Kazuhiko Kaneda

DNA methylation: its contribution to systemic lupus erythematosus.

AbstractRecent studies on epigenetics, including the methylation of DNA and the enzymes regulating methylation, seem likely to contribute to understanding the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). In fact, the relationship between DNA methylation and SLE has long been the subject of investigation. To obtain a deeper understanding of the role of DNA methylation in the onset of SLE, we reviewed the findings reported in the literature and our own data about DNA methylation and SLE. Various studies have indicated the possible importance of DNA methylation, especially hypomethylation, in the aetiology of SLE. Epigenetic studies may provide clues for elucidating the pathogenesis of SLE and for developing new strategies to treat this disease.

Reactivity of Anti-Proliferating Cell Nuclear Antigen (PCNA) Murine Monoclonal Antibodies and Human Autoantibodies to the PCNA Multiprotein Complexes Involved in Cell Proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the “protein machinery” for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.

Churg–Strauss syndrome (CSS) in a patient receiving pranlukast

Pranlukast is a cysteinyl leukotriene receptor I antagonist (LTRAs) approved for treatment of asthma in Japan since 1995. Compared to other LTRAs, such as zafilukast and montelukast, only few cases with Churg-Strauss syndrome (CSS) have been reported in association with treatment with pranlukast. We describe a 17-year-old Japanese male patient who developed CSS with a 13 month history of mild asthma receiving pranlukast for 11 months without systemic and/or inhaled corticosteroid administration prior to development of CSS. From the aspect of temporal relationship between treatment with pranlukast and development of CSS, a direct induction of CSS by pranlukast is suggested in our case.

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases.

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.

A case of aplastic anemia in a patient with systemic lupus erythematosus

Abstract A case of aplastic anemia with a 16-year history of systemic lupus erythematosus (SLE) is described. The diagnosis of aplastic anemia was established by bone marrow biopsy. Aplastic anemia is an extremely rare complication of SLE. The pathogenesis of aplastic anemia associated with SLE remains to be clarified.

Detection of serum IgE class anti-SSA antibodies in mothers with foetal loss

We previously reported a close relationship between serum immunoglobulin (Ig)E and anti-SSA antibody levels in mothers with foetal loss. Here, we investigated the existence of IgE class anti-SSA antibodies (IgE anti-SSA antibody) and the relationship of such antibodies with foetal loss. Serum samples from 24 women who were positive for IgG class anti-SSA antibody (IgG anti-SSA antibody) were examined for IgE anti-SSA antibody by enzyme-linked immunosorbent assay (ELISA). Then, a retrospective analysis of the relationship between the IgE anti-SSA antibody positivity and the frequency of foetal loss was performed. Using our ELISA system, IgE anti-SSA antibodies were detected in the serum samples, and the frequency of foetal loss was increased among the mothers with higher IgE anti-SSA antibody titers. Our results indicate that IgE anti-SSA antibodies may be a useful marker for the risk of foetal loss in mothers with anti-SSA antibodies.

Analysis of the Structure of Proteasome‐Proliferating Cell Nuclear Antigen (PCNA) Multiprotein Complex and Its Autoimmune Response in Lupus Patients

We have recently found that the PCNA multiprotein complexes (PCNA complexes) associated with cell proliferation can be purified using monoclonal antibodies (mAbs) to PCNA.1 We attempted to analyze the structure of the complexes and found that proteasome, which had been known as an ATP-dependent proteolytic enzyme involved in antigen presentation on class I major histocompatibility molecules, was one of the elements of the PCNA complex. We also found that there was an autoimmune-response linkage between PCNA and proteasome.

Analysis of autoantibodies to cell cycle-associated antigens

Abstract To determine the specificities of autoantibodies targeting cell cycle-associated antigens in rheumatic diseases, we studied 30 sera which were obtained from patients visiting our hospital and which exhibited a variegated speckled pattern in indirect immunofluorescence (IF). The immunoreactivities of the sera were analyzed by Western blotting (WB) and IF. Various reactivities to cellular components were observed in IF. The sera reacted with proteins of various molecular weights in WB. Serum OH from a patient with rheumatoid arthritis showed fine speckled nucleoplasmic and nucleolar staining at interphase, discrete dot staining associated with chromosomes and the midbody at mitosis, and reacted with a 34-kD polypeptide in WB. The target antigen was different from proliferating cell nuclear antigen (PCNA). The 34-kD antigen was characterized by IF using double staining procedures and cell synchronization. The results showed that the expression of the antigen was mainly observed between the late G1 and M-phases. This study indicated that various cell-cycle-associated antigens were recognized in sera from patients with rheumatic diseases, and suggested that a 34-kD antigen recognized by serum OH was a novel cell cycle-associated autoantigen.

Activated peripheral blood mononuclear cells detected in lupus patients using cDNA coding for proliferating cell nuclear antigen

Abstract The expression of proliferating cell nuclear antigen (PCNA) mRNA in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) was measured by dot blot hybridization using a PCNA cDNA, and correlated with the percentage of PCNA-positive cells detected immunohistochemically using a monoclonal anti-PCNA antibody. PCNA-positive PBMCs were detected in 72.2% of SLE patients (n = 36), which is significantly more than among healthy controls. In addition, among those in whom PCNA expression was detected, the percentage of PBMCs expressing PCNA was significantly higher in SLE patients (mean 2.5% vs. 0.15%). The level of PCNA mRNA was increased in PBMCs from 83.3% of SLE patients, and was significantly correlated with the percentage of PCNA-positive cells (r = 0.54, P < 0.01) and with the disease activity score (r = 0.56, P < 0.01). A longitudinal study of two SLE patients confirmed that PCNA mRNA expression and the percentages of PCNA-positive cells varied in parallel with disease activity. Thus, an analysis of activated PBMCs from SLE patients using PCNA cDNA may be a useful method by which to estimate SLE disease activity.