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  • XL-888 In order to investigate their effects in vitro theref

    2022-11-08

    In order to investigate their effects in vitro, therefore, we first used TE671 cells, which have provided a useful source of human AChR for in vitro and radioimmunoassay studies. We found no marked effect of either MuSK-MG or SNMG sera on AChR numbers or on AChR subunit expression. This appears to contrast with the work of Guyon et al., 1994, Guyon et al., 1998 and Poea et al. (2000) who reported that more clinically severe MG patients, including SNMG patients, showed an upregulation of some AChR genes both in patient-derived muscle biopsies, obtained from sternocleidomastoid muscle samples, and in in vitro experiments on TE671 cells. However, these findings were more prominent in AChR-MG than AChR-antibody negative MG patients, and the MuSK antibody status of the SNMG patients was not available at that time. Our results also contrast with a recent study by Boneva et al. (2006) which demonstrated that some MuSK-MG sera can alter morphology, growth and cell proliferation of TE671 XL-888 when incubated for up to 72 h. Their sera also reduced mRNA expression of RhoA and cdc42 and of AChR α and β and of rapsyn — all important players in the formation of AChR clusters and in the maintenance of the postsynaptic membrane at the NMJ. Interestingly, this effect was seen with only about 50% of MuSK-MG sera and results with the other MuSK-MG sera were not distinguishable from controls suggesting that the effect may not be related to the MuSK antibody. Even in incubations of up to 48 h, we did not find any evidence suggesting an effect of MuSK-MG sera on cell numbers of TE671 cells; the reason for this discrepancy is unclear. It is difficult in any case to compare our results, since there is no evidence that they heated and dialysed their sera as we did here, and the MuSK antibody titres were generally lower than those which we find (eg. McConville et al., 2004). On the other hand, the upregulation of atrogin-1 expression (Boneva et al., 2006) parallels our findings of upregulation of another atrophy-related gene product, MuRF-1, by MuSK-MG sera in C2C12 myotubes (Benveniste et al., 2005) and supports our clinical findings that MuSK-MG is associated with muscle atrophy in MRI studies (Farrugia et al., 2006). The TE671 cells, however, cannot be differentiated to form myotubes, and do not express rapsyn, a component that is crucial in the signalling cascade and clustering pathway. The C2C12 myotube system allowed us to study the effect of MuSK antibodies on agrin-induced AChR clusters. We showed that MuSK-MG sera, but not SNMG sera, reduced the number of AChR clusters, as previously reported (Hoch et al., 2001). This decline in cluster numbers was associated with an increase in cluster length and no overall effect on AChR numbers. Altering the duration of exposure to agrin or to the serum or both did not make any substantial difference to the results. Quantitative mRNA measurements showed a trend towards reduced AChR α subunit mRNA in MuSK-treated cells but no other changes. These results, therefore, suggest that either the MuSK antibodies do not affect AChR numbers, or that some key component is missing in these cells. The recently identified Dok-7 protein, that is a binding partner for MuSK, could be such a protein (Beeson et al., 2006). Therefore, from our in vitro model of synaptogenesis, it remains unclear how MuSK antibodies cause MG but our data are not in disagreement with other recent findings. There is evidence to suggest that MuSK antibodies do not activate complement and do not act via complement-mediated destruction (McConville et al., 2004, Shiraishi et al., 2005). In rat muscle, silencing of MuSK RNA, using RNAi demonstrated changes in the NMJs with severe fragmentation of AChR clusters and loss of the integrity of the postsynaptic membrane but only after 6 weeks (Kong et al., 2004). We know that there are limitations in the use of the C2C12 cells in that they express the foetal isoform of the mouse AChR, and the AChRs within the agrin-induced clusters have a higher turnover rate than at the adult NMJ (Fambrough et al., 1979). This suggests that the in vitro model is insufficient to demonstrate the effects of MuSK antibodies since there is a limit to the duration of treatment of cells in culture, and the density and stability of the AChRs are different. Active immunisation models in mice or rabbits immunised with recombinant MuSK have found evidence of clinical weakness and altered neuromuscular junction morphology (Jha et al., 2006, Shigemoto et al., 2006). Sera from these animals inhibited agrin-induced AChR aggregation in C2C12 myotubes (Shigemoto et al., 2006). Further active as well as passive immunisation animal models are crucial to help us unravel further the pathogenicity of these antibodies.