Yasuhiko Hirabayashi

Functional SNPs in CD244 increase the risk of rheumatoid arthritis in a Japanese population

Rheumatoid arthritis is a chronic autoimmune inflammatory disease with a complex genetic etiology. Members of the signaling lymphocyte activation molecule (SLAM) family carry out pivotal functions in innate immunity and in conventional lymphocytes. We identified a linkage disequilibrium block associated with rheumatoid arthritis in the chromosome 1q region containing multiple SLAM family genes. In this block, the association peaked at two functional SNPs (rs3766379 and rs6682654) in CD244 in two independent rheumatoid arthritis cohorts from Japan (P = 3.23 × 10−8 and P = 7.45 × 10−8). We also identified a Japanese cohort with systemic lupus erythematosus that had a similar genotype distribution as the rheumatoid arthritis cohorts. We demonstrated that the rheumatoid arthritis–susceptible alleles of rs3766379 and rs6682654 and their haplotype increased their expression in luciferase and allele-specific transcript quantification assays. CD244 is a genetic risk factor for rheumatoid arthritis and may have a role in the autoimmune process shared by rheumatoid arthritis and systemic lupus erythematosus.

The Endoplasmic Reticulum Stress-Inducible Protein, Herp, Is a Potential Triggering Antigen for Anti-DNA Response

Anti-dsDNA Abs are highly specific indicators of systemic lupus erythematosus (SLE) and play a pathogenic role in lupus nephritis. Human anti-dsDNA Abs are most likely generated by an Ag-driven mechanism. However, the Ag responsible for triggering anti-dsDNA Ab production has not been identified. To search for proteins that are cross-reactive with anti-dsDNA Abs, we screened a cDNA library from a patient with SLE with single-chain Fv of O-81 human anti-ss/dsDNA mAb by using a two-hybrid system. Homocysteine-induced ER protein (Herp), an endoplasmic reticulum (ER) stress-inducible ER membrane protein, was identified and shown to bind to original O-81 Ab and human lupus anti-dsDNA Abs. Some IgG purified from patients with active SLE by Herp-immobilized affinity chromatography bound to dsDNA. BALB/c mice immunized with Herp showed IgG anti-dsDNA Abs, IgG anti-nucleosome Abs, and glomerular IgG deposition. Herp reactivity was strongly positive in a proportion of PBLs from patients with active SLE, but undetectable in those from healthy controls. Moreover, activation of caspases was observed in the Herp-positive cells, implying that ER stress-induced apoptosis likely occurs in patients with active SLE. Herp is exposed on blebs of ER stress-induced apoptotic cells, suggesting that Herp can be recognized by immune cells. These results indicate that Herp mimics structural determinants of DNA immunologically and can be immunogenic in vivo. Thus, Herp represents a candidate autoantigen for anti-DNA Abs. This study may help explain how common environmental factors induce the production of anti-DNA Abs and contribute the development of SLE.

Intakes of Vitamin B6 and Dietary Fiber and Clinical Course of Systemic Lupus Erythematosus: A Prospective Study of Japanese Female Patients

Background Intakes of selected vitamins and dietary fiber may influence the clinical course of systemic lupus erythematosus (SLE). Using a cohort study method, we investigated the associations of dietary intake of vitamin B6 and B12, folate, and dietary fiber with the risk of active disease and atherosclerotic vascular events in SLE. Methods The study included female SLE patients in the Miyagi Lupus Cohort, which was founded in 1995. Dietary nutrients at baseline were estimated by a semiquantitative food frequency questionnaire. The association of each nutrient intake with the risk of active disease was investigated in 216 patients who had inactive disease at baseline. The association with atherosclerotic vascular events was assessed in 196 women who had inactive disease and no history of atherosclerotic diseases at baseline. Results Forty-three cases of active disease were identified during 9966 person-months of follow-up (1995–1999). During 19 575 person-months of follow-up (1995–2005), 20 atherosclerotic vascular events were documented. The Cox proportional hazards model revealed an inverse association between vitamin B6 intake and the risk of active disease (hazard ratio for the highest as compared with the lowest tertile, 0.41; 95% confidence interval, 0.18–0.97; P for trend = 0.04). An inverse association was also found for dietary fiber intake (P for trend = 0.01). However, no significant association was observed between intakes of these nutrients and the risk of atherosclerotic vascular events. Conclusions Higher intake of vitamin B6 and dietary fiber may prevent the occurrence of active disease in SLE.

Clonal frequency analysis of B cells producing pathogenic anti-DNA antibody-associated idiotypes in systemic lupus erythematosus

In order to identify the mechanism responsible for autoantibody production in systemic lupus erythematosus (SLE), B cell repertoires associated with anti-DNA idiotypes were explored by a limiting dilution analysis using Epstein-Barr virus (EBV) transformation methods and ELISA spot assays. The frequencies of B cell clones producing antibodies to DNA and to conventional antigens, tetanus toxoid, dinitrophenyl, or keyhole limpet hemocyanin were higher in active SLE compared to those in inactive SLE and in normal subjects. In addition, there was a disproportionate increase in anti-DNA antibody- and anti-DNA idiotype (Id)-producing clones at the precursor cell levels as well as at the mature cell level. On the other hand, numbers of anti-Id clones against anti-DNA-Id, termed 0-81 Id, were markedly increased at inactive stages of the disease but not at active stages. These were confirmed by serial studies in some patients with SLE. These results support a two-step mechanism for autoantibody production, in which initial polyclonal activation is followed by an antigen-driven process, and indicate an alteration of the precursor B cell repertoire in SLE, which may also associate with a preferential expansion of anti-DNA clones.

Sustainable Efficacy of Switching From Intravenous to Subcutaneous Tocilizumab Monotherapy in Patients With Rheumatoid Arthritis

To evaluate the efficacy and safety of switching from intravenous (IV) tocilizumab (TCZ) to subcutaneous (SC) TCZ monotherapy in rheumatoid arthritis patients.

Antibodies to Proteinase 3 Prime Human Oral, Lung, and Kidney Epithelial Cells To Secrete Proinflammatory Cytokines upon Stimulation with Agonists to Various Toll-Like Receptors, NOD1, and NOD2

ABSTRACT Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies, the detection of which in serum can be used in the diagnosis of Wegeners granulomatosis (WG). Proteinase 3 (PR3) is a major target antigen of ANCA in WG patients, and the interaction of PR3 ANCA with leukocytes causes a debilitating autoimmune disease. The first signs and symptoms in WG patients are observed in the oral cavity, lungs, and kidneys. Human epithelial cells generally do not secrete proinflammatory cytokines upon stimulation with pathogen-associated molecular patterns (PAMPs). In this study, anti-PR3 antibodies (Abs) and PR3 ANCA-containing sera from WG patients endowed human oral, lung, and kidney epithelial cells with responsiveness to PAMPs in terms of the production of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor alpha. Protease-activated receptor-2 (PAR-2) agonist peptides mimicked the priming effects of PR3 ANCA against PAMPs. Furthermore, the anti-PR3 Ab-mediated cell activation was significantly abolished by RNA interference targeting PAR-2 and NF-κB. This is the first report of priming effects of anti-PR3 Abs (PR3 ANCA) on epithelial cells. The results suggest that anti-PR3 Abs (PR3 ANCA) prime human epithelial cells to produce cytokines upon stimulation with various PAMPs, and these mechanisms may be involved in severe chronic inflammation in WG.

Somatic mutation in autoantibody-associated VH genes of circulating IgM+IgD+ B cells.

Naive B cells expressing IgM and IgD on their surface have no or little somatic mutations in V genes. We have demonstrated that the human IgM+IgD+B cell clone (0 – 81), which expresses nephritogenic idiotypes, produces IgM anti‐DNA antibodies which show monospecificity to DNA. Using a DNA probe which specifically links to the VH gene of antibody 0 – 81, we identified the counterpart germ‐line V gene of 0 – 81, V3‐7, which appears to be used by pathogenic autoantibodies in humans. Clone 0 – 81, which may belong to naive B cells in terms of cell phenotype, uses a somatically mutated V3‐7 gene. We further studied DNA sequences of V3‐7 genes in circulating IgM+IgD+B cells from normal subjects and patients with systemic lupus erythematosus (SLE). The results revealed that rearranged V3‐7 genes in IgM+IgD+B cells from patients with SLE contained somatically mutated sequences at significantly increased frequencies. These data indicate an abnormal maturation of B cells in autoimmune states that may be associated with an escape of self‐reactive B cells from the elimination process in the germinal center.

Gastrojejunostomy and Duodenojejunostomy for Megaduodenum in Systemic Sclerosis Sine Scleroderma: Report of a Case

Systemic sclerosis is an autoimmune disease that causes sclerotic changes primarily in the skin and in other organs. Systemic sclerosis sine scleroderma (ssSSc) is a variant of the disease in which visceral involvement occurs in the absence of skin thickening [1]. Most patients with systemic sclerosis have digestive tract complications, 90% of which involve esophageal diseases such as gastroesophageal reflux disease (GERD) and Barrett esophagus [2–6]. The rate of small intestinal complications in systemic sclerosis, although lower than that of esophageal complications, is estimated to be about 50%. In systemic sclerosis, more common small intestinal complications include intestinal pseudoobstruction with dilatation of the small intestine and small intestinal diverticula [1–5]. Megaduodenum is relatively rare as a small

Chemically Modified Ribozyme to V Gene Inhibits Anti-DNA Production and the Formation of Immune Deposits Caused by Lupus Lymphocytes

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 × 106 PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.

A Single-Stranded DNA-Cross-Reactive Immunogenic Epitope of Human Homocysteine-Inducible Endoplasmic Reticulum Protein

The mechanism involved in generating anti‐DNA antibodies (Abs) remains unclear, as DNA is poorly immunogenic. Molecular mimicry between DNA and non‐DNA substances has been implicated as a possible mechanism. We previously reported that homocysteine‐inducible endoplasmic reticulum protein (Herp), which is induced by endoplasmic reticulum stress, is recognized by anti‐double‐stranded DNA (dsDNA) IgG from patients with systemic lupus erythematosus and that immunization with Herp elicits anti‐dsDNA Abs in BALB/c mice. In this study, we observed that anti‐single‐stranded DNA (ssDNA) Abs were also generated in Herp‐immunized BALB/c mice and established an anti‐Herp monoclonal antibody (mAb), HT4, which specifically cross‐reacted with ssDNA. The epitope of the HT4 mAb on Herp, ‘EPAGSNR’, was identified by screening a synthetic peptide library. The binding of the HT4 mAb to the peptide was competitively inhibited by ssDNA. Immunization of the epitope peptide elicited anti‐ssDNA Abs in BALB/c mice. These results indicate that the epitope exists in a human self‐protein, mimics ssDNA and shows antigenicity for anti‐ssDNA Abs in normal mice. Anti‐ssDNA Abs are often found in patients with drug‐induced lupus erythematosus. Treatment with representative drugs that cause drug‐induced lupus (chlorpromazine, procainamide and hydralazine) induced Herp expression and apoptosis in HeLa cells. These findings suggest that molecular mimicry between Herp and ssDNA is involved in anti‐ssDNA Ab production in drug‐induced lupus.