Our results using RT PCR confirm

Our results using RT-PCR confirm earlier findings (Wilisch et al., 1999, Bruno et al., 2004) including the presence of both the P3A+ and P3A− isoforms of the α-subunit (Beeson et al., 1990). The lack of detection of the ε-subunit mRNAs in some thymomas, and lack of α-, β-, δ- and γ-subunit mRNAs by RPA, even in samples positive by RT-PCR, suggests that the levels of these subunits are relatively low in all cases. The ε-subunit mRNAs that we detected by RPA are very unlikely to have derived from myoid cells, which are rare in most thymomas (Rosai and Levine, 1976). Our failure to detect ε by RPA in normal thymus, which contains myoid cells that express fetal AChR (Schluep et al., 1987), is consistent with this and implies, therefore, over-expression in thymomas. Conversely, the α-, β-, δ- and γ-AChR subunits that we detected in thymoma samples by RT-PCR might well have derived from myoid cells in adjacent normal thymic tissue (Leite et al., 2007). Thymomas show strong associations with autoimmune disorders (Buckley et al., 2001) and generate and export T cells (Muller-Hermelink and Marx, 2000). In theory, the tumors could either be directly selecting/sensitizing T cells against self-antigens that they express (Nagvekar et al., 1998) or failing to tolerize against antigens that they do not express (Marx and Muller-Hermelink, 2005). The over-expression of the ε-subunit in thymomas argues in favor of the former, as does the preference for adult AChR shown by serum tcY-NH2 from the two patients with the highest levels of ε-subunit mRNA. In the other five, the more prevalent antibodies against fetal AChR, which are common in MG, might be the end-result of determinant spreading (Vincent et al., 1998) initiated by earlier responses to the ε-subunit. Indeed, several features of the AChR ε distinguish it from the other subunits and suggest a special potential for breaking tolerance in MG. Firstly, transcription of the ε-subunit is under tight regulatory control, and is normally restricted to the subsynaptic nuclei of muscle fibers (Sanes and Lichtman, 2001), suggesting that aberrant over-expression in thymomas could be immunologically ‘dangerous'. Secondly, since its expression is so minimal in the normal thymus, it may tolerize incompletely with the result that patients would have some circulating ε-subunit-specific T cells that could colonize the thymoma. Thirdly, as the ε-subunit appears to be produced in excess over the other AChR subunits in thymomas, it is much more likely to be degraded rapidly than to be incorporated into whole AChR molecules, with the result that the resulting peptides would be available for presentation to developing T cells, either by the neoplastic epithelial cells (Gilhus et al., 1995) or the abundant adjacent dendritic cells. It has already been shown that the ε-subunit includes at least one important helper T cell epitope (Hill et al., 1999) and T cells from MG patients have been found to recognize epitopes unique to the ε-subunit (Ragheb et al., 2005). The autoimmune regulator (AIRE) plays a crucial role in driving the expression of self-antigens in medullary thymic epithelial cells. It has been shown to play a role in controlling thymic expression of the AChR α-subunit from the CHRNA locus, and possibly setting the threshold for self-tolerance versus autoimmunity for the AChR (Giraud et al., 2007). However, AIRE expression is down-regulated in human thymoma. Whereas the presence of AIRE is responsible for activating a number of loci, and its loss correspondingly reduces their expression (Ströbel et al., 2007), for other gene loci AIRE may negatively regulate expression (Johnnidis et al., 2007). It may be that AIRE has a contrasting role in controlling thymic expression from the CHRNE locus. The relationship with tumour type is intriguing. In type A or AB thymomas the epithelial cells frequently show ‘medullary’ or ‘spindle’ morphology, and thymocytes may be focally abundant (type AB or ‘mixed’ thymoma) or absent (type A). In type B the pathology resembles disorganized thymic cortex with polygonal epithelial cells and abundant (B1 and B2) or some (B3) developing T cells; about 50% of type B thymomas are invasive. Around 30% of all patients with type B thymomas develop MG, but curiously only 5–25% of patients with A or AB thymomas (Ströbel et al., 2004). One could hypothesise that, in order to induce an immune response, higher levels of AChR ε-subunit expression are required in A/AB thymomas than in type B2 tumours, which provide a more immunogenic environment. Subsequent determinant spreading of the response to other AChR epitopes expressed by myoid cells in the adjacent thymus would generally occur leading to the typical serum antibodies found in most patients with MG.