To promote an understanding of autoimmunity in BD, we surveyed autoAgs in patients with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE-WB, out of which eight spots were identified. They are enolase-1, cofilin-1, vimentin, Rho-GDI β protein, tubulin-like protein, and actin-like proteins. The autoAbs to one of the identified proteins, cofilin-1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin-1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%) of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin-1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs.
Autoantibodies to proliferating cell nuclear antigen (PCNA) are specifically, if rarely, present in systemic lupus erythematosus (SLE) patient sera. Even SLE patients lacking PCNA reactivity often show reaction to PCNA-binding protein. Here, immunoreactivity to chromatin assembly factor-1 (CAF-1), an essential molecule for DNA replication and a PCNA-binding protein, was compared for the sera of SLE patients, normal healthy controls (NHCs) and other disease controls, and in autoimmune sera reactive to standard autoantigens, by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoblotting. CAF1 and IRF1 expression in SLE and NHC peripheral mononuclear cells were compared by quantitative real-time polymerase chain reaction. Serum interferon-γ-inducing protein-10 and anti-double-stranded (ds)DNA antibody levels were measured by ELISA. Increased CAF-1 autoimmune reactivity was recognized in SLE or serum anti-dsDNA antibody-positive patients. Significantly greater central nervous system (CNS) involvement (aseptic meningitis) and serum anti-dsDNA antibody titers were present more often in anti-CAF-1 antibody-positive than antibody-negative SLE patients. IFN-γ positively regulated CAF-1 expression in vitro and was associated with anti-CAF-1 antibody production in SLE. Thus, a novel anti-CAF-1 autoantibody is frequently found in patients with SLE and is a useful biomarker for diagnosis, especially in cases with CNS involvement. Aberrant IFN-γ regulation appears to play an important role in anti-CAF-1 antibody production in SLE.
Heat-induced cell death and apoptosis were studied with respect to intracellular ATP. Studies on the relationship between hyperthermic cell-killing at 44 degrees C and cellular ATP levels in four cell lines grown as monolayers and six cell lines grown in suspension showed good correlations between cellular ATP levels and the sensitivity to heat. D(0) values (the dose required to reduce survival in the linear portion of the response by 63%) linearly increased with an increase in cellular ATP levels. No such changes in sensitivity to heat were observed between the cells cultured at different cell densities, regardless of the change in the cellular ATP level. These results suggest that cellular intrinsic ability to supply ATP rather than the level of pooled ATP per se is responsible for the thermal response. Heat-induced apoptosis in L5178Y cells was observed following treatment at 42 degrees C for 70 min, 44 degrees C for 20 min or 47 degrees C for 3 min, which corresponded to surviving fractions of 25, 0.6 and 0.8%, respectively, but not at 47 degrees C for 20 min, indicating that mild heat shock induced apoptosis. 2-deoxyglucose (2DG) and 2,4-dinitrophenol (DNP) increased the sensitivity to heat and affected the mode of cell death. Cells treated with 2DG and DNP (2DG/DNP) were heated at 42 degrees C for 20 min, and then incubated at 37 degrees C for up to 2h in the presence or absence of 2DG/DNP. In the absence of 2DG/DNP, the cellular ATP level recovered to 76% of the control level and DNA ladder formation was observed, whereas in the presence of 2DG/DNP, the cellular ATP level was further decreased (3-7% of the control) and no DNA fragmentation was detected. These results suggest that the inhibition of ATP synthesis is closely associated with the enhancement of sensitivity to heat and that ATP is required for the induction of apoptosis.
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