest X-ray. Computed tomography (CT) showed multiple tumours in both lungs, the liver, and the spleen. The right lung tumour was 8 cm in diameter with a cavity. Biopsy of the lung and liver revealed lymphomatoid granulomatosis (LG) and diffuse large B-cell lymphoma (DLBCL). These lesions spontaneously regressed after withdrawal of methotrexate without any therapy for the lymphoma. This is the first report of self-limiting LG in a patient, complicated with methotrexate-treated RA.

A multicenter study of leukocytapheresis in rheumatoid arthritis.

OBJECTIVE: To evaluate the efficacy and safety of leukocytapheresis (LCAP) in patients with rheumatoid arthritis (RA) that is refractory to disease modifying antirheumatic drugs (DMARDs), we conducted a prospective, multicenter, open-label clinical trial. METHODS: We enrolled 38 active RA patients, including 32 patients who showed an inadequate response to > or = 2 DMARDs and 6 patients with rapidly progressive RA. All patients continued drug therapy and were treated with 5 LCAP sessions conducted at 1-week intervals. The clinical response was evaluated at baseline before starting LCAP and at 4 weeks after the completion of all the LCAP sessions using the American College of Rheumatology (ACR) criteria and the 28-joint disease activity score (DAS28) of the European League Against Rheumatism (EULAR). RESULTS: Of the 35 patients who fulfilled the study's eligibility criteria, 24 (69%), 10 (29%), and 23 (66%) patients achieved 20% (ACR20), 50% (ACR50), and DAS28-C-reactive protein (CRP) EULAR improvement, respectively. The mean DAS28-CRP score of the 35 patients decreased significantly from 5.99 +/- 0.92 at baseline to 4.54 +/- 1.39 after treatment. Comparison analysis of the ACR20 responders and non-responders to LCAP revealed that 22 of 24 responders (92%) concomitantly received methotrexate, whereas significantly fewer, that is, 6 of 11 non-responders (55%) received methotrexate. Less frequent and transient mild-to-moderate adverse events, including nausea and headache, were seen in 12 of 189 LCAP sessions (6.3%). CONCLUSION: These results demonstrate the usefulness of LCAP in combination with DMARDs, particularly methotrexate, as an effective and safe treatment for refractory RA.

High diagnostic performance of ELISA detection of antibodies to citrullinated antigens in rheumatoid arthritis.

OBJECTIVE: We investigated the rheumatoid arthritis (RA) diagnostic performances of anti-cyclic citrullinated peptide antibody (anti-CCP) and antifilaggrin antibody (AFA) in comparison with RF and matrix metalloproteinase-3 (MMP-3). METHODS: We used a second generation enzyme-linked immunosorbent assay (ELISA) kit for the detection of anti-CCP. We constructed recombinant human filaggrin, which was citrullinated in vitro by human peptidylarginine deiminase, and subsequently used it as the coating antigen for AFA-ELISA. A total of 549 RA patients and 208 other rheumatic disease patients were included in the study. RESULTS: The specificities of anti-CCP (88.9%) and AFA (94.7%) were superior to those of RF (81.7%) and MMP-3 (49.5%). The sensitivity of anti-CCP (87.6%) was superior to all others. However, the sensitivity of AFA (68.7%) was inferior to those of RF (69.8%) and MMP-3 (75.7%). Furthermore, receiver operating characteristic curves of anti-CCP and AFA passed closer to the upper left corner than those of RF and MMP-3, and the areas under the curves (AUC) of AFA and anti-CCP were significantly larger. In addition, the AUC of anti-CCP was significantly larger than that of AFA. CONCLUSION: ELISA detection of antibodies to citrullinated antigens, especially a second generation anti-CCP, showed higher discriminative ability than other assays, including RF, and would be useful to aid the diagnosis of RA in clinical practice.

Inhibition of L-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614.

T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors.

Identification of 142 single nucleotide polymorphisms in 41 candidate genes for rheumatoid arthritis in the Japanese population.

Single-nucleotide polymorphisms (SNPs) can make an important contribution to our understanding of genetic backgrounds that may influence medical conditions and ethnic diversity. We undertook a systematic survey of genomic DNA for SNPs located not only in coding sequences but also in non-coding regions (e.g., introns and 5' flanking regions) of selected genes. Using DNA samples from 48 Japanese patients with rheumatoid arthritis (RA) as templates, we surveyed 41 genes that represent candidates for RA, screening a total of 104 kb of DNA (30 kb of coding sequences and 74 kb of non-coding DNA). Within this 104 kb of genomic sequences we identified 163 polymorphisms (1 per 638 bases on average), of which 142 were single-nucleotide substitutions and the remainder, insertions or deletions. Of the coding SNPs, 52% were non-synonymous substitutions, and non-conservative amino acid changes were observed in a quarter of those. Sixty-nine polymorphisms showed high frequencies for minor alleles (more than 15%) and 20 revealed low frequencies (<5%). Our results indicated a greater average distance between SNPs than others have reported, but this disparity may reflect the type of genes surveyed and/or the relative ethnic homogeneity of our test population.

Growth retardation in mice lacking the proteasome activator PA28gamma.

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated alpha, beta, and gamma; PA28alpha and PA28beta form the heteropolymer mainly located in the cytoplasm, whereas PA28gamma forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28alpha and PA28beta is involved in the processing of intracellular antigens, but the function of PA28gamma remains elusive. To investigate the role of PA28gamma in vivo, we generated mice deficient in the PA28gamma gene. The PA28gamma-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28gamma(+/-) or PA28gamma(+/+) mice. We also investigated the effects of the PA28gamma deficiency using cultured embryonic fibroblasts; cells lacking PA28gamma were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28alpha/beta nor the subcellular localization of PA28alpha was affected in PA28gamma(-/-) cells. These results indicate that PA28gamma functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28alpha nor PA28beta compensates for the PA28gamma deficiency.

Autoantibodies to T cell costimulatory molecules in systemic autoimmune diseases.

To determine whether antilymphocyte Abs to T cell costimulatory molecules are generated in patients with autoimmune diseases and, if they exist, to clarify the mechanism of their production and pathological roles, we investigated the presence of autoantibodies to CTLA-4 (CD152), CD28, B7-1 (CD80), and B7-2 (CD86) in serum samples obtained from patients with various autoimmune diseases and from normal subjects using recombinant fusion proteins. In ELISAs, anti-CD28, anti-B7-1, and anti-B7-2 Abs were rarely seen, whereas anti-CTLA-4 Abs were detected in 8.2% of the patients with systemic lupus erythematosus, 18.8% of those with rheumatoid arthritis, 3.1% of those with systemic sclerosis, 31.8% of those with Behçet's disease, 13.3% of those with Sjögren's syndrome, and 0% of healthy donors. This reactivity was confirmed by immunoblotting. More importantly, the purified anti-CTLA-4 Abs reacted with CTLA-4 expressed on P815 cells by flow cytometry. In addition, we found at least three epitopes on the CTLA-4 molecule. Furthermore, among the patients with Behçet's disease, uveitis was seen significantly less frequently in the anti-CTLA-4 Ab-positive patients. Taken collectively, these data indicate that anti-CTLA-4 autoantibodies are generated in systemic autoimmune diseases by an Ag-driven mechanism and may modulate the immune response in vivo by binding to CTLA-4 on T cells.

Cell surface expression of HLA-E molecules on PBMC from a TAP1-deficient patient.

It was recently revealed from studies on TAP-deficient cell lines that HLA-E molecules are associated with nonamer peptides derived from certain HLA class I leader sequences and are expressed on the cell surface in a TAP-dependent manner. We have previously reported a homozygous TAP1 gene mutation in a HLA class I-deficient patient. In the present report, we demonstrate HLA-E molecule expression on the surface of the peripheral blood mononuclear cells (PBMC) of the TAP1-deficient patient. The HLA-E expression level on the monocytes of the patient was as high as that in healthy donors, whereas the HLA-E expression level on the lymphocytes of the patient was slightly lower. On the other hand, HLA-E expression was not detected on KMW-B2 cells, an Epstein-Barr virus (EBV)-transformed B-cell line derived from the lymphocytes of the TAP1-deficient patient. These data suggest the existence of TAP-dependent and -independent pathways for the surface expression of HLA-E molecules.

Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome.

Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient.