Although weakness produced by EAMG in mice is often not obvious (25) and the hang-time test requires sensitization of animals with pancuronium bromide, in this investigation the effect was so profound that this step proved unnecessary. the human disease, and strongly suggest that in disease flares complement inhibitors might have therapeutic value. Introduction Myasthenia gravis (MG) is a syndrome characterized by fatiguing skeletal muscle weakness. A large body of research on MG patients LY-2584702 tosylate salt and on experimental autoimmune MG (EAMG) in animals has shown that the disease is Ab-mediated, producing loss of or compromised function of skeletal muscle nicotinic acetylcholine receptors (AChRs). Three mechanisms have been implicated: (a) autoantibodies against AChR cross-link surface AChR and induce their endocytosis, resulting in their depletion from the postjunctional membrane; (b) the autoantibodies themselves interfere directly with AChR function by blocking acetylcholine-binding sites; and (c) the autoantibodies contribute to destruction of the endplate with consequent AChR loss (1C4). Several lines of evidence indicate that complement activation resulting from autoantibody binding to AChR is a key effector mechanism in the pathogenesis of MG. C3 activation fragments, C9, and the membrane attack complex (MAC) can be detected at motor endplates in patients and EAMG animals (5C7). Depletion of C3 by cobra venom factor protects rats against passively or actively induced disease (8C9). As a result of diminished AChR density following initial induction, animals become resistant to a second induction of passive transfer EAMG because of insufficient Ab deposition to activate complement or induce other effector responses (10C11). Administration of anti-C6 Ab prevents the development of EAMG in rats (12). In actively induced EAMG, C5-deficient mice develop less severe disease (13). Finally, treatment with soluble CR1, a complement inhibitor, can protect against EAMG in rats LY-2584702 tosylate salt (14). These data, taken together, indicate that at the motor endplate, C3b deposition and MAC assembly with consequent membrane perturbation damage the postsynaptic surface of the neuromuscular junction, compromising neuromuscular transmission. Host tissues are protected from autologous complement-mediated injury by cell surface regulators that function intrinsically in their plasma membranes [reviewed in ref. 15]. These regulators consist of the decay-accelerating factor (DAF or CD55), the membrane cofactor protein (MCP or CD46), and the membrane inhibitor of reactive lysis (MIRL or CD59). Collectively, these control proteins accelerate the decay of autologous C3 convertases ( and ) and C5 convertases ( and ) that inappropriately assemble on self cell surfaces (16), promote the cleavage of uncomplexed autologous cellCbound C4b and C3b fragments (17), and inhibit the formation of autologous MACs, which brings about cell lysis (18C20). In this study, we examined the role of DAF in protecting against AChR damage in passively induced EAMG in mice. To accomplish this, we used DAF knockout mice (21). Mice differ from humans LY-2584702 tosylate salt in that there are two genes rather than one. While the second gene, termed gene (22). Previous studies have shown that neuromuscular DAF protein in mice derives from the gene (21). Consequently, we used mice targeted at this gene. We found that following anti-AChR Ab administration, mice devoid of neuromuscular DAF protein became dramatically sicker than their littermates. Greater postjunctional membrane damage was documented by electron microscopy (EM) in the mice, more marked reduction of AChR levels was measured by specific immunoradiometric assay, and more C3 deposition specifically directed at motor endplates was found by immunohistological staining. The results strongly suggest that DAF plays a critical role in protecting the motor endplate and its surface AChR molecules against autoantibody-initiated, complement-mediated injury. Methods Daf1 knockout mice. littermates were used at 8C10 weeks of age. Induction of EAMG. EAMG was passively induced using rat anti-mouse muscle AChR mAb McAb-3 (a gift of Vanda Lennon, Mayo Clinic, Rochester, Minnesota, USA), which binds to mouse skeletal muscle AChR (23) (see Discussion). At time zero, 50 l of purified McAb-3 (4.6 mg/ml) or ascites fluid LY-2584702 tosylate salt containing an equivalent amount of mAb was injected intraperitoneally. Assessment of muscle weakness. Weakness was quantitated as described by Karachunski et al. (24) by hanging mice three times from a grid and measuring the time it took for them to release their hold and fall (holding time). Although weakness produced by EAMG in mice is often not obvious (25) and the hang-time test requires sensitization of animals with pancuronium bromide, in this investigation the effect was so profound that this step proved unnecessary. Twenty-four hours after Ab administration, the mice were placed upside-down on a grid placed 3 feet above ground level and the holding time was measured. Mice were EGR1 then kept under close observation and sequentially analyzed at 24 and 48 hours, and videotape documentation was performed. Some animals received intraperitoneal edrophonium injections to evaluate them for a neuromuscular transmission defect. Assay of mouse muscle.