Ординатура / Офтальмология / Английские материалы / Advances in Understanding Mechanisms and Treatment of Infantile Forms of Nystagmus_Leigh, Devereaux_2008

the role of extraocular proprioception. J AAPOS. 1999;3:166–182.

12.Hertle RW, Dell’Osso LF, FitzGibbon EJ, Thompson D, Yang D, Mellow SD. Horizontal rectus muscle tenotomy in patients with infantile nystagmus syndrome: a pilot study. J AAPOS. 2004;8:539–548.

13.Dell’Osso LF. Extrocular muscle tenotomy, dissection and suture: an hypothetical therapy for congenital nystagmus. J Pediat Ophth Strab. 1998;35:232–233.

14.Büttner-Ennever JA, Horn AKE. The neuroanatomical basis of oculomotor disorders: the dual

motor control of extraocular muscles and its possible role in proprioception. Curr Opin Neurol. 2002;15:35–43.

15.Hertle RW, Chan CC, Galita DA, Maybodi M, Crawford MA. Neuroanatomy of the extraocular muscle tendon enthesis in macaque, normal human, and patients with congenital nystagmus. J AAPOS. 2002;6:319–327.

16.Wang Z, Dell’Osso LF, Zhang Z, Leigh RJ, Jacobs JB. Tenotomy does not affect saccadic velocities: support for “small-signal” gain hypothesis. Vision Res. 2006;46:2259–2267.

ABSTRACT

Extraocular muscle (EOM) is nearly always involved in myasthenia gravis. The reasons are multifaceted, but the authors contend that an intrinsic low level of cell surface complement inhibitors is a key factor. Complement inhibitors have been shown to protect the neuromuscular junction (NMJ) from attack by acetylcholine receptor (AChR) antibodies. The EOMs of rodents have a low level of expression of complement regulator genes, and their NMJs have low protein expression, which would put them at greater risk for the antibodymediated, complement-dependent pathology of myasthenia gravis. Preliminary studies support that EOM suffers greater complement damage in experimentally acquired myasthenia gravis. Among ocular myasthenic patients, the serum concentration of AChR antibody is low or absent, which suggests that EOMs are more susceptible to antibody injury. Taken together, these observations support the complement hypothesis of EOM susceptibility to myasthenia gravis, which states that extraocular muscle susceptibility to myasthenia gravis is caused by a low level of intrinsic complement regulators at their neuromuscular junctions.

Myasthenia gravis (MG) is a neuromuscular transmission disorder caused by antibody-mediated complement injury to the neuromuscular junction (NMJ).1-3 The resultant damage to the NMJ leads to loss of acetylcholine receptors (AChR) and simplification of the postsynaptic architecture.4 Patients with MG demonstrate a

distinct predilection for involvement of extraocular muscle (EOM). While the authors have, in previous reviews, presented arguments for functional and physiological reasons as to why this might be the case,5,6 in this chapter we present mounting evidence that a low level of complement-inhibitory proteins may be the predominant contributor to the differential involvement of EOM by MG. If correct, this contention has significant therapeutic ramifications. Complement inhibitors are being evaluated in human trials, and their application to MG in general, and its ocular complications in particular, may be of significant benefit to patients. This chapter provides a brief review of the pathophysiology of MG, followed by a discussion of possible explanations for the differential involvement of EOM by MG. The authors then present their observations to support the “complement hypothesis” as an explanation for the propensity of EOM manifestations produced by MG.

PATHOPHYSIOLOGY OF MG

Since the proposals by Simpson and Nastuck in the 1950s that MG was an autoimmune disorder, MG has become one of the few disorders to fulfill strict criteria for an antibody-mediated autoimmune disorder:

(a) antibody is identified at the NMJ, the primary site of pathology; (b) when injected into experimental animals, antibody—essentially exclusively the comple- ment-fixing antibody fraction, from sera of MG patients, or antibody directed against the muscle AChR—causes weakness and fatigue—the hallmarks of human MG—and results in electrophysiological

evidence of a neuromuscular transmission defect;

(c) immunization of animals with purified AChR leads to autoantibody formation, weakness, and pathological findings of MG; and (d) plasma exchange, which removes antibody, reduces clinical manifestations of MG.1-3

AChR antibody affects neuromuscular transmission by three mechanisms: (1) a process of antigenic modulation, which leads to the accelerated degradation of AChR; (2) block of AChR function; and (3) activation of complement-mediated disruption of the NMJ. Antigenic modulation refers to the ability of an antibody to cross-link two AChRs, which leads to a signal that accelerates endocytosis and degradation of the surface AChR.7 The ability of certain AChR antibodies to produce antigenic modulation is a function of not only all IgG having two antigen binding sites but also the close proximity of AChR on the postsynaptic muscle surface. Therefore, the epitope location on the AChR offers the possibility for a single AChR antibody to bind to two AChR. AChR antibody of MG patients accelerates the degradation rate of the AChR in vivo and in cultured muscle cells.8 AChR antibody may block the acetylcholine (ACh) binding site, and experimentally acquired MG (EAMG) produced in animals by infusion of such antibodies causes rapidly developing, severe weakness.9 However, in humans, such antibodies do not appear to be clinically important.1-3

The unifying mechanism by which AChR antibody causes neuromuscular transmission failure is the reduction of endplate potential to below the required level for achievement of a propagated action potential. Under normal conditions, the endplate potential is well above this level. The endplate potential is a function of

(a) the quantal release of ACh, (b) the conduction properties and density of the postsynaptic AChR, and

(c) the acetylcholinesterase (AChE) activity concentrated at the NMJ.4,10 The postsynaptic folds form a high-resistance pathway that focuses endplate current flow on voltage-gated sodium channels, which are concentrated at the bottom of the folds, and MG produces a loss of postsynaptic folds.11,12 Compromise of AChR activation decreases the endplate potential, but the potential may still activate an action potential. However, if release of ACh is reduced by repetitive activity, the endplate potential may fall below the threshold needed to trigger the action potential. This difference between threshold and endplate potential is termed the safety factor.

A new autoantigen has been identified as a cause of a subset of so-called seronegative MG patients, or patients with clinical and electrophysiological evidence of MG but an absence of AChR antibody. The muscle-specific kinase (MuSK) protein is localized to the NMJ and plays a role in clustering of AChR to the postsynaptic surface. Hoch et al.13 found that about

one-third of patients with seronegative MG have antibodies directed against MuSK, and that these antibodies impair clustering of AChR. Since then, there has been a rapid proliferation of discovery that identifies MuSK antibody patients as having a predominance of weakness involving the bulbar musculature (but rarely isolated ocular myasthenia) and being relatively treatment resistant. Immunization of animals with purified MuSK has produced disease with a neuromuscular transmission defect.14 Whether or not MuSK-related MG is induced by complement mechanisms has not been established.

EXTRAOCULAR MUSCLE

SUSCEPTIBILITY TO MG

The functional and physiological reasons for the differential involvement of EOM by MG can be enumerated as follows: (a) even a slight reduction of EOM force generation may misalign the visual axes and produce dramatic symptoms; (b) the NMJ of EOM are stimulated at much higher frequencies than those of other skeletal muscles, which, if they function in a similar manner to the NMJ of other skeletal muscle, would be expected to make them more vulnerable to fatigue; (c) anatomic and physiological properties of EOM fibers and their NMJ suggest susceptibility to neuromuscular transmission block; and (d) the autoimmune pathology may contribute to targeting of EOM by MG.5,6

The most obvious explanation for EOM susceptibility is the requirement for perfect alignment of the visual axes to assure clear vision; if they are not aligned, then the dramatic visual complaints of diplopia, dizziness, visual confusion, or blurring occur.15 Such symptoms will bring patients to medical attention early in the course of disease. In contrast, a small reduction of force by a limb muscle may not be symptomatic. In addition, proprioceptive feedback producing compensatory changes that limit clinical manifestations is likely more prominent in other skeletal muscle compared to the ocular motor system.15

The ocular motor neurons have firing stimulation frequencies. Fast EOM motor units appear to normally function at 100 Hz or above, while nonocular motor neurons function at such levels only briefly.16-18 Based on the discussion of the pathophysiology of MG, one would expect such high stimulation rates to put EOM junctions at risk for neuromuscular transmission fatigue. The normally high frequency of stimulation of EOM may be expected to occur at the price of a low safety factor, and therefore any reduction of endplate potential produced by MG would impair neuromuscular transmission.

The NMJs of EOM have structural features that would be expected to place them at risk for transmission failure. Eighty percent of EOM fibers have a single point of neuronal contact, similar to all other skeletal muscles19,20 These are the so-called singly innervated fibers (SIF). Ultrastructural analysis reveals less prominent synaptic folds.19 From this observation, one would predict fewer AChRs and sodium channels on the postsynaptic membrane. A reduction in AChR, sodium channels, and quantal content would reduce the safety factor for transmission at EOM SIFs, putting them at risk for neuromuscular transmission failure. Khanna et al.21 have gone so far as to hypothesize that differential susceptibility among the EOM junctions could explain the observation of “ultra-fast saccades” of MG patients, despite the simultaneous observation of intrasaccadic fatigue. A single study of miniature endplate potential amplitudes surprisingly showed that EOM and leg-muscle junctions have similar levels, which suggests that AChR density and other membrane properties are similar.22 No measures of safety factor at EOM NMJs have been performed. The observation that nearly all neuromuscular transmission disorders affect the ocular muscle would support the idea that physiological reasons are important in their frequent involvement in MG. However, it is important to appreciate that not all patients with MG, or congenital myasthenia patients, have ocular motility defects. Therefore, physiological reasons are not likely to be the only reason for preferential involvement of EOM by MG.

About 20% of EOM fibers are innervated at multiple points, and these are the multiply innervated fibers (MIF).19,20 The NMJ of the MIF are smaller than those of the SIF, and they lack postsynaptic folding. The contractile force of the MIF is directly proportional to the membrane depolarization caused by the endplate potential—they contract in tonic fashion, in contrast to the twitch contraction of the SIF. A safety factor does not exist for the MIF NMJ.23 Any reduction of endplate potential induced by AChR loss will decrease contractile force of the MIF.

The molecular organization differs slightly between EOM and other skeletal muscle fibers. Alpha-dystro- brevin and syntrophin beta 1 are members of dys- trophin-related protein complex localized to the NMJ; however, they are found extrasynaptically at some EOM fibers.20 The potential difference in the expression pattern may contribute to differences in junctional folds. Altered expression of certain structural proteins of NMJ, specifically rapsyn upregulation, reduces disease severity of EAMG, probably by an increase in the stability of the AChR at the NMJ.24 Although alterations in rapsyn expression are not known in EOM, the lack of junctional folds is evidence that the NMJ of

EOM may be less structurally stable due to the underlying molecular organization.

Preferential immunologic targeting of EOM synapses has been proposed as an explanation for EOM susceptibility.25,26 Sera of some MG patients binds only the MIF synapses, and use of EOM as a source of AChR for AChR antibody assays leads to higher rates of autoantibody detection, which suggests EOM has unique antigenic targets.27,28 Adult EOM uniquely expresses the fetal AChR at MIF and certain SIF end- plates.29-31 Therefore, the fetal AChR would be a target for differential antibody attack of EOM NMJ. However, patients with ocular myasthenia have neither antibodies directed primarily toward fetal AChR nor specific T-cell responses toward fetalAChR epitopes.32,33 The fetal AChR does not appear to be a specific target. Up to half of ocular myasthenics have nondetectable antibodies to AChR, while 90% of generalized MG patients are seropositive for AChR antibodies. Perhaps the ocular MG patients have antibodies directed at non-AChR antigen.

OVERVIEW OF THE COMPLEMENT SYSTEM AND MG

Complement protects the host against invading pathogens by distinct mechanisms, which include cell lysis of pathogens, opsonization with complement fragments, chemotaxis of inflammatory cells, and formation of the membrane attack complex (MAC).34,35 MAC is a multimeric protein complex that produces cell lysis (in the case of MG, localized destruction of the NMJ). In adaptive immune response, complement is the effector system for the primary and secondary antibody responses of B cells. Complement activation is regulated by a series of about 30 plasma and membrane proteins participating in classical, alternative, and lectin pathways. For our discussion, the classical pathway is relevant because of its activation by complement-fixing IgG antibodies that target an antigen, which for MG is primarily the AChR.

Extensive data support the theory that the complement cascade is the primary mechanism mediating AChR loss at the NMJ in MG. First, C3 activation fragments and the MAC are detected at the NMJ in patients and EAMG animals.11,36,37 Second, if complement is depleted by cobra venom, then EAMG cannot be induced by either infusion of AChR antibody or immunization of rodents with purified AChR.38 Third, administration of treatments that inhibit complement activity, such an antibody that binds and inhibits the C6 component or the soluble complement receptor 1, protects rodents from EAMG.39,40 Fourth, in EAMG caused by AChR immunization, mice deficient of C5

or C4 have less severe disease.41,42 Collectively, these data support the proposition that complement deposition and MAC assembly are critical in destruction of the NMJ that causes the defect of neuromuscular transmission.

Host tissues are protected from autologous comple- ment-mediated injury by a system of cell-associated and serum-regulatory proteins.43,44 During spontaneous or antibody-initiated activation of the complement cascade, nascent complement components condense with free hydroxyl and amino groups on biological membranes. Because the reactions occur on host cell surfaces as well as on target surfaces, and because, once bound, these fragments serve as assembly points for subsequent components and are the central amplification enzymes of the cascade, their activities on host cells must be strictly controlled.

The cell-associated regulators include decay-accel- erating factor (DAF, CD55), the membrane cofactor protein (MCP or CD46), and the membrane inhibitor of reactive cell lysis (MIRL, CD59).45 DAF, MCP, complement receptor 1-related gene/protein y (Crry), and complement receptor 1 (CR1) are inhibitors of C3 and C5 convertases. CD59 prevents the binding of C9

to C8 and acts as an inhibitor of membrane attack complex (MAC) formation. Collectively, these proteins accelerate the decay of autologous C3 convertases that inappropriately assemble on self cell surfaces,45 promote the cleavage of uncomplexed autologous cell-bound complement components,46 and inhibit the formation of MAC.47-49 DAF forms complexes out of certain complement components. MCP, anchored to the membrane by a polypeptide domain, functions at the level of C3 convertases. CD59 blocks the uptake of the terminal components of the cascade and the assembly of MAC. CD59, as is DAF, is anchored to the cell surface membrane.48,50,51 In mice, the rodent-specific membrane regulator Crry has activity similar to human MCP, but also has functions that overlap with that of DAF.52

In a series of experiments, we have investigated the importance of complement regulator proteins in EAMG. Mice that are deficient in DAF develop profound weakness when AChR antibodies are administered, while, in marked contrast, wild-type mice show no obvious signs of weakness.53 We extended this investigation to evaluate the respective roles of each of the regulators in protection of the NMJ. We confirmed

the original report and demonstrated that mice having an absence of the CD59 had greater evidence of disease, as determined by complement deposition at the NMJ and degree of destruction of NMJ ultrastructure, than normal mice.54 Mice with a deficiency of both DAF and CD59 had such severe weakness that even reduced doses of AChR antibody administration (as compared to the original study) required immediate euthanasia. The protective effect of complement regulatory proteins in EAMG has been confirmed by Morgan et al.55

THE “COMPLEMENT HYPOTHESIS” TO EXPLAIN EXTRAOCULAR MUSCLE SUSCEPTIBILITY TO MG

In a fundamental look at the differences between EOM and other skeletal muscle, gene-expression studies have been performed on rats and mice to assess specific markers that create divergence in tissue type.56-59 Using DNA microarray and serial analysis of gene expression, the results identified significant numbers of differentially expressed genes in EOM, compared to

Figure 14.3 Neuromuscular junction (NMJ) of extraocular muscle from an experimentally acquired myasthenia gravis rat. The NMJ shows a wide synaptic cleft and electron-dense material within the synaptic cleft, indicating severe injury.

other muscle ranging in number from approximately 100 to 350 genes. The studies indicate expression differences, compared to other skeletal muscle, of genes involved in intermediary metabolism, excitation– contraction coupling, structural organization, transcriptional regulation, and myogenesis. DAF was discovered to have a significantly lower expression level in EOM.57 This observation was the first suggestion that EOM may not benefit from DAF’s protection in the antibodymediated, complement-dependent disease MG.

Complement regulatory proteins are concentrated at the NMJ of other skeletal muscle (Fig. 14.1) (also see color insert), but appear not to be concentrated to the same degree at EOM NMJ. Further, when EAMG is produced, the complement regulatory genes demonstrate a marked down-regulation in expression, and limited to nonexistent up-regulation of complement regulators is observed at the NMJ of EOM. 60 Therefore, EOM does not appear to benefit from having concentrations of complement regulators at the NMJ.

We have indirect evidence from preliminary studies that there is a greater degree of complement deposition at EOM NMJ. We ablated serum-complement activity by use of an antibody directed against the C5 component of complement, and thereby induced EAMG. In the same animals (Fig. 14.2A) one cannot detect complement deposition at the non-EOM NMJ, but complement is found at the EOM NMJ (Fig. 14.2B) (also see color insert). This observation supports that a lack of complement inhibitors at the EOM junctions allow complement deposition, even when systemic complement is inhibited. We investigated complement deposition and NMJ damage in EAMG induced by AChR administration and found greater complement component (C3) deposits at EOM NMJ than diaphragm NMJ, as well as a greater degree of ultrastructural injury

(Fig. 14.3). The result supports the concept that EOMs are subject to greater complement-mediated injury than the NMJ of other muscle.

Among ocular myasthenic patients, the serum concentration of AChR antibody is lower (or absent) than in patients with generalized MG. Although correlation of antibody concentration and severity of weakness is not absolute,61,62 it appears that lower titers of antibody are more capable of inducing injury to EOM than other skeletal muscle NMJ. This would be the case if human EOM expressed the complement regulators at low levels.

FUTURE DIRECTIONS

For a clinician, the visual disability produced by MG remains a challenge to treat because of its unpredictable response to therapy and the poor side-effect profile of corticosteroids, the mainstay of immunosuppressive treatment.6 If the complement hypothesis proves correct, complement-inhibitor treatment may be particularly beneficial for the ocular manifestations of human MG. Such treatments are on the horizon. Antibody against the C5 component of complement (Eculuzimab) has exhibited short-term safety in several human disorders, including acute myocardial infarction,63 coronary artery bypass graft surgery,64 and lung transplantation.65 The drug has demonstrated long-term safety and efficacy in paroxysmal nocturnal hemoglobinuria.66 Treatment trials of complement inhibitors for MG would provide proof of concept for the complement hypothesis of EOM susceptibility.

References

1.Conti-Fine BM, Milani M, Kaminski HJ. Myasthenia gravis: past, present, and future. J Clin Invest. 2006;116:2843–2854.

2.Vincent A. The neuromuscular junction and neuromuscular transmission. In: Karpati G, Hilton-Jones D, Griggs RC, eds. Disorders of Voluntary Muscle, 7th ed. Cambridge, UK: Cambridge University Press; 2001:142–167.

3.Vincent A. Unravelling the pathogenesis of myasthenia gravis. Nat Rev Immunol. 2002;2: 797–804.

4.Hughes BW, Kusner LL, Kaminski HJ. Molecular architecture of the neuromuscular junction. Muscle Nerve. 2006;33:445–461.

5.Ubogu EE, Kaminski HJ. Preferential involvement of extraocular muscle by myasthenia gravis. Neuroophthalmology. 2001;25:219–228.

6.Kusner LL, Puwanant A, Kaminski HJ. Ocular myasthenia: diagnosis, treatment, and oathogenesis. Neurologist. 2006;12:231–239.

7.Merlie JP, Heinemann S, Lindstrom JM. Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies. J Biol Chem. 1979;254:6320–6327.

8.Drachman D, Angus CW, Adams RN, Kao I. Effect of myasthenic patients’ immunoglobulin on acetylcholine receptor turnover: selectivity of degradation process. Proc Natl Acad Sci U S A. 1978;75:3422–3426.

9.Gomez CM, Richman DP. Anti-acetylcholine receptor antibodies directed against the alpha-bun- garotoxin binding site induce a unique form of experimental myasthenia. Proc Natl Acad Sci U S A. 1983;80:4089–4093.

10.Wood SJ, Slater CR. Safety factor at the neuromuscular junction. Prog Neurobiol. 2001;64:393–429.

11.Sahashi K, Engel AG, Lambert EH, Howard FM Jr. Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis. J Neuropathol Exp Neurol. 1980;39:160–172.

12.Ruff RL, Lennon V. End-plate voltage-gated sodium channels are lost in clinical and experimental myasthenia gravis. Ann Neurol. 1998;43:370–379.

13.Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A. Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat Med. 2001;7:365–368.

14.Shigemoto K, Kubo S, Maruyama N, et al. Induction of myasthenia by immunization against muscle-spe- cific kinase. J Clin Invest. 2006;116:1016–1024.

15.Leigh RJ, Zee DS. The Neurology of Eye Movements. 4th ed. New York, NY: Oxford University Press; 1999:385–451.

16.Fuchs A, Scudder C, Kaneko C. Discharge patterns and recruitment order of identified motoneurons and internuclear neurons in monkey abducens nucleus. J Neurophysiol. 1988;60: 1874–1895.

17.Fuchs A, Becker W, Ling L, Langer T, Kaneko C. Discharge patterns of levator palpebrae superioris motoneurons during vertical lid and eye movements in the monkey. J Neurophysiol. 1992;68: 233–243.

18.Goldberg SJ, Meredith MA, Shall MS. Extraocular motor unit and whole-muscle responses in the

lateral rectus muscle of the squirrel monkey. J Neurosci. 1998;18:10629–10639.

19.Spencer RF, Porter JD. Biological organization of the extraocular muscles. Prog Brain Res. 2005; 151:43–80.

20.Khanna S, Richmonds C, Kaminski H, Porter J. Molecular organization of the extraocular muscle

neuromuscular junction: partial conservation of and divergence from skeletal muscle prototype.

Invest Ophthalmol Vis Sci. 2003;44:1918–1926.

21.Khanna S, Liao K, Kaminski H, Tomsak R, Joshi A, Leigh R. Ocular myasthenia revisited: insights

from pseudo-internuclear ophthalmoplegia.

J Neurol. 2007;254:1569–1574.

22.Mosier DR, Siklós L, Appel S. Resistance of extraocular motoneuron terminals to effects amyotropic lateral sclerosis sera. Neurology. 2000; 54:252–255.

23.Jacoby J, Chiarandini DJ, Stefani E. Electrical properties and innervation of fibers in the orbital layer of rat extraocular muscles. J Neurophysiol. 1989;61:116–125.

24.Losen M, Stassen MH, Martinez-Martinez P, et al. Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis. Brain. 2005;128:2327–2337.

25.Kaminski HJ, Maas E, Spiegel P, Ruff RL. Why are eye muscles frequently involved by myasthenia gravis? Neurology. 1990;40:1663–1669.

26.Oda K, Shibasaki H. Antigenic difference of acetylcholine receptor between single and multiple form endplates of human extraocular muscle. Brain Res. 1988;449:337–340.

27.Hayashi M, Kida K, Yamada I, Matsuda H, Tsuneishi M, Tamura O. Differences between ocular and generalized myasthenia gravis: binding characteristics of anti-acetylcholine receptor antibody against bovine muscles. J Neuroimmunol. 1989;21:227–233.

28.Vincent A, Newsom-Davis J. Acetylcholine receptor antibody characteristics in myasthenia gravis. I. Patients with generalized myasthenia or disease restricted to ocular muscles. Clin Exp Immunol. 1982;49:257–265.

29.Horton RM, Manfredi AA, Conti-Tronconi BM. The “embryonic” gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle. Neurology. 1993;43:983–986.

30.Kaminski HJ, Kusner LL, Block CH. Expression of acetylcholine receptor isoforms at extraocular muscle endplates. Invest Ophthalmol Vis Sci. 1996;37:345–351.

31.Kaminski HJ, Kusner LL, Nash KV, Ruff RL. The γ-subunit of the acetylcholine receptor is not expressed in the levator palpebrae superioris. Neurology. 1995;45:516–518.

32.MacLennan C, Beeson D, Buijs A-M, Vincent A, Newsom-Davis J. Acetylcholine receptor expression in human extraocular muscles and their susceptibility to myasthenia gravis. Ann Neurol. 1997;41:423–431.

33.Wang Z, Diethelm-Okita B, Okita D, Kaminski H, Howard J, Conti-Fine B. T-cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis. J Neuroimmunol. 2000;108:29–39.

34.Kohl J. Self, non-self, and danger: a complementary view. Adv Exp Med Biol. 2006;586:71–94.

35.Walport MJ. Complement. N Engl J Med. 2001; 344:1058–1066.

36.Sahashi K, Engel AG, Linstrom JM, Lambert EH, Lennon VA. Ultrastructural localization of immune complexes (IgG and C3) at the end-plate in experimental autoimmune myasthenia gravis. J Neuropathol Exp Neurol. 1978;37:212–223.

37.Nakano S, Engel AG. Myasthenia gravis: quantitative immunocytochemical analysis of inflammatory cells and detection of complement membrane attack complex at the end-plate in 30 patients. Neurology. 1993;43:1167–1172.

38.Lennon VA, Seybold ME, Lindstrom JM, Cochrane C, Ulevitch R. Role of complement in the pathogenesis of experimental autoimmune myasthenia gravis. J Exp Med. 1978;147:973–983.

39.Biesecker G, Gomez CM. Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6. J Immunol. 1989;142:2654–2659.

40.Piddlesden SJ, Jiang S, Levin JL, Vincent A, Morgan BP. Soluble complement receptor 1 (sCR1) protects against experimental autoimmune myasthenia gravis. J Neuroimmunol. 1996;71: 173–177.

41.Christadoss P. C5 gene influences the development of murine myasthenia gravis. J Immunol. 1988;140:2589–2592.

42.Tuzun E, Scott BG, Goluszko E, Higgs S, Christadoss P. Genetic evidence for involvement of classical complement pathway in induction of

experimental autoimmune myasthenia gravis. J Immunol. 2003;171:3847–3854.

43.Janeway C, Travers P, Walport M, Shlomchik M. Innate immunity. In: Janeway C, Travers P, Walport M, Shlomchik M, eds. Immunobiology: The Immune System in Health and Disease. New York, NY: Garland Publishing; 2001:35–91.

44.Miwa T, Song WC. Membrane complement regulatory proteins: insight from animal studies and relevance to human diseases. Int Immunopharmacol. 2001;1:445–459.

45.Medof ME, Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med. 1984;160:1558–1578.

46.Seya T, Turner J, Atkinson J. Purification and characterization of a membrane protein (gp45-70)

that is a cofactor for cleavage of C3b and C4b. J Exp Med. 1986;163:837–855.

47.Davies A, Simmons DL, Hale G, et al. CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J Exp Med. 1989;170:637–654.

48.Holguin MH, Fredrick LR, Bernshaw NJ, Wilcox LA, Parker CJ. Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest. 1989;84:7–17.

49.Harada R, Okada N, Fujita T, Okada H. Purification of 1F5 antigen that prevents complement attack on homologous cell membranes. J Immunol. 1990;144:1823–1828.

50.Medof ME, Walter EI, Roberts WL, Haas R, Rosenberry TL. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry. 1986;25:6740–6747.

51.Davitz M, Low M, Nussenzweig V. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C(PI-PLC). J Exp Med. 1986; 163:1150–1161.

52.Lin F, Fukuoka Y, Spicer A, et al. Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knockout mouse and site-specific monoclonal antibodies. Immunology. 2001;104:215–225.

53.Lin F, Kaminski H, Conti-Fine B, Wang W, Richmonds C, Medof M. Enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection. J Clin Invest. 2002;110:1269–1274.

287–293.

55.Morgan BP, Chamberlain-Banoub J, Neal JW, Song W, Mizuno M, Harris CL. The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice. Clin Exp Immunol. 2006;146:294–302.

56.Porter JD, Khanna S, Kaminski HJ, et al. Extraocular muscle is defined by a fundamentally distinct gene expression profile. Proc Natl Acad Sci U S A. 2001;98:12062–12067.

57.Cheng G, Porter JD. Transcriptional profile of rat extraocular muscle by serial analysis of gene expression. Invest Ophthalmol Vis Sci. 2002;43: 1048–1058.

58.Fischer MD, Gorospe JR, Felder E, et al. Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol Genomics. 2002;9:71–84.

59.Fischer MD, Budak MT, Bakay M, et al. Definition of the unique human extraocular muscle allotype by expression profiling. Physiol Genomics. 2005;22:283–291.

60.Kaminski HJ, Li Z, Richmonds C, Lin F, Medof ME. Complement regulators in extraocular muscle and experimental autoimmune myasthenia gravis. Exp Neurol. 2004;189:333–342.

61.Howard FJ, Lennon V, Finley J, Matsumoto J, Elveback L. Clinical correlations of antibodies that bind, block, or modulate human acetylcholine receptors in myasthenia gravis. Ann N Y Acad Sci. 1987;505:526–538.

62.Limburg PC, The TC, Hummel-Teppel E, Oosterhuis H. Anti-acetylcholine receptor antibodies in myasthenia gravis. I. Relation to clinical

parameters in 250 patients. J Neurol Sci. 1983;58: 357–370.

63.Patel MR, Granger CB. Pexelizumab: a novel therapy for myocardial ischemia-reperfusion. Drugs Today (Barc). 2005;41:165–170.

64.Fitch JC, Rollins S, Matis L, et al. Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Circulation. 1999;100:2499–2506.

65.Keshavjee S, Davis RD, Zamora MR, de Perrot M, Patterson GA. A randomized, placebo-controlled trial of complement inhibition in ischemia-reper- fusion injury after lung transplantation in human beings. J Thorac Cardiovasc Surg. 2005;129:423–428.

66.Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355:1233–1243.