Kohei Nagasawa

Association of tumor necrosis factor receptor type II polymorphism 196R with systemic lupus erythematosus in the Japanese: Molecular and functional analysis

OBJECTIVEnTo investigate whether a polymorphism(s) or mutation(s) in the tumor necrosis factor receptor II (TNFRII) gene is involved in the pathogenesis of systemic lupus erythematosus (SLE).nnnMETHODSnAll 10 exons of the TNFRII gene were analyzed by exon-specific polymerase chain reaction-single-strand conformation polymorphism, followed by nucleotide sequencing of exons that displayed aberrant bands. To analyze the function of the TNFRII polymorphisms, the full-length TNFRII complementary DNA of each allele was transfected in HeLa cells and then studied for specific binding of 125I-TNFalpha, as well as interleukin-6 (IL-6) production and cytotoxic activity after treatment with recombinant human TNFalpha.nnnRESULTSnWe identified 4 polymorphisms, at codons 56, 181, 196, and 232. The latter 2 had amino acid substitutions M196R and E232K, respectively. Only the 196R allele was significantly associated with SLE in our 105 Japanese SLE patients, with an allele frequency of 20.5%, compared with 12.6% in 99 healthy controls (P = 0.0335). More importantly, using TNFRII-transfected HeLa cells, we demonstrated significantly increased IL-6 production by 196R TNFRII compared with 196M TNFRII. The cytotoxic activity induced by 196R TNFRII was also increased compared with that of 196M TNFRII. This increase was achieved without affecting the binding affinity of TNFalpha to TNF-RII, as demonstrated by the finding that specific TNFalpha binding to the HeLa transfectants of 196R and 196M TNFRII was similar, with Kd values of 3.12 x 10(-10)M and 4.34 x 10(-10)M, respectively.nnnCONCLUSIONnThese results suggest that 196R TNFRII, which transduces the signals of TNFalpha more effectively than does 196M TNFRII, is involved in the pathogenesis of SLE.

Retinal disease in patients with systemic lupus erythematosus

OBJECTIVE To investigate the incidence of retinopathy in systemic lupus erythematosus (SLE) and to clarify its significance in relation to other clinical manifestations. METHODS A cross sectional study on lupus retinopathy was made in 69 patients with SLE. One expert ophthalmologist examined the ocular fundi of the lupus patients without any information of their disease state. Clinical and laboratory findings in the patients with retinopathy and those without were compared. RESULTS Retinopathy was found in 7/69 (10%) patients. The findings included haemorrhages, vasculitis, cotton wool spots, and hard exudates, all of which were considered to reflect vascular damage. Retinopathy was found to be associated with the presence of anticardiolipin antibody (p<0.05) and with central nervous system lupus (p<0.01). The patients with retinopathy had higher levels of serum creatinine than the patients without retinopathy (p<0.01). The disease activity of lupus, as assessed by the maximum SLE disease activity index (SLEDAI) score of the patients, was also significantly higher in the patients with retinopathy (p<0.03). CONCLUSION Incidence of retinopathy in SLE was similar to that in previous reports and it may reflect tissue microangiopathy, particularly associated with vasculitis or anticardiolipin antibodies, or both.

Role of TLR4/MD-2 and RP105/MD-1 in Innate Recognition of Lipopolysaccharide

TLR4 and RP105 are unique members of the Toll-like receptor (TLR) family molecules. They are associated with small molecules called MD-2 and MD-1, respectively, to form heterodimers (TLR4/MD-2 and RP105/MD-1) and function as recognition/signaling molecules of lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria. Analysis of transfectant cell lines and gene-targeted mice revealed that both MD-2 and MD-1 are involved in the recognition of LPS as well as in the regulation of intracellular distribution and the surface expression of TLR4 and RP105, respectively. Since RP105 or MD-1-deficient mice show a reduced but not complete lack of LPS responsiveness, there may be functional associations between TLR4/MD-2 and RP105/MD-1. In addition, there was an increased frequency of RP105-negative B-lymphocytes in the peripheral blood in several rheumatic diseases, such as systemic lupus erythematosus, suggesting the involvement of RP105 in the pathophysiology of autoimmunity. Further analysis of the structure and function of TLR4/MD-2 and RP105/MD-1 will provide a better understanding of the pathophysiology, and a chance to develop evidence-based treatments for septic shock syndrome and autoimmunity.

B cells lacking RP105, a novel B cell antigen, in systemic lupus erythematosus

OBJECTIVEnRP105 is a leucine-rich repeat (LRR) protein found on all mature mouse B cells. Its function is poorly defined, although it has been suggested that RP105 activates B cells to make them resistant to apoptosis. The human homolog of RP105 has been reported, but knowledge of its function is limited. We explored the expression and the function of the human homolog of murine RP105 on B cells in patients with systemic lupus erythematosus (SLE).nnnMETHODSnThe expression of RP105 and various markers on B cells in patients with SLE was analyzed using monoclonal antibodies and flow cytometry. Susceptibility to corticosteroid-induced apoptosis was examined by annexin V binding, and the production of immunoglobulin by RP105-negative B cells was examined by intracellular staining of IgG.nnnRESULTSnAs in mice, virtually all B cells in the peripheral blood of normal humans expressed the RP105 molecule. However, a significant proportion of circulating B cells (15.9%) in SLE patients were RP105 negative. Serial analyses of B cells in 7 SLE patients revealed that RP105-negative B cells markedly decreased in parallel with a reduction in disease activity (from 35.2% to 3.3%; P = 0.000003). The SLE Disease Activity Index and serum levels of IgG also correlated with the percentage of RP105-negative B cells. The phenotype of RP105-negative B cells was defined as CD95-positive, CD86-positive, CD38-bright, IgD-negative, IgM-dull, indicating that the cells were highly activated, as further suggested by the detection of intracellular IgG. RP105-negative B cells were clearly distinct from CD5-positive B1 cells. In vitro experiments indicated that RP105-negative B cells were susceptible to corticosteroid-induced apoptosis.nnnCONCLUSIONnThese findings suggest that loss of RP105 is associated with B cell activation and increased disease activity in SLE patients.

Interferon regulatory factor 5 is critical for the development of lupus in MRL/lpr mice

OBJECTIVEnInterferon regulatory factor 5 (IRF-5) is a transcription factor that mediates intracellular signals activated by engagement of Toll-like receptors (TLRs). IRF5 polymorphisms are associated with an increased or decreased risk of systemic lupus erythematosus (SLE) in various human populations, but the precise role of IRF5 in SLE development is not fully understood. This study was undertaken to examine the role of IRF5 in the development of murine lupus.nnnMETHODSnWe crossed gene-targeted IRF5-deficient (IRF5(-/-) ) mice with MRL/MpJ-lpr/lpr (MRL/lpr) mice and examined the progeny for survival, glomerulonephritis, autoantibody levels, immune system cell populations, and dendritic cell function.nnnRESULTSnIRF5(-/-) MRL/lpr mice survived longer than control IRF5(+/+) MRL/lpr mice and displayed only very mild glomerulonephritis. Autoantibodies to SLE-related nuclear antigens were lower in IRF5(-/-) MRL/lpr mouse serum, and numbers of activated CD4+ T cells were reduced in the spleen. Splenic DCs from IRF5(-/-) MRL/lpr mice produced lower levels of inflammatory cytokines when treated in vitro with TLR-7 or TLR-9 ligands or immune complexes. Interferon-α production in response to CpG was also decreased.nnnCONCLUSIONnOur results show that IRF5 is a crucial driver of lupus development in mice, and indicate that IRF5 may be an attractive new target for therapeutic intervention to control disease in SLE patients.

Anticentromere Antibody as a Risk Factor for Cancer in Patients with Systemic Sclerosis

Abstract: This study has estimated the cancer risk among patients with systemic sclerosis (SSc) using a population-based analysis. Using the inpatient and outpatient registries for patients at Kyushu University Hospital between 1982 and 1996, standardised incidence rates (SIRs) (ratio of observed-to-expected cancers) were calculated in 43 patients with SSc, 24 patients with polymyositis (PM) and 17 patients with dermatomyositis (DM). Risk factors predisposing to cancers were also investigated in the SSc patients. Compared with the Japanese general population, the SIR for developing cancer in SSc patients was 5.1 (95% confidence interval (CI), 1.7–10.8), while the SIRs for cancer in the PM and DM groups were 4.7 (95% CI, 1.5–10.3) and 61.2 (95% CI, 46.8–77.6), respectively. A statistically significant risk factor for cancers in the SSc patients was positivity for anticentromere antibody (ACA) (p<0.05), while the erythrocyte sedimentation rate, serum lactate dehydrogenase concentration, serum γ-globulin concentration, titre of antinuclear antibody and positivity for antitopoisomerase I antibody were not associated with cancer in SSc. Our population-based study confirms the increased risk of cancer among patients with SSc in Japan and provides new evidence that positivity for ACA should be considered as a risk factor for cancer in future monitoring of patients.

Therapeutic response of patients with adult Still’s disease to biologic agents: multicenter results in Japan

ObjectiveThe efficacy of biologics in treating adult Still’s disease (ASD) is suggested, but the information is still lacking and the validation is insufficient. To determine the efficacy of several biologic agents in refractory ASD in Japan, a multicenter survey was performed.MethodClinical data on 16 ASD patients who had been treated with at least 1 of the biological agents (total 24 occasions) were collected retrospectively.ResultsInfliximab was used in 9 cases, etanercept in 4, and tocilizumab in 11. Half of the patients that had been treated initially with infliximab or etanercept were changed to another biologics. Tocilizumab was effective in cases switched from another 2 drugs. Tocilizumab showed efficacy in treating both systemic and arthritic symptoms and showed apparent steroid-sparing effect and the highest continuation rate.ConclusionTocilizumab may be a promising biologic agent in refractory ASD.

Analysis of Fas ligand gene mutation in patients with systemic lupus erythematosus

OBJECTIVEnTo investigate the possible association of a Fas ligand (FasL) gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE).nnnMETHODSnFor amplification of the introns of the FasL gene, long polymerase chain reaction (PCR) using exon-based primers was utilized, followed by partial sequencing to construct exon-specific oligonucleotide primers for the analyses of FasL genomic DNA in SLE patients. Structural defects were studied by use of a composite analysis of reverse transcriptase-PCR/single-strand conformational polymorphism (SSCP) analysis of messenger RNA (mRNA) transcripts of the FasL gene in 35 SLE patients and PCR/SSCP analysis of FasL genomic DNA in 143 SLE patients.nnnRESULTSnThe sizes of the introns were approximately 0.6 kb for intron 1, 4.3 kb for intron 2, and 1.3 kb for intron 3. By SSCP analysis, we did not identify any mutations or polymorphisms in the FasL mRNA transcripts or in any of the 4 exons or areas of the introns adjacent to the exons.nnnCONCLUSIONnUsing the same methods used in the present studies (PCR/SSCP), one group of investigators identified a structural defect of the FasL molecule in 1 of 75 SLE patients evaluated. Among the 143 SLE patients in the present study, however, we did not identify any mutations or polymorphisms of the FasL gene. Our results suggest that a FasL defect is not the major contributing factor in the pathogenesis of SLE.

Cyclosporin A treatment for Japanese patients with severe adult-onset Still’s disease

For over 10xa0years there have been no clinical studies about adult-onset Still’s disease (AOSD) in Japan. We aimed to investigate recent clinical features and treatment of AOSD and to evaluate the efficacy of cyclosporin A (CyA) in the treatment of AOSD. The data from 34 patients with AOSD who were admitted to our hospital between 1994 and 2007 were analyzed retrospectively. Of several immunosuppressive agents, the efficacy of CyA given to seven patients was precisely evaluated. Clinical features observed in this study did not differ from those in our previous study, and serum ferritin levels were elevated in all the patients. Among immunosuppressive agents CyA, used concomitantly with corticosteroids (CS) for seven patients with severe AOSD, proved to be very effective. The disease was led to remission promptly by CyA in six patients, and all the patients except one experienced no recurrence. These results suggest that CyA can be one of the potent candidates to be used next to CS for patients with AOSD that is resistant to CS.

Analysis of CD40 ligand gene mutations in patients with primary biliary cirrhosis

An elevated immunoglobulin (Ig)M concentration in serum is a common and distinctive feature of primary biliary cirrhosis (PBC). Little is known, however, about the mechanism of hyper-IgM in PBC. CD40 ligand (CD40L) has a crucial role in immunoglobulin class switching in B cells. Mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome. To identify mutations in the gene for CD40L in PBC patients, we analyzed CD40L gene mutations, using reverse transcription (RT)-PCR single-strand conformation polymorphism (SSCP) analysis. No mutations were detected in cDNA from any of 24 PBC patients by the RT-PCR-SSCP technique. These data suggest that other, unidentified mechanisms are involved in hyper-IgM in PBC patients.