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.

The natural biodegradation of seven polycyclic aromatic hydrocarbons (PAHs) by native microorganisms was studied in five soils from Normandy (France) from diffusely polluted areas, which can also pose a problem in terms of surfaces and amounts of contaminated soils. Bioavailability tests using cyclodextrin-based extractions were performed. The natural degradation of low molecular weight (LMW) PAHs was not strongly correlated to their bioavailability due to their sorption to geosorbents. Conversely, the very low degradation of high molecular weight (HMW) PAHs was partly correlated to their poor availability, due to their sorption on complexes of organic matter and kaolinites or smectites. A principal component analysis allowed us to distinguish between the respective degradation behaviors of LMW and HMW PAHs. LMW PAHs were degraded in less than 2-3 months and were strongly influenced by the relative percentage of phenanthrene-degrading bacteria over total bacteria in soils. HMW PAHs were not significantly degraded, not only because they were less bioavailable but also because of a lack of degrading microorganisms. Benzo[a]pyrene stood apart since it was partly degraded in acidic soils, probably because of a catabolic cooperation between bacteria and fungi.

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.

No abstract

Affinity capillary electrophoresis (ACE) is a robust tool for the study of noncovalent biomolecular interactions and to determine the binding constants. It is advantageous due to the speed of analysis, the high and reproducible separation efficiencies, the low consumption of analytes, the ability to study several interactions at the same time, and to cover a wide range of affinity. The use of an ion trap-mass spectrometer as a sensitive and specific detector, coupled on-line with a classical UV detector, permits extracting simultaneously the electropherograms corresponding to each ionic species. The mass spectra, acquired by scanning the results of a first separation due to ACE, were assimilated into a virtual two-dimensional (2-D) gel. We developed a software application, which was designed to create and analyze these virtual 2-D gels. The validity of this new analytical tool for probing biomolecular interactions has been demonstrated on mixtures of antibiotics of the vancomycin group and several dipeptide substrates. Using the dynamic equilibrium affinity electrophoresis approach, we have shown that molecular components interacting with a low affinity are easily located on the virtual 2-D gels, and that binding constants and stoichiometry of the interactions can be assessed. As the binding constants derived from ACE-electrospray ionization-mass spectrometry (ESI-MS) are unreliable, they must only be determined with the UV detector.

Antigenic cross-reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross-reactivity of SLE-related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody-secreting B-lymphocytes. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW x BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp-2 cells as assessed by indirect immunofluorescence assay. Proteins from HL-60 extract were separated by 1-D and 2-D PAGE. Western blotting with mAb 4B7 after SDS-PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2-D PAGE. After trypsin in-gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) Reflector or quadrupole-time of flight (Q-TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS-A RNP. Thus, these results showed that 2-D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross-reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B-cell tolerance observed in SLE.

Hydrocarbon contamination, which can alter the physiochemical and biological properties of the subsurface, can be monitored by using geochemical analyses and integrated geophysical techniques. Electrical resistivity tomography, induced polarization, ground‐penetrating radar, and self‐potential methods were used in this work to characterise the hydrocarbon contamination and investigate the geoelectrical properties of a site impacted by an aged hydrocarbon plume. Throughout the investigation, geoelectrical measurements supported the conductive plume model and consistently recorded low‐resistivity anomalies and high‐chargeability values in the contaminated aquifer. Furthermore, the low‐resistivity anomalies were found to be coincident with regions of ground‐penetrating radar attenuated reflections and significant negative self‐potential anomalies associated with oxidation–reduction processes. These findings were supported by the geochemical measurements, which revealed depleted concentrations of terminal electron acceptors (TEAs) and elevated amounts of ions and total dissolved solids, which could be attributed to bacterial biodegradation of hydrocarbons. The study showed that biological alterations of hydrocarbon‐induced subtle changes in the pore water biogeochemistry, which consequently modified the geophysical properties of the contaminated sediments. Based on these observations, the extent of groundwater contamination was delineated according to the geophysical contrast between the contaminated and clean zones. The combination of different geophysical methods constrained by geochemical point measurements provided insight on the different processes that might have modified the soil and groundwater biogeochemical properties.

Antigenic cross-reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross-reactivity of SLE-related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody-secreting B-lymphocytes. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW x BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp-2 cells as assessed by indirect immunofluorescence assay. Proteins from HL-60 extract were separated by 1-D and 2-D PAGE. Western blotting with mAb 4B7 after SDS-PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2-D PAGE. After trypsin in-gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) Reflector or quadrupole-time of flight (Q-TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS-A RNP. Thus, these results showed that 2-D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross-reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B-cell tolerance observed in SLE.