Methylation of arginine residues has been implicated in many cellular activities like mRNA splicing, transcription regulation, signal transduction and protein-protein interactions. Protein arginine methyltransferases are the enzymes responsible for this modification in living cells. The most commonly used methyltransferase in man is protein arginine methyltransferase 1 (PRMT1). Since methylation processes appear to interfere in the emergence of several diseases, including cancer, we investigated the localisation of the protein in cancer tissue and, for the first time, the relation that possibly exists between the expression of PRMT1 gene and breast cancer progression. We used tumour specimens from 62 breast cancer patients and semi-quantitative RT-PCR to determine the expression of PRMT1 gene and was found to be associated with patient's age (p = 0.002), menopausal status (p = 0.006), tumour grade (p = 0.03), and progesterone receptor status (p = 0.001). Survival curves revealed that PRMT1-v1 status-low expression relates to longer disease-free survival (DFS; p = 0.036). To the contrary, PRMT1-v2 status is not associated neither with the clinical or pathological parameters nor with DFS (p = 0.31). PRMT1-v3 was not statistically significantly expressed in breast cancer tissue. Selected cancer and normal breast samples were stained for PRMT1. In both normal and cancerous breast tissues, staining was in the cytoplasm and only in rare cases the cell nucleus appeared stained. Present results show a potential use for this gene as a marker of unfavourable prognosis for breast cancer patients.

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Six hundred and twelve monoclonal Ig (MIg) were studied for their antibody activity against the following autoantigens: actin, tubulin, thyroglobulin, myosin, myoglobin, fetuin, albumin, transferrin, and double-stranded DNA (dsDNA). Of these 612 MIg, 36 (i.e., 5.75%) were shown to possess antibody activity. Thirty-two of these 36 (5.22% of the total) were mainly directed against actin. The four others were directed, respectively, against tubulin, myosin, thyroglobulin, and dsDNA. The interaction of the MIg with the respective antigen was demonstrated by immunoenzymatic methods with monospecific antisera and by blotting experiments. Furthermore, this interaction in the 12 cases studied was mediated by the dimeric fragment F(ab')2 of the MIg. The MIg with antitubulin, antithyroglobulin, and anti-dsDNA activities were exclusively inhibited by their homologous antigens. Those with antiactin activity were predominantly inhibited by actin and also by tubulin and thyroglobulin. The one binding to myosin was, for the most part, inhibited by myosin and also significantly by actin and tubulin. Retrospective clinical analysis was possible for 31/36 patients. Twenty- six of 31 had malignant lymphoplasmocytic disorders. The five others were followed for miscellaneous disorders without overt signs of multiple myeloma (MM) or Waldenstrom's macroglobulinemia (WM). The correlation between the antibody activity of the MIg and the clinical features is discussed. These results indicate that a high proportion of MIg possess antibody activity against actin (5.22%). This incidence contrasts sharply with the positive reactions found toward all the other antigens tested: only one each for dsDNA, tubulin, thyroglobulin, and myosin, and none against myoglobin, fetuin, albumin, and transferrin. The significance of these results and the relationship between MIg and natural antibodies are discussed.

SUMMARYThyroglobulin (Tg) is a target autoantigen in autoimmune thyroid diseases, such as Graves' disease (GD) and Hashimoto's thyroiditis. In a previous study we identified three 20mer Tg peptides bearing epitopes of autoantibodies associated with GD (TgP15, TgP26 and TgP41: sequences 2339-2358, 2471-2490 and 2651-2670 of human Tg, respectively). In the present study, we investigated the antigenicity of the above peptides in experimental immunization with Tg, the immunogenicity of antigenic peptides and the possibility of intramolecular B-cell epitope spreading during peptide immunization. For this purpose, two rabbits were injected with human Tg in CFA six times, every three weeks. Two control animals were injected only with CFA. Testing of antisera and of affinity-purified antibodies, by ELISA against the three peptides, revealed reactivity only to TgP41. This synthetic peptide was subsequently administered to two rabbits, in its free form (100 m g in CFA six times, every two weeks). A strong serological response was developed not only against TgP41, but also to intact human and rabbit Tg. Immunization with TgP41 induced intramolecular B-cell epitope spreading, i.e. production of antibodies to sites on Tg other than that corresponding to TgP41, as revealed by immunoadsorption and competitive ELISA. Histopathological studies did not reveal any infiltration in thyroid glands. We conclude that peptide TgP41 encompasses not only an epitope of disease-associated autoantibodies, but also a dominant immunogenic epitope of experimentally induced Tg-specific antibodies, able to drive B-cell epitope spreading.

SUMMARYWe have previously shown that the human thyroglobulin (hTg) 20-mer peptide p2340 (aa 2340± 2359) contains an epitope recognized by Tg-reactive B cells in patients with Graves' disease. The presence of several E k -binding motifs within p2340 prompted us to examine whether this peptide can stimulate a T-cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2 k ) mice. The peptide was found to be immunogenic at the T-cell level since it induced speci®c proliferative responses as well as interleukin-2 and interferon-g secretion in secondary cultures of peptide-primed lymph node cells (LNC). The p2340-speci®c proliferation was blocked almost completely by an E k -speci®c monoclonal antibody (mAb) but was unaffected by a control A kspeci®c mAb. Peptide-primed LNC did not respond to intact hTg and conversely, LNC primed in vivo with hTg did not respond to p2340 in culture, suggesting that p2340 contains non-dominant Tcell epitope(s). Direct subcutanaeous challenge of AKR/J mice (n 9) with p2340 in adjuvant, elicited mild to moderate EAT (in®ltration index of 1±2) and strong p2340-speci®c immunoglobulin G responses in all mice tested. These data delineate a new thyroiditogenic sequence within the carboxyl terminal region of hTg.