Objective. To identify a new autoantigen/ autoantibody population in rheumatoid arthritis (RA) sera.Methods. Following a population-based recruitment effort, 255 patients with very early arthritis (median disease duration 4 months) were studied using different clinical, biologic, and radiologic assessments. After a followup period of 1 year, patients were classified as having RA (n ‫؍‬ 145), non-RA rheumatic diseases (n ‫؍‬ 70), and undifferentiated arthritis (n ‫؍‬ 40). Patients' sera were analyzed by one-dimensional (1D) and 2D Western blotting. The recognized 50-kd protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). RA serum reactivities were evaluated against the recombinant protein synthesized by an in vitro coupled transcription-translation system.Results. On 1D Western blots, 36 of the 145 RA sera bound to a 50-kd polypeptide. On 2D Western blots, anti-50-kd؉ RA sera recognized a triplet of isoelectric point 6.5-7.0 and a molecular mass of 50 kd. The 3 spots of the triplet were analyzed by MALDI-TOF MS and were shown to correspond to human ␣-enolase. A goat anti-enolase antiserum, which recognized a band comigrating with the 50-kd antigen on 1D Western blots, gave a labeling pattern on 2D Western blots similar to that observed with anti-50-kd؉ RA sera. Among the 36 RA sera that identified ␣-enolase in protein maps, only 8 recognized the recombinant (unmodified) ␣-enolase. The specificity of anti-␣-enolase antibodies for RA was 97.1%. Half of the anti-␣-enolase-positive RA patients were negative for both rheumatoid factor and antifilaggrin antibodies. The presence of anti-␣-enolase antibodies was the greatest predictive factor of radiologic progression in the first 66 RA patients included.Conclusion. Autoantibodies to ␣-enolase, an enzyme of the glycolytic pathway, are present in the sera of patients with very early RA and have potential diagnostic and prognostic value for RA.

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Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White × BXSB)F1 (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, α-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-α-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.

We have compared the protein maps of agar-entrapped Pseudomonas aeruginosa cells to those of free counterparts grown in the presence or absence of the immobilized-cell gel support. Principal component analyses (PCAs) were used to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis. PCA of the data matrix (923 rows x 6 columns) in which spot density values were standardized horizontally extracted three principal components (PCs) with eigenvalues higher than 1, accounting together for 71.6% of the variability in the data. Principal component 1 (PC1) opposed free (F) and agar-entrapped (AE) cultures, with a low contribution of agar-released, free (ARF) cultures to PC1. Inversely, the contribution of ARF cultures to PC2 was high, opposing those of AE and F cultures. Component 3 was related to the duration of incubation. Only 10% of total proteins were upregulated in AE cells during the first 18 h of incubation, the number of underexpressed peptides balancing that of overexpressed ones. Downregulation clearly became the dominant tendency when the incubation time was extended to 48 h. These results demonstrate that AE and ARF bacteria are physiologically different from F organisms.