René Louis Humbel

Autoimmunity to the cell cycle-dependent centromere protein p330d/CENP-F in disorders associated with cell proliferation

p330d/CENP-F is a novel proliferation-associated and cell cycle-dependent centromere autoantigen which appears to play a very important role in mitotic progression. As an initial step in exploring the clinical and biological significance of autoantibodies to this protein, we evaluated the clinical histories of 26 patients producing these antibodies. The antibodies were detected by both indirect immunofluorescence microscopy (IIF) and Western blotting. All the sera contained anti-p330d/CENP-F IgG antibodies, with an average titer by IIF of 1:6,917 (range 1:160 to 1:20,480). Most of the patients had disorders associated with abnormal or increased cell proliferation at the time the anti-p330d/CENP-F antibodies were detected. These included cancers of various types (14), chronic liver disease (3), chronic rejection of renal allografts (2), and Crohns disease (1). The average IIF titer of the anti-p330d/CENP-F antibodies in the patients with cancer, 1:10,103, was significantly higher than the average titer in non-cancer patients, 1:3,200 (P = 0.008). Autoimmunity to p330d/CENP-F appeared not to be associated with rheumatic diseases, in particular scleroderma, since only three of the 26 patients had rheumatic disease and the antibodies were not detected by IIF in a group of 351 patients with scleroderma and related disorders. Our findings, although retrospective and limited to a relatively small number of patients, point to the hypothesis that autoimmunity to p330d/CENP-F could be related to events involving increased or abnormal cell proliferation.

Anti-Ro52 reactivity is an independent and additional serum marker in connective tissue disease

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease. Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing. Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases. Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

Stratégie d’étude des anticorps anti-nucléaires

RésuméLe terme d’anticorps anti-nucléaires (ANA), consacré par l’usage, est trop restrictif puisque certains de ces anticorps reconnaissent des antigènes cytoplasmiques. Un très grand nombre d’ANA a été identifié au cours des 30 dernières années.Seul un nombre restreint de ceux-ci est spécifiquement associé à des maladies précises, connues sous le vocable de connectivites. Toutefois, des ANA peuvent assui être rencontrés dans d’autres affections, un syndrome infectieux ou inflammatoire, une néoplasie, une maladie spécifique d’organe, en particulier les maladies du foie comme l’hépatite auto-immune et certaines cholangiopathies. Enfin, il ne faut pas oublier l’apparition d’ANA survenant lors de la prise de certains médicaments. La spécificité de ces anticorps est tout à fait différente de celle que l’on observe dans les connectivites.

Anticorps anti-cytoplasmiques dans les polymyosites

En ConclusionL’immunofluorescence sur cellules HEp2 demeure la technique de choix pour la recherche des anticorps anti-cytoplasmiques spécifiques de la polymyosite. De nombreux réactifs ont été développés qui permettent l’identification des anticorps les plus fréquemment rencontrés, anti-Jo1, PL7 et PL12. Les anti-SRP se reconnaissent également sur les coupes de tissus, mais l’aspect est très voisin de celui que l’on observe avec les anti-ribosomes. L’identification des anti-ribosomes est actuellement facille à réaliser, celle des anti-SRP reste encore limitée à certains laboratoires disposant des antigènes correspondants.

Detection of rheumatoid arthritis-specific anti-filaggrin antibodies by line immunoassay shows complementarity to RF and corresponds to the AFA-blot using natural antigen

Anti-filaggrin autoantibodies (AFA) are highly specific markers for rheumatoid arthritis (RA) and can be detected by immunoblotting using human epidermal protein extracts. Furthermore, it was demonstrated that citrullination of the filaggrin epitopes is crucial for epitope recognition and that citrullinated peptides are also recognized by these specific autoantibodies. Based on these data, a line immunoassay (LIA) was developed containing as individual markers in vitro citrullinated recombinant filaggrin and two citrullinated synthetic peptides. Firstly, a comparison was made between this prototype LIA and the AFA blot using natural filaggrin. A blind serum panel consisting of 25 early RA, 25 longstanding RA, and 25 disease controls was selected. Results showed a similar performance of both tests at a specificity level of 95%, while the LIA proved significantly better (P = 0.035) than the AFA blot at 99% specificity. At the latter specificity level, 2 out of 17 RF negative samples were retrieved on LIA but not on Western blot. The LIA was further evaluated on sera obtained from 335 RA patients and 254 patients with non-RA rheumatological disorders in a retrospective study. The overall sensitivity of the LIA including three markers (LIA3) was 65.1% versus 61.8% if only the reactivity towards the citrullinated peptides was considered (LIA2). The specificity of LIA3 was 97.6% versus 98.4% for LIA2, which correlates with an estimated positive predictive value (PPV) of 87.3% for LIA3 and 90.7% for LIA2. Thirty-seven percent (30/81) of the RF-negative RA samples proved LIA2-positive, while 52 out of 55 RF positive control samples were negative for anti-filaggrin. Higher specificity and sensitivity was obtained for LIA2 in comparison with anti-RA33 immunoblot, whereas good agreement could be observed with anti-keratin antibody (AKA) testing. In conclusion, anti-filaggrin autoantibodies can be readily detected by a LIA test based on citrullinated peptides, resulting in a high specificity and hence high PPV for RA. The assay can serve as a user-friendly alternative for AKA immunofluorescent and immunoblot techniques. Together with the RF complementarity, this test provides a valuable tool in the differential diagnosis of RA.