The immunopathogenesis of Miller Fisher syndrome

Journal of Neuroimmunology 100 Ž1999. 3–12 www.elsevier.comrlocaterjneuroim

Review

The immunopathogenesis of Miller Fisher syndrome Hugh J. Willison ) , Graham M. O’Hanlon UniÕersity Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF, Scotland, UK Received 1 September 1999; received in revised form 9 September 1999; accepted 9 September 1999

Abstract Over the past decade, remarkable progress has been made in our understanding of the pathogenesis of Miller Fisher syndrome ŽMFS., a clinical variant of Guillain Barre´ syndrome ŽGBS.. MFS comprises the clinical triad of ataxia, areflexia and ophthalmoplegia. It is associated with acute-phase IgG antibodies to GQ1b and GT1a gangliosides in over 90% of cases which are highly disease specific. Like GBS, MFS is a post-infectious syndrome following diverse infections, but particular attention has been paid to its association with Campylobacter jejuni enteritis. Serostrains of C. jejuni isolated from infected patients bear ganglioside-like epitopes in their lipopolysaccharide core oligosaccharides, which elicit humoral immune responses exhibiting molecular mimicry with GQ1brGT1a gangliosides. These antibodies are believed to be the principal cause of the syndrome and physiological studies aimed at proving this have focused on the motor-nerve terminal as a potential site of pathogenic action. This review describes these findings and formulates a pathogenesis model based on our current state of knowledge. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Miller Fisher syndrome; Guillain Barre´ syndrome; Gangliosides; Campylobacter jejuni; Neuromuscular junction; Molecular mimicry

1. Introduction Miller Fisher syndrome ŽMFS. is a clinical variant of the Guillain Barre´ syndrome ŽGBS., an acute post-infectious paralytic illness caused by inflammatory disruption of peripheral nerve integrity and function ŽRopper, 1994; Hahn, 1998.. In contrast to the generalised and often severe weakness and sensory loss that occurs in GBS, the manifestations of MFS are restricted to limb ataxia, tendon reflex loss and extraocular muscle paralysis with affected cases usually making a good recovery. The clinical entity that Miller Fisher described in 1956 ŽFisher, 1956. has more than held its place in the neuroimmunological firmament despite its relative rarity. MFS only accounts for 5%–10% of GBS cases, the incidence of the latter syndrome being 1–2 per 100,000 ŽHughes and Rees, 1997.. Indeed, through a series of remarkable advances achieved during the 1990s, MFS has emerged as the archetypal anti-ganglioside antibody mediated human neuropathy and is providing valuable insights into the pathogenesis of its more serious counterpart, GBS. This review will describe the last decade of progress and formulate a pathophysiological model based on our current understanding of MFS. )

Corresponding author. Tel.: q44-141-201-2464; fax: q44-141-2012993; e-mail: [email protected]

2. Autoimmune serology and clinical phenotypes In 1992, Chiba et al. first reported the presence of anti-GQ1b ganglioside antibodies in MFS, a finding which has since been substantiated in many other studies ŽChiba et al., 1992; Willison et al., 1993b; Yuki et al., 1993; Carpo et al., 1998.. Serological findings in a typical patient population are shown in Fig. 1 and relevant ganglioside structures are shown in Fig. 2. Anti-GQ1b antibodies in MFS almost invariably cross-react with the structurally similar ganglioside, GT1a although rare exceptions may exist ŽChiba et al., 1993; Ilyas et al., 1998.. Up to 50% of MFS sera also demonstrate reactivity with other gangliosides containing a disialosyl epitope ŽFig. 3., such as GD3, GD1b and occasionally GT1b ŽWillison et al., 1994.. In MFS, oropharyngeal weakness may also be present and some evidence suggests this is preferentially associated with anti-GT1a seroreactivity, whereas ophthalmoplegia is associated with anti-GQ1b seroreactivity ŽMizoguchi et al., 1994; O’Leary et al., 1996; Kashihara et al., 1998; Koga et al., 1998a.. Clinical latitude is required when considering the definition of MFS: some anti-GQ1b antibody positive cases commence typically and then proceed to a more confluent GBS-like picture with limb weakness. The reverse pattern may also occur in that cases of generalised GBS in which ophthalmoplegia is present also have anti-

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H.J. Willison, G.M. O’Hanlonr Journal of Neuroimmunology 100 (1999) 3–12

Fig. 1. Anti-GQ1b antibodies in MFS. ;90% of MFS cases have elevated titres of anti-GQ1b IgG antibodies. Low titres of anti-GQ1b IgG may be found in GBS cases but are absent from patients with multiple sclerosis ŽMS., other neurological diseases ŽOND. and healthy controls ŽNORM..

GQ1b antibodies ŽRopper, 1994; Ter Bruggen et al., 1998.. In Bickerstaff’s brain-stem encephalitis, a MFS-like condition with superimposed CNS dysfunction manifested by coma and pyramidal tract abnormalities, anti-GQ1b antibodies are also found ŽYuki, 1995; Kikuchi et al., 1997.. This latter condition continues to fuel the long standing debate about the relative contribution of PNS and CNS pathophysiology to the clinical manifestations of MFS ŽGoldberg-Stern et al., 1994; Najim Al-Din et al., 1994; Urushitani et al., 1995; Fargas et al., 1998; Kuwabara et al., 1999; Yeh et al., 1999.. The anti-GQ1b antibody marker thus sensitively identifies a cluster of closely related syndromes which share in common the presence of ophthalmoplegia; equally significant is the complete absence of anti-GQ1b antibodies from other normal and disease control groups, indicating a high level of specificity for this disease association. In addition to motor involvement, the vast majority of MFS cases also have sensory ataxia, due to loss of proprio-

Fig. 2. Schematic representation of structures of some polysialylated gangliosides. Antibodies to one or more of these structures are commonly associated with MFS.

ceptive input from peripheral sense organs. The site at which this defect arises is debated but may be at least in part at the level of the dorsal root ganglion. It is possible that in some cases the cerebellum may also be targeted directly ŽKornberg et al., 1996., and reactive epitopes have also been observed on the intrafusal apparatus of the muscle spindle ŽWillison et al., 1996; Fig. 5.. The antiganglioside antibody specificity responsible for the ataxia may also be broadly directed against disialylated ganglioside structures, including GD1b, GD3, GQ1b and GT1a. Patients with acute sensory ataxic neuropathy Ži.e., MFS without ophthalmoplegia or other motor manifestations. may have antibodies to GD1b andror GD3 without antiGQ1brGT1a cross-reactivity ŽWillison et al., 1994; O’Leary and Willison, 1997.. Although difficult to demonstrate in clinical serological studies performed to date, one might predict that GBS patients with prominent ataxia and sensory loss should also have anti-GD1b antibodies, thus supporting a central role for GD1b as one target antigen for large fibre sensory involvement in both acute and chronic autoimmune neuropathy. In addition to GD1b, some ataxic neuropathy cases may have antibodies to GQ1balpha, which is concentrated in lamina 1 and 3 of the dorsal horn ŽKusunoki et al., 1993a; Tagawa et al., 1997.. In MFS, serum anti-ganglioside antibody titres are at their peak at clinical presentation and decay rapidly in most cases concomitant with clinical recovery, being undetectable as early as 1 month after onset ŽWillison and Veitch, 1994; Mizoguchi, 1998.. Although IgM, IgA and IgG anti-GQ1b antibodies are seen, the IgG response is most frequently measured for clinical diagnosis and usually persists for longer and at higher titre than the IgMrIgA response ŽKoga et al., 1998b; Schwerer et al., 1999..

Fig. 3. Thin layer chromatography immuno-overlay of anti-ganglioside antibody-containing sera showing the typical patterns of reactivity seen in MFS. Five purified gangliosides comprising GD3, GT1a, GD1b, GT1b and GQ1b Ž1 mg of each per lane. are immunostained as follows: lane 1, serum Ždiluted 1r500. from a patient with CANOMAD which reacts with all five chromatographed gangliosides; lane 2, serum Ž1r200. from an MFS patient which reacts only with GQ1b and GT1a; lane 3, serum Ž1r200. from a patient with acute ataxic neuropathy without ophthalmoplegia or other motor involvement, reacting only with GD3 and GD1b; lane 4, serum Ž1r200. from a MFS case which reacts with GQ1b and GT1a, GD3 and very weakly with GD1b and GT1b Žtaken from Willison et al., 1994..

H.J. Willison, G.M. O’Hanlonr Journal of Neuroimmunology 100 (1999) 3–12

Another temporal pattern for anti-disialylated ganglioside antibodies occurs in rare circumstances in which IgM antibodies appear and persist indefinitely. These IgM antibodies are monoclonal Žalso termed paraproteins or monoclonal gammopathies. and arise from a single clone of B lymphocytes whose proliferative control is dysregulated ŽIlyas et al., 1985; Arai et al., 1992; Obi et al., 1992; Yuki et al., 1992; Willison et al., 1993a.. IgM paraprotein levels in affected individuals are remarkably stable over many years and rarely progress to frank B cell malignancy. This persistently elevated level of circulating anti-disialylated ganglioside IgM antibody is accompanied by a chronic clinical syndrome with a similar regional pattern to MFS, which we have called CANOMAD Ž chronic ataxic neuropathy with ophthalmoplegia, monoclonal M-protein, cold agglutinins and anti-disialosyl antibodies. ŽWillison et al., 1996.. The shared clinical similarities between the acute and chronic syndromes linked by anti-disialosyl antibodies provides very strong, albeit circumstantial evidence that they are central to pathogenesis.

3. Relationship to preceding infections As is the case with GBS, the temporal pattern of clinical onset, nadir and spontaneous recovery that occurs in MFS is highly suggestive of an acute phase primary immune response, peaking 10 to 14 days after an infectious event, followed by gradual decay of the immune response, as so clearly outlined by Dale McFarlin in his review of this subject in 1990 ŽMcFarlin, 1990.. MFS follows a wide variety of infections including Campylobacter jejuni Ž C. jejuni . enteritis and upper respiratory tract viral infections. With respect to C. jejuni, considerable information now supports the principle of molecular mimicry between GQ1brGT1a and C. jejuni lipopolysaccharide ŽLPS. and lipo-oligosaccharide ŽLOS. core oligosaccharides Žcore OSs. as central to the induction of this response ŽJacobs et al., 1995, 1997a,b; Neisser et al., 1997; Yuki, 1997, 1998.. Campylobacter isolates from MFS cases have been studied by spectroscopic analysis of LPS structure and by immunohistological probing with human and murine sera and monoclonal antibodies reactive with GQ1b and related structures ŽFig. 4.. These structural studies have demonstrated that GD3- and GT1alike oligosaccharides are present in GBS and MFS-associated C. jejuni core OSs ŽSalloway et al., 1996; Penner and Aspinall, 1997; Shin et al., 1997.. In immunochemical studies, GQ1b cross-reactive epitopes have been demonstrated on core OSs, although the entire GQ1b oligosaccharide structure has not been structurally identified. We have recently shown that immunisation of the mouse with GT1a-containing LPS can produce a serum anti-GQ1b antibody response, and it has been possible to derive monoclonal antibodies from these mice which react with

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Fig. 4. Molecular mimicry between core oligosaccharide structures of disialylated gangliosides and C. jejuni lipopolysaccharide ŽAspinall et al., 1994; Salloway et al., 1996.. Abbreviations as follows: NeuAc s N-acetyl neuraminic acid; Gal s galactose; GalNAc s N-acetyl galactosamine; X sGlc Ž1™1. ceramide Žgangliosides. or the remaining core OSrlipid A ŽLPSs..

GQ1b, GT1a, GD3 and other disialylated gangliosides ŽGoodyear et al., 1999.. With respect to other preceding infectious events, the immunological circumstances responsible for generating the anti-GQ1b response is unknown. Since viruses do not encode glycoslyating enzymes, but use host glycosylation pathways for their glycoprotein synthesis, the mechanisms by which molecular mimicry between gangliosides and viral components might occur are less clear than with bacterial infection such as C. jejuni. However, viral proteins may become glycosylated with ganglioside-mimicking oligosaccharides in the respiratory epithelium or other tissues, acquire ganglioside containing membranes while budding, or bind gangliosides directly. As a result of viral proteinroligosaccharide association, the immunological milieu for antigen uptake by B cells specific for the oligosaccharide with subsequent peptide presentation to T helper cells could be established. However, this hypothesis has yet to be demonstrated experimentally. B cell activation occurring in such a manner would result in the induction of an anti-GQ1brGT1a directed humoral immune response capable of undergoing T helper cell directed class switching and affinity maturation. Indeed, anti-GQ1b antibodies that occur in MFS are polyclonal and of IgM, IgA and IgG classes ŽWillison and Veitch, 1994.. Furthermore, in addition to the production of class switched antibodies, the IgG response is subclassrestricted, being dominated by IgG1 andror IgG3 antibodies. This is an unusual subclass pattern for human anticarbohydrate antibodies which are predominantly IgM and if switching occurs, IgG2 . Some evidence suggests that the Ig-subclass pattern may depend on the nature of the preceding infection ŽSchwerer et al., 1999.. Although no human IgG mAbs have been studied Žnone are currently available., Ig variable region analysis of anti-ganglioside IgM mAbs cloned from human neuropathy cases show extensive somatic mutation suggesting an antigen-driven response ŽPaterson et al., 1995; Willison et al., 1996.. Both the presence of the IgG1r3 subclasses and the somatic mutation do provide indirect evidence to indicate a dependence on T-cell help ŽTD. in the induction of anti-gang-

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H.J. Willison, G.M. O’Hanlonr Journal of Neuroimmunology 100 (1999) 3–12

lioside antibodies rather than a purely T-independent ŽTI. origin as is usually associated with oligosaccharide antigens, including LPS. This interesting subject area requires further experimental analysis since it is likely that the pathogenicity of anti-ganglioside antibodies in acute syndromes such as MFS is related to their higher affinity and class-switching, in comparison to lower affinity IgM antibodies associated with chronic, low grade clinical syndromes.

4. Localisation of MFS-associated gangliosides in peripheral nerve It would be reasonable to predict that the pattern of clinical symptoms in anti-ganglioside antibody mediated neuropathies should correlate broadly with the proportional distribution of target gangliosides throughout the PNS. In other words, affected regions in MFS should be composed of more ‘GQ1b-like’ gangliosides relative to unaffected ones. This surprisingly complex issue has been addressed in several studies of relevance to MFS. Chiba et al. Ž1993. first noted in an immunohistochemical study using an anti-GQ1b specific mAb, that the extraocular cranial nerves had high levels of immunoreactive GQ1b at nodes of Ranvier. They also showed that the nerve trunks supplying the human extraocular muscles has a relatively high content of GQ1b compared with other cranial or spinal nerves when analysed biochemically by total lipid extraction and TLC ŽChiba et al., 1997.. However, from the same study, it was clear that GQ1b is also present in significant amounts at sites unaffected by MFS, and MFS-like antiganglioside antibodies have been shown to bind to the nodes of Ranvier in other nerves ŽWillison et al., 1996; Goodyear et al., 1999.. Thus, the absolute tissue distribution Ži.e., presence or absence. of gangliosides appears to be insufficient as the sole explanation for regional localisation of the clinical pathology. The two commonly used analytical approaches, illustrated above, for defining ganglioside distribution Ži.e., biochemical and immunohistological. each have their mer-

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its and failings. Although biochemical analyses of tissue extracts can identify significant differences in the ganglioside and lipid composition of different nerves, the approach has limitations. The size of a structure that can be cleanly dissected from its surroundings is large and of heterogeneous cellular composition, only a small proportion comprising purely neural elements. Furthermore, information is lost about factors which may influence the nature and development of antibody mediated injury, such as the microscopic distribution and functional organisation of gangliosides within membranes. While immunohistological studies can provide good information about microscopic ganglioside distribution, caution must also be exercised in the interpretation of results. When using whole MFS sera or Ig fractions, the signal from non-anti-ganglioside antibody components of the sera ambiguously confounds interpretation, and it is thus preferable to use affinity purified antisera or monoclonal antibodies. Furthermore, many anti-ganglioside antibodies are not monospecific but may cross-react with structurally similar gangliosides and other glycoconjugate antigens, making interpretation of ganglioside localisation difficult. Some anti-ganglioside antibodies may be part of the polyreactive antibody repertoire, thereby exaggerating this problem further. The antigen density and the surrounding lipid environment can also markedly influence the ability of antiganglioside antibodies to bind; thus failure to detect a ganglioside by immunohistology does not necessarily indicate its biochemical absence ŽLloyd et al., 1992; Kremer et al., 1997; Pestronk et al., 1997.. Tissue sectioning may expose gangliosides which normally occupy cryptic sites Že.g., within compact myelin. and immunohistology can thus misrepresent the ganglioside array which would be visible to circulating antibodies in physiological environments. Furthermore, since gangliosides can be heterogeneously distributed within a membrane, the ganglioside-rich microdomains described above may allow for good immunohistological detection, whereas a ganglioside which is evenly distributed throughout a membrane may have the same total tissue concentration in biochemical evaluation, yet not be detectable by immunohistology.

Fig. 5. Immunolabelling studies of the human and rodent peripheral nervous system by anti-ganglioside antibodies with MFS-associated reactivity. HaRCE, a human IgM, was affinity purified from the serum of a CANOMAD patient ŽWillison et al., 1996.. CGM3 is a mouse monoclonal IgM cloned from a mouse inoculated with Campylobacter LPS ŽGoodyear et al., 1999.. Both antibodies bound polysialylated gangliosides on TLC, and labelled a variety of structures within the PNS. Ža–c. Human dorsal-root ganglion labelled with anti-neurofilament ŽNF. antibody Ža., and HaRCE Žb. overlayed in Žc.. The anti-ganglioside antibody produces granular staining of the neuronal cytoplasm, and an area extending beyond the NF cytoskeleton, presumably the cytoplasmic membrane. Bar s 20 mm. Žd–f. Similar cytoplasmic staining was observed in mouse trigeminal ganglion neurons, labelled for NF Žd., and the anti-GD3 antibody CGM3 Že. overlayed in Žf.. Bar s 50 mm. Žg–i. The mouse neuromuscular junction ŽNMJ. labelled with anti-NF and a-bungarotoxin which binds to the postsynaptic ACh receptors Žg. and HaRCE Žh. overlayed in Ži.. Strong binding of HaRCE is observed at the presynaptic terminal. Bar s 20 mm. Žj. The NMJ Žarrows. of specialised polyinnervated muscle fibres within the rat extraocular muscle are labelled by HaRCE Žred.. The pre-terminal axon is labelled with anti-NF Žgreen.. Bar s 20 mm. Žk. Teased fibres from mouse sciatic nerve labelled with the anti-GM1 ligand Cholera toxin ŽCT; green. and CGM3 Žred.. The paranodal region of the left-hand fibre is labelled with CT. CGM3 weakly binds the myelin surface, but there is strong labelling of the nodal axon. At the level of the Schwann cell ŽSC. nucleus Žasterisk; right-hand fibre., there is labelling of SC cytoplasmic elements. Bar s 20 mm. Žl. The nodal staining by HaRCE Žred. extends beyond the specialised axonal region containing sodium channels Žgreen.. Bar s 20 mm. Žm. The rat muscle spindle capsule is stained by CT Žgreen., while HaRCE strongly labels the region immediately around the intrafusal muscle fibres Žasterisks.. Bar s 20 mm.

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The pathogenic role of anti-ganglioside antibodies is likely to depend not only on the density and distribution of gangliosides but the extent to which the target gangliosides are involved in modulating the function of neuronal proteins or membrane systems. Within phospholipid membranes, gangliosides may form microdomains or ‘‘functional rafts’’, into which proteins such as growth factor receptors or ion channels are specifically included or excluded ŽSimons and Ikonen, 1997., and it appears that gangliosides can be intimately involved in the normal functioning of such proteins ŽThomas and Brewer, 1990; Mutoh et al., 1995.. As such, the relative contribution of these factors may vary from site to site, and between anti-ganglioside antibodies of differing reactivity. In addition, some membrane systems may be more susceptible to low grade complement mediated attack than others, in part dependent upon the distribution of complement regulatory proteins. The calcium binding properties of gangliosides have been demonstrated in several model membrane systems, and it is possible that one role is to chelate extracellular calcium; a function which may be of particular relevance to nerve terminal injury ŽPlomp et al., 1999.. In addition to the structural and anatomical factors which might influence the development of neuropathic change, the accessibility of target gangliosides to circulating antibodies is important. For example, an explanation for the rarity of CNS involvement in MFS is the protection from autoimmune attack afforded by the preserved blood-brain barrier, rather than the absence of GQ1b in CNS neural membranes. As stated above, interpretation of both biochemical and immunohistological studies require caution. With this background in mind, key points about the tissue distribution of gangliosides of relevance to MFS are summarised below and illustrated in Fig. 5. In the dorsal root ganglion, polysialylated gangliosides, especially those with an Ž a 2–8. configured disialosyl group ŽGD1b, GT1b, GQ1b, GD3, GD2., are the most prominent gangliosides in cultured DRG neurons ŽCalderon et al., 1995. and antibodies recognising these gangliosides are able to bind andror lyse such cells ŽOhsawa et al., 1993.. Many studies have also shown anti-disialosyl antibody Žincluding GD1b. staining of DRG neurons in histological sections from several species, including man ŽO’Hanlon et al., 1996, 1998; Kusunoki et al., 1997; Maehara et al., 1997; Vriesendorp et al., 1997; Fig. 5.. In oculomotor cranial nerves, GQ1b is particularly enriched at nodes of Ranvier ŽChiba et al., 1993; Kusunoki, 1995.. Polysialylated gangliosides can be also be detected by antibody and tetanus toxin binding studies at the nodes in somatic nerves ŽGanser and Kirschner, 1984; Kusunoki et al., 1993b, 1997; Scherer, 1996; Willison et al., 1996.. Tetanus toxin binding gangliosides Že.g., GD1b, GT1b, GQ1b. have been identified on paranodal and internodal axolemma ŽGanser and Kirschner, 1984., and the adaxonal membrane ŽMolander et al., 1997.. Similarly, an antibody

reactive with disialosyl gangliosides bound to the internodal axolemma andror adaxonal Schwann cell cytoplasm ŽWillison et al., 1996; Goodyear et al., 1999; Fig. 5.. The NMJ may be particularly vulnerable to autoimmune attack in MFS as it lies outside the blood–nerve barrier, is rich in gangliosides, and is dependent upon rapid turnover of membrane for normal functioning. Many bacterial toxins, including botulinum and tetanus toxins, depend on binding to gangliosides with subsequent uptake into the NMJ for their proteolytic action ŽWillison and Kennedy, 1993; Kozaki et al., 1998; Schengrund, 1999.. Histological analyses of NMJ have demonstrated the binding of antibodies reactive to polysialylated gangliosides ŽWillison et al., 1996; Goodyear et al., 1999; Plomp et al., 1999.. This was also the case for the specialised en grappe end-plates of polyinnervated muscle fibres found within the extraocular muscles ŽFig. 5.. Muscle spindles are proprioceptive transducers located within the muscle, and form an integral part of the gamma reflex loop. They contain specialised muscle fibres which have motor innervation, and which are encircled by a sensory ending. The neural components and intrafusal muscle fibres of spindles are labelled by antibodies to disialylated gangliosides ŽWillison et al., 1996; Fig. 5.. Thus, muscle spindles may be an important target in MFS, injury to which could account for stretch reflex changes and impairment of proprioception ŽKuwabara et al., 1999..

5. Pathogenic effects of anti-GQ1b antibodies in model systems of MFS No direct evidence for a primary pathogenic role for anti-GQ1b antibodies is forthcoming from human studies, although the circumstantial clinical evidence for such a role seems overwhelming. Several experimental studies in animals do, however, provide very strong supportive evidence that anti-GQ1b and other anti-disialylated ganglioside antibodies can mediate pathological changes. It had been widely viewed that anti-GQ1b antibodies in MFS would be most likely to cause the clinical motor manifestations via segmental demyelination of extraocular and craniobulbar nerve trunks in a similar pattern to that classically described in GBS. In vitro conduction studies on isolated, desheathed mouse sciatic nerve have demonstrated antidisialosyl antibody binding and complement fixation at nodes of Ranvier occurring in the absence of conduction abnormalities, thus indicating that in this model, the site is relatively resistant to acute physiological failure ŽPaparounas et al., 1999.. This is not, however, the case at the NMJ which we have focused on as a potential site of injury. In collaborative studies with Vincent and Newsomdavis started in the early 1990s, we reported that anti-GQ1b positive MFS sera and IgG caused a temporary and moderate increase of spontaneous quantal acetylcholine ŽACh. release at NMJs of mouse hemi-diaphragm preparations, as

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assessed by a rise in miniature end plate potential frequencies, and subsequently induced nerve terminal paralysis ŽRoberts et al., 1994.. We then performed passive immunisation studies in the mouse using a human affinity purified and cloned CANOMAD-associated IgM mAb and demonstrated extensive in vivo deposits of IgM at motor nerve terminals in conjunction with electrophysiological evidence of nerve terminal dysfunction ŽWillison et al., 1996.. In a series of more extensive studies performed in collaboration with Plomp and Molinaar, the in vitro effects of MFS sera, MFS IgG fractions and a human monoclonal anti-GQ1b IgM antibody on mouse NMJs were re-examined ŽPlomp et al., 1999.. Here we demonstrated that anti-GQ1b antibodies bind at NMJs where they induced massive quantal release of ACh from nerve terminals and eventually blocked neuromuscular transmission in a purely pre-synaptic fashion closely resembling the effect of the paralytic neurotoxin a-latrotoxin. Furthermore, through the use of different complement-deficient sera, we showed the effect of anti-GQ1b antibodies was entirely dependent on activation of complement components. Since neither classical pathway activation nor the formation of membrane attack complex was required, the effect could be due to involvement of the alternative pathway and intermediate complement cascade products such as C3a and C5a. In a series of studies performed by Buchwald et al. Ž1995; 1998a; b. using a perfused macropatch clamp electrode technique on the phrenic nerve hemidiaphragm preparation, IgG from anti-GQ1b positive as well as antiGQ1b negative MFS patients blocked evoked ACh release and depressed the amplitude of post-synaptic potentials, indicating both a pre- and post-synaptic blocking effect. This effect was fully reversible by washing out the perfusate and occurred in an entirely complement-independent manner. Although the details of these observations may seem at variance with the electrophysiological results of Plomp, the studies collectively indicate that the motor nerve terminal and NMJ should be seriously viewed as one of the potential targets for antibody mediated attack in MFS. Clinical electrophysiological studies could investigate this site more thoroughly in affected patients, as recently reported ŽUncini and Lugaresi, 1999.. Similarly, the terminal motor nerve may be a pathogenic site in more generalised paralytic syndromes, as occurs in acute motor axonal neuropathy ŽHo et al., 1997.. Most recently, we have further investigated the role of anti-GQ1b antibodies, arising as a result of C. jejuni infection with cross-reactive LPS core oligosaccharides, in mediating these effects as follows. To determine whether structural mimicry resulted in pathogenic autoantibodies, we immunised mice with GT1arGD3-like C. jejuni LPS and thereafter cloned mAbs that reacted with both the immunising LPS and GQ1brGT1arGD3 gangliosides. In immunohistological studies, the mAbs bound to ganglioside-rich sites including the NMJ ŽGoodyear et al., 1999.. In ex vivo electrophysiological studies in the phrenic

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nerve-hemidiaphragm preparation, application of antibodies either ex vivo or in vivo via passive immunisation induced massive quantal release of ACh, followed by neurotransmission block in an identical fashion to the MFS sera and Ig fractions, and the CANOMAD mAb. Again the effects were complement-dependent and associated with extensive deposits of IgM and C3c at nerve terminals. We have since conducted studies at both the light and electron microscopic levels which show morphological destruction of the nerve terminal in this model ŽO’Hanlon et al., unpublished observations.. In addition to strengthening the electrophysiological data using human Ig and mAbs, these data also provide strong support for the molecular mimicry hypothesis as a mechanism for the induction of cross-reactive pathogenic anti-gangliosiderLPS antibodies in MFS. What has yet to be demonstrated is an in vivo paralytic syndrome in an animal model, induced either by passive immunisation with anti-ganglioside antibody or active immunisation with LPS or gangliosides. Despite the reliable animal model for T cell mediated experimental allergic neuritis, attempts to induce an in vivo antibody-mediated model of MFSrGBS have been frustratingly unsuccessful. One outstanding exception, however, is the ataxic neuropathy model in the rabbit induced by immunisation with the disialylated ganglioside GD1b, pioneered by Kusunoki et al. Ž1996; 1997; 1999. and Hitoshi et al. Ž1999.. In this model, a proportion of rabbits become severely ataxic ) 30 days after being repeatedly immunised with GD1b dissolved in keyhole limpet haemocyanin and emulsified in Freund’s adjuvant. All rabbits develop anti-GD1b antibodies but the vulnerability to ataxia appears to preferentially require seroreactivity with the disialosyl epitope on GD1b rather than solely the terminal GalŽb1–3.GalNAc epitope common to GD1b, GM1 and asialo-GM1. Pathological changes comprise axonal degeneration in the dorsal root and dorsal column of the spinal cord and neuronal degeneration in the dorsal root ganglion. In immunohistology using mouse mAb and rabbit polyclonal antisera, staining of DRG neurons and paranodal myelin in nerve roots occurs with GD1b reactive antibodies but not with those solely reactive with GM1. These observations provide very strong support for the view that GD1b antibodies are at least one of the specificities capable of mediating dorsal root Žganglion. injury. This is consistent with clinical reports of patients with acute and chronic ataxic neuropathies in whom serum antibodies react preferentially with GD1b. However, antibodies to other disialylated gangliosides, including GQ1b, GQ1balpha and GT1a may also be capable of mediating injury in this site. 6. Conclusions Miller Fisher syndrome is the best understood of the acute inflammatory neuropathies in terms of the pathogenic cycle from origin to effects of anti-ganglioside antibodies. A simplistic working model proposes that anti-GQ1b anti-

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bodies, which usually also react with structurally similar disialosyl-containing gangliosides, arise as part of a primary immune response to infectious organisms bearing cross-reactive oligosaccharide determinants. These antiGQ1brdisialosyl antibodies are able to gain access to and then bind selectively to GQ1brdisialosyl enriched sites in the nervous system Že.g., motor nerves innervating extraocular muscles and the dorsal root ganglion., thereby dictating the regionality of the clinical features seen in the syndrome. Once bound to neural membranes, anti-GQ1b antibodies initiate complement-mediated, pro-inflammatory injury leading to the loss of structurerfunction expressed clinically as craniobulbar weakness and ataxia. As the primary immune response decays, the syndrome recovers spontaneously, provided irreversible neural injury has not occurred. Many of the detailed components of this pathogenic cycle remain unanswered, particularly the host factors that control an individual’s susceptibility to the development of anti-GQ1b antibodies on exposure to the mimicking microbial epitope Ži.e., the loss of tolerance., and the sites and mechanismŽs. by which neural injury occurs. Equally pressing is the need to extend the MFS paradigm into the generalised neuropathy, GBS and into other neuroimmunological diseases, including multiple sclerosis, that could have a greater anti-glycolipid antibody mediated component than is currently recognised. References Arai, M., Yoshino, H., Kusano, Y., Yazaki, Y., Ohnishi, Y., Miyatake, T., 1992. Ataxic polyneuropathy and anti-pr2 IgM-kappa m-proteinemia. J. Neurol. 239, 147–151. Aspinall, G.O., Mcdonald, A.G., Pang, H., Kurjanczyk, L.A., Penner, J.L., 1994. Lipopolysaccharides of Campylobacter jejuni serotype-O19 — structures of core oligosaccharide regions from the serostrain and 2 bacterial isolates from patients with the Guillain-Barre-syndrome. Biochemistry 33, 241–249. Buchwald, B., Weishaupt, A., Toyka, K.V., Dudel, J., 1995. Immunoglobulin G from a patient with Miller-Fisher syndrome rapidly and reversibly depresses evoked quantal release at the neuromuscular junction of mice. Neurosci. Lett. 201, 163–166. Buchwald, B., Toyka, K.V., Zielasek, J., Weishaupt, A., Schweiger, S., Dudel, J., 1998a. Neuromuscular blockade by IgG antibodies from patients with Guillain-Barre syndrome: a macro-patch-clamp study. Ann. Neurol. 44, 913–922. Buchwald, B., Weishaupt, A., Toyka, K.V., Dudel, J., 1998b. Pre- and postsynaptic blockade of neuromuscular transmission by Miller-Fisher syndrome IgG at mouse motor nerve terminals. Eur. J. Neurosci. 10, 281–290. Calderon, R.O., Attema, B., DeVries, G.H., 1995. Lipid composition of neuronal cell bodies and neurites from cultured dorsal root ganglia. J. Neurochem. 64, 424–429. Carpo, M., Pedotti, R., Lolli, F., Pitrola, A., Allaria, S., Scarlato, G., Nobileorazio, E., 1998. Clinical correlate and fine specificity of anti-GQ1b antibodies in peripheral neuropathy. J. Neurol. Sci. 155, 186–191. Chiba, A., Kusunoki, S., Shimizu, T., Kanazawa, I., 1992. Serum IgG antibody to ganglioside GQ1b is a possible marker of Miller Fisher syndrome. Ann. Neurol. 31, 677–679. Chiba, A., Kusunoki, S., Obata, H., Machinami, R., Kanazawa, I., 1993.

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