RICHARDP.BUNGEMEMORIALLECTUREANDREVIEW

The immunobiology of Guillain-Barre¤ syndromes

Hugh J.Willison

Division of Clinical Neurosciences, Southern General Hospital, Glasgow, Scotland, UK

Abstract This presentation highlights aspects of the immunobiology of the Guillain-

Barre syndromes (GBS), the world’s leading cause of acute autoimmune neuromuscular

paralysis. Understanding the key pathophysiological pathways of GBS and developing

rational, specific immunotherapies are essential steps towards improving the clinical out-

come of this devastating disorder. Much of the research into GBS over the last decade has

focused on the forms mediated by anti-ganglioside antibodies, and we have made sub-

stantial progress in our understanding in several related areas. Particular highlights include

(a) the emerging correlations between anti-ganglioside antibodies and specific clinical

phenotypes, notably between anti-GM1/anti-GD1a antibodies and the acute motor axonal

variant and anti-GQ1b/anti-GT1a antibodies and the Miller Fisher syndrome; (b) the identi-

fication of molecular mimicry between GBS-associated Campylobacter jejuni oligosacchar-

ides and GM1, GD1a, and GT1a gangliosides as a mechanism for anti-ganglioside antibody

induction; (c) the development of rodent models of GBS with sensory ataxic or motor

phenotypes induced by immunisation with GD1b or GM1 gangliosides, respectively. Our

work has particularly studied the motor nerve terminal as a model site of injury, and

through combined active and passive immunisation paradigms, we have developed murine

neuropathy phenotypes mediated by anti-ganglioside antibodies. This has been achieved

through use of glycosyltransferase and complement regulator knock-out mice, both for

cloning anti-ganglioside antibodies and inducing disease. Through such studies, we have

proven a neuropathogenic role for murine anti-ganglioside antibodies and human GBS-

associated antisera and identified several determinants that influence disease expression

including (a) the level of immunological tolerance to microbial glycans that mimic self-

gangliosides; (b) the ganglioside density in target tissue; (c) the level of complement

activation and the neuroprotective effects of endogenous complement regulators; and

(d) the role of calcium influx through complement pores in mediating axonal injury. Such

studies provide us with clear information on an antibody-mediated pathogenesis model for

GBS and should lead to rational therapeutic testing of agents that are potentially suitable

for use in humans.

Key words: autoantibodies, Campylobacter jejuni, complement regulator, gangliosides,

glycosyltransferase, Guillain-Barre syndrome, membrane attack complex, neuromuscular

synapse, neuropathy, tolerance

IntroductionAutoimmune neuropathies are a diverse group of

paralytic syndromes characterised by inflammation in

the peripheral nervous system (PNS), in turn initiated by

a range of quite distinct immunopathological

events. Understanding the immunological mechanisms

Address correspondence to: Prof. Hugh J. Willison, Division ofClinical Neurosciences, Institute of Neurological Sciences, SouthernGeneral Hospital, Glasgow G51 4TF, Scotland, UK. Tel: þ44-141-201-2464; Fax: þ44-141-201-2993; E-mail: h.j.willison@clinmed.gla.ac.uk

Journal of the Peripheral Nervous System 10:94–112 (2005)

� 2005 Peripheral Nerve Society 94 Blackwell Publishing

underlying any pathological responses is clearly crucial to

selecting the correct strategies for novel therapeutic inter-

ventions, and this is particularly germane to the Guillain-

Barre syndromes (GBS) where the relative involvement of

T- and B-cell responses is continually debated. In recent

years, great progress has been made in our understand-

ing of this group of disorders. Out of many avenues

explored, one of the most promising has been the dis-

covery and analysis of antibody responses to peripheral

nerve gangliosides and their microbial mimics, the rela-

tionships these have to different clinical phenotypes of

GBS, and our increasing insights into their mechanisms of

action. Despite this rapidly advancing progress, consider-

able gaps in our knowledge persist. The focus of this

lecture is on gangliosides and their corresponding auto-

antibodies in GBS. I will first provide a brief background to

the field, summarise some of the highlights, identify and

analyse areas of remaining weakness, and then present

some data to support novel therapeutic approaches that

could capitalise on the latest experimental findings.

The Clinical ProblemGBS is the prototypic acute inflammatory disorder

and the foremost cause of post-infectious neuromuscu-

lar paralysis worldwide, with a global incidence of

approximately 1.5/105 across all age groups (Hughes

and Rees, 1997; Hahn, 1998). The lifetime likelihood of

any one individual acquiring the disease is thus approxi-

mately 1 : 1000. Onset is rapid, and approximately 20%

of cases lead to total paralysis, requiring prolonged

intensive therapy with mechanical ventilation. The ther-

apeutic window for GBS is short, and the current opti-

mal treatment with whole plasma exchange or

intravenous immunoglobulin (Ig) therapy lacks immuno-

logical specificity and only halves the severity of the

disease (Visser et al., 1999; 2004; Raphael et al., 2001;

Hughes et al., 2004a; 2004b). The patients left severely

disabled (approximately 12% survivors unable to walk

after 1 year) or dead (UK mortality of 5–10%) represent

a major social and economic burden (Buzby et al., 1997).

Thus, there is an incentive to understand GBS patho-

genesis as a prerequisite to developing and instituting

effective, contemporary immunotherapies.

Clinical and Pathological Patterns of GBSDespite the all-embracing eponym, GBS’s clinical

pathophysiology is long recognised as being highly com-

plex (Hartung et al., 1995a; 1995b). GBS is an acute

phase illness occurring 10–14 days after trivial infections,

comes and goes rapidly (within 4 weeks), and leaves

variable residual injury. The acute, monophasic nature

of GBS provides crucial clues to the immunopathological

background and quite clearly distinguishes GBS from

most other pro-inflammatory insults that lead to neuro-

pathy. The commonest form of GBS arises from seg-

mental demyelination of peripheral nerve (acute

inflammatory demyelinating polyneuropathy [AIDP]),

executed by macrophage-mediated stripping of the mye-

lin sheath (Hafer-Macko et al., 1996b). At least a propor-

tion of this injury appears to be mediated by antibody and

complement deposition on Schwann cell and myelin

membranes, although the putative antigenic target(s) in

AIDP remain elusive, as discussed below. Clinically, this

demyelinating process is widespread, affecting most

myelinated limb, axial and lower cranial motor and sen-

sory nerves, but curiously sparing myelinating axons

innervating extraocular muscles that are so sensitively

affected in Miller Fisher syndrome (MFS). Resting intra-

neural Schwann cells proliferate and migrate into the

lesion sites to remyelinate denuded axons, producing a

good recovery in most cases. In AIDP, demyelinating

pathology may be extensive throughout the length of

the nerve, especially in proximal nerve roots and the

distal intramuscular nerve segments where the blood–

nerve barrier (BNB) is weak (Olsson, 1968). In agree-

ment, clinical electrophysiological studies indicate that

sites throughout the nerve can be affected but often

point towards proximal (absent or delayed F-wave laten-

cies) or distal (prolonged distal motor latencies) as domi-

nant sites of nerve impairment. It would seem intuitively

likely that the rate of recovery from AIDP should be

independent of the site of demyelination because there

is no evidence that the remyelinating capacity of

Schwann cells varies along the length of the nerve.

Axons are generally unaffected in AIDP, although may

suffer so-called bystander injury, the mechanisms for

which remain unclear and deserve further study.

In contrast to AIDP, the primary target for immune

attack in the GBS variant, acute motor (and sensory)

axonal neuropathy (AMAN, AMSAN) is the axolemmal

membrane (Feasby et al., 1986; McKhann et al., 1993).

Again, this inflammatory process occurs predominantly

either in the nerve roots or distal nerve terminals

(Hafer-Macko et al., 1996a; Ho et al., 1997a;

Kuwabara et al., 2003). Immune attack can lead to

reversible axonal conduction block due to reversible

axonal injury (at best) or complete axonal transaction

(at worst). Wallerian degeneration (Wld) will occur dis-

tal to the site if axons are transected; otherwise, mye-

lin is unaffected. Depending on the site of transection

(proximal or distal), axonal recovery may be poor or

good, owing to the distance over which regeneration

is effective (Ho et al., 1997a; 1997b). This is important

clinically because extensive radicular involvement in

AMAN generally leads to a catastrophic, permanent

injury. In contrast, very distal axonal injury would also

induce a severe acute axonal syndrome, but one in

which rapid reinnervation with functional recovery

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

95

could readily occur (Ho et al., 1997a; Kuwabara et al.,

2003).

Formes Frustes of GBS Also Exist asRegionalVariants

The regional variants of GBS only paralyse specific

areas of the body, such as the eyes or face, or the

afferent sensory and autonomic systems (Ropper,

1994). The most widely studied of these variants is the

MFS (Fisher, 1956; Willison and O’Hanlon, 1999). Our

understanding of MFS was revolutionised following

Chiba and Kusunoki’s discovery of the anti-GQ1b anti-

body marker (Chiba et al., 1992; 1993). Since the syn-

drome was first described in 1956 as the discrete clinical

triad of ophthalmoplegia, ataxia, and areflexia, anti-GQ1b

antibody testing has allowed MFS to evolve nosologi-

cally and now encompasses closely related formes

frustes, mainly characterised by acute cranial motor neu-

ropathies with ataxia – the anti-GQ1b antibody syn-

dromes (Odaka et al., 2001; Paparounas, 2004). In the

Bickerstaff’s encephalitis, MFS-like features occur in

conjunction with brain stem involvement, comprising

pyramidal tract signs and impaired consciousness, and

two thirds of cases are anti-GQ1b antibody positive

(Bickerstaff and Cloake, 1951; Odaka et al., 2003). The

close relationship between GBS and MFS is considered

because it should direct our search towards common

underlying immunopathological mechanisms. Thus,

some MFS cases merge into confluent GBS with

respiratory and limb involvement, and similarly, some

GBS cases also evolve to develop an MFS pattern of

clinical involvement – in these overlapping cases, anti-

GQ1b antibodies are generally detected. MFS variants

include solitary ophthalmoplegia or ataxia and orophar-

yngeal weakness without ophthalmoplegia (O’Leary

et al., 1996). Viewed in its broadest contemporary

sense, MFS could be considered as a PNS/CNS overlap

syndrome with extremely variable degrees of involve-

ment of particular central or peripheral anatomical sites

in individual cases, with all permutations being highly

associated with anti-GQ1b and anti-GT1a antibodies

(O’Leary et al., 1996; Odaka et al., 2001).

The selective affliction of cranial and in particular

extraocular nerves in the anti-GQ1b antibody syn-

dromes is believed to be due to the enrichment of

the target antigen(s) in affected sites (Chiba et al.,

1997). However, there are clearly additional levels of

complexity influencing the clinical phenotype that

remain undiscovered. What is notable by its absence

is involvement of extraocular motor nerves in AIDP,

arguing that the AIDP antigen(s) may analogously be

relatively enriched in limb and axial myelin (in compar-

ison with extraocular motor nerve myelin) as opposed

to being a generic myelin antigen(s). This might

mitigate against the major structural proteins of myelin

being dominant AIDP antigens. A chronic ataxic neuro-

pathy has also been reported that may be associated

with relapsing ophthalmoplegia in some cases; such

patients often have anti-ganglioside antibodies, includ-

ing anti-GQ1b, which are persistently present, usually

occurring as IgM paraproteins (Willison et al., 2001).

Anti-NerveAntibodies in GBS: LessonsFrom Rodent Models

Great progress has been made in correlating clinical

phenotypes with serological profiling of anti-nerve antibo-

dies, covering a wide spectrum of peripheral nerve anti-

gens. The myelin protein-specific T and B cell-mediated

rodent model of GBS (experimental allergic neuritis [EAN])

was described 50 years ago and has yielded many impor-

tant experimental insights into peripheral nerve inflamma-

tion (Spies et al., 1995; Rostami, 1997; Gabriel et al., 1998;

Taylor and Pollard, 2003). However, translational studies

identifying equivalent immune responses in human neuro-

pathy cases have been more modest (Gabriel et al., 2000;

Ritz et al., 2000; Yan et al., 2000; Favereaux et al., 2003;

Latov and Renaud, 2004). Whether further research will

overcome this remains to be seen.

In contrast, the progress in identifying myelin and

axonal glycolipids as antigens has been somewhat

more forthcoming. Interestly that ganglioside-/glycolipid-

induced rabbit EAN was also first described several

decades ago (Nagai et al., 1976; Saida et al., 1979),

but in contrast to myelin protein-induced EAN, was

then virtually neglected for approximately 20 years,

and has now been revived with compelling data that

reinforce many recent clinical findings. Thus, a series

of recent rabbit immunisation studies have led to the

generation of models of anti-GD1b antibody-associated

ataxic neuropathy and anti-GM1 antibody-associated

motor axonal neuropathy that mimic many of the clin-

ical and pathological features of the human syndromes

(Kusunoki et al., 1996; Yuki et al., 2001; 2004).

These studies have provided some of the most

compelling evidence that anti-ganglioside antibodies

and their effector pathways can induce clinically rele-

vant phenotypes. In this respect, ganglioside-induced

EAN has come full circle. The return of researchers,

principally in Japan, to these early rabbit studies was

driven in large part by the clinical identification of anti-

ganglioside antibodies in GBS cases, starting in 1988

(Ilyas et al., 1988) and escalating in scope ever since. A

wealth of clinical serological associations have now been

described and the topic reviewed regularly (Yuki, 2001;

Willison and Yuki, 2002). In summary, in human disease,

anti-GM1, anti-GD1a, anti-GM1b, and anti-GalNAcGD1a

antibodies are highly associated with AMAN (Ogawara

et al., 2000) and anti-GQ1b, anti-GT1a, anti-GD3, and

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anti-GD1b with MFS and chronic ataxic neuropathy

(Chiba et al., 1993; Willison et al., 2001). Anti-GM1 IgG/

IgM antibodies are also associated with AIDP and

chronic demyelinating clinical phenotypes, the latter

with or without concomitant axonopathy, as seen in

multifocal motor neuropathy (Pestronk and Choksi,

1997). However, considerable debate still remains as to

the relative extent of demyelinating and axonal pathol-

ogy that can be associated with anti-GM1 antibody syn-

dromes. An exciting new finding has suggested that

gangliosides assembled in complexes (in this case

GM1 and GD1a) (Kaida et al., 2004) might provide higher

avidity targets for GBS-associated autoantibodies than

single ganglioside species. Although it has long been

known that accessory lipids play an important role in

enhancing or attenuating ganglioside–antibody interac-

tions, this provocative finding greatly raises the complex-

ity of examining sera for the new autoantibody

specificities, including the elusive AIDP antigen(s).

Gangliosides and Structural Mimics onMicrobial Glycans

One highly fruitful area has been the discovery of a

range of ganglioside and glycolipid mimics on microbial

glycans (Yuki et al., 1993; Yuki, 2001). There are

approximately 50 structurally distinct gangliosides

synthesised through step-wise addition of monosac-

charides by Golgi glycosyltransferases in complex

developmental, spatial, and cell-specific patterns

(Kolter et al., 2002). Gangliosides are enriched in neural

tissues and primarily localised to raft domains of the

extracellular leaflet of plasma membranes, especially

at synapses, where they are available for anti-

ganglioside antibody binding (Ledeen, 1978; Ledeen and

Yu, 1982; Ledeen et al., 1998; Ogawa-Goto and Abe,

1998). A simplified scheme of the major ganglioside

structures is shown in Figure 1. The carbohydrate moi-

eties of gangliosides are structural mimics of microbial

glycans, including the lipo-oligosaccharides (LOS) of

Campylobacter jejuni (Aspinall et al., 1994; Yuki et al.,

1997; Sheikh et al., 1998; Prendergast and Moran,

2000; Moran et al., 2002) and Haemophilus influenzae

(Mori et al., 1999; Ju et al., 2004). Evidence from

human and animal studies indicates a key role for this

molecular mimicry in GBS pathogenesis (Goodyear

et al., 1999; Bowes et al., 2002; Yuki et al., 2004). In

this model, well documented for GM1, GD1a, and

GQ1b, the acute phase anti-LOS/ganglioside complement-

fixing IgG antibodies that arise eradicate infections but

also bind to peripheral nerve gangliosides where they

induce autoimmune injury. Potential pathogenic role(s)

exist for other known and unknown glycolipids

enriched in Schwann cell, myelin, and axonal mem-

branes, such as galactocerebroside, LM1, and

sulfatides that also have microbial glycan mimics

(Yuki, 2001; Willison and Yuki, 2002).

ImmunologicalTolerance Exists toSelf-Ganglioside Structures

One of the curiosities of GBS is that such a small

proportion of infections or vaccinations triggers the

illness. Thus, 99% of humans infected with ganglio-

side-mimicking strains of C. jejuni neither develop anti-

LOS/ganglioside antibodies nor GBS (Nachamkin,

2001). The immunological factors that regulate this

unresponsiveness to challenge with microbial mimics

of self-glycan structures are poorly understood, but

it seems likely that B-cell tolerance is an important

component. Anti-LOS/ganglioside antibodies exist

within the natural antibody repertoire, acting as

innate defence against bacteria. Being carbohydrates,

gangliosides elicit T cell-independent (TI) humoral

responses (Martin et al., 2001; Zubler, 2001) and can-

not be presented by major histocompatibility complex

molecules (Ishioka et al., 1993) (except via CD1 pre-

sentation [Brigl and Brenner, 2004; Watts, 2004] ).

Anti-ganglioside antibodies exist as low-affinity IgM

isotypes in normal subjects (Willison et al., 1993;

Mizutamari et al., 1994; Casali and Schettino, 1996).

To prevent autoimmune reactions, their level and affi-

nity are controlled by tolerance (Cornall et al., 1995;

Fagarasan and Honjo, 2000). In GBS, the appearance

of high titre anti-ganglioside antibodies is a clear failure

of tolerance. B-cell tolerance to TI ganglioside antigens

is poorly understood and remains a major research

goal in the GBS field. Aspects are analogous to organ

transplantation paradigms involving Gal(a1–3)Gal anti-

gens as studied in a1–3galactosyltransferase knock-

out (KO) mice (Kawahara et al., 2003; Galili, 2004)

and in the ABO blood group system (Fan et al., 2004;

Fehr and Sykes, 2004).

We and others anticipated that the expression of

gangliosides in tissues outside the nervous system

that can be sensed by newly developing and pre-exist-

ing B cells (bone marrow and spleen, respectively)

would be an important regulator of tolerance, as sug-

gested for anti-Gal(a1–3)Gal (Yang et al., 1998). Thus,

we showed that mice lacking complex gangliosides in

any tissue (i.e., the GalNAcT KO mice that only

express GM3 and GD3, see Fig. 1) develop exagger-

ated humoral responses to gangliosides compared

with wild-type (WT) controls when challenged with

C. jejuni LOS (Bowes et al., 2002) or gangliosides

(Lunn et al., 2000). An example of the differential anti-

body responses to GD1a ganglioside and GD1a-bearing

C. jejuni LOS in GalNAcT WT and KO mice that clearly

illustrates this principle is shown in Figure 2.

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Isolating and Characterising MurineMonoclonal Anti-GangliosideAntibodies

An offshoot of these experiments aimed at

bypassing tolerance to microbial glycans has been

the creation of high-affinity IgG responses to ganglio-

sides that are suitable for cloning top quality anti-gang-

lioside antibodies – a long-awaited goal for these

poorly immunogenic structures. In addition to over-

coming any tolerogenic factors, to circumvent any TI

restriction on affinity maturation and class switching

(including that which might occur in glycosyltransferase

KO mice), we have developed hapten-carrier

immunisation protocols using ganglioside–protein

conjugates and LOS/ganglioside liposome-encapsulated

proteins to provide the necessary T cell-dependent

(TD) environment required to produce the magnitude

of immune responses seen in Figure 2. Using these

methods, we and others have generated long-lived IgG

memory responses to gangliosides and to LOS in gly-

cosyltransferase KO mice. Using such an approach,

we have cloned approximately 50 IgG/IgM monoclonal

antibodies specific for discrete ganglioside epitopes for

use in a wide range of pathogenesis and therapeutic

studies (Boffey et al., 2004; Willison et al., 2004).

Ugcg–/– Cer

Cer

Cer

Cer

CerGA2

A simplified scheme of the ganglioside biosynthetic pathway

CerGA1

CerGM1b

Cer 0 series

a series

b series

GD1c

CerGT1a

CerGQ1b

CerGD1a

CerGT1b

CerGM1a

CerGD1b

CerGM2

CerGD2

GM1

HS:19(GM1+, GD1a+) and HS:4 HS:19(GM1+, GT1a+)

Lipid A

Galactose Glucose NeuNAc Cer CeramideGalNAcKey

Lipid A Lipid A Lipid A Lipid A

HS:10

Examples of Campylobacter jejuni LOS stuctures

Candidate oligosaccharide fragments for inhibition/immunoadsorption studies

GD1a GQ1b

Cer

GlcCer

A

B

C

LacCer

GD3s–/–

GM3

GD3

GalNAcT–/–

Figure 1. A schematic representation of the key structures and enzymes in ganglioside biosynthesis, relevant glycanfragments, and core structures identified in Campylobacter jejuni lipo-oligosaccharides. Note that GalNAc transferase(GalNAcT) and GD3 synthase (GD3s) deficiency result in the loss of all complex and b-series gangliosides, respectively.

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

98

These antibodies have been powerful tools in enhan-

cing our understanding of pathogenic pathways that

model the human disorders.

One area of particular interest is in understanding

structure–specificity relationships amongst antibodies

and their binding partner glycans. Thus, Ig variable (V)

region sequencing, affinity, and glycan-binding studies

on the above IgG/IgM mouse monoclonal antibodies

have allowed us to investigate whether (a) specificity is

dictated by particular V-gene usage and (b) class

switching from IgM to IgG is associated with affinity

maturation. Interestly that the minor alteration in spe-

cificity from terminal disialosyl residues (on GQ1b,

GT1a, and GD3) to internal disialosyl residues (on

GD1b and GT1b) is mutually exclusively associated

with a change from murine VH7183.3b to VH10.2b

gene segment usage (Boffey et al., 2004; Boffey,

unpublished data). V-gene mutation rates and affinity

measurements (using Biacore surface plasmon

resonance) indicate that affinity maturation for most

AG monoclonal antibodies is modest, even amongst

class-switched monoclonal antibodies isolated from

glycosyltransferase KO mice (Townson, unpublished

data). Collectively, these murine data indicate that

diverse antibody specificities encoded by restricted

sets of VH/VL genes reside within the natural antibody

repertoire and can be expanded by bacterial LOS once

released from tolerance. This is not necessarily asso-

ciated with affinity maturation; nevertheless, such anti-

bodies can readily injure the PNS, as described below.

The observations we have made with murine anti-

bodies has shifted our predictions about the likely shape

of the human anti-ganglioside antibody repertoire in

GBS, which is not well understood because few have

been cloned to date apart from long-lived IgM antibo-

dies (Paterson et al., 1995). It is known that the antibody

response in GBS has class-switched from IgM to the

TD complement-fixing IgG1 and IgG3 isotypes (Willison

and Veitch, 1994). This would usually co-occur with

affinity maturation of a range of clones within the

B-cell repertoire. However, we now suspect that the

repertoire may be considerably more clonally restricted

and unmutated in GBS than previously thought and are

currently investigating this area. In support of the rela-

tively low affinity for GBS-associated antibodies are our

findings that (a) human IgG anti-GQ1b antibodies can be

readily displaced from antigen by polyclonal human IgG

(IVIg therapy) (Jacobs et al., 2003) and (b) they slowly

elute off immuno-affinity columns in the presence of

physiological strength buffers (Willison et al., 2004).

This has important implications for several therapeutic

strategies aimed at inhibition of antibody–glycan inter-

actions or selective ablation of ganglioside-specific

B-cell pools, as outlined in Figure 3.

The Sites and Mechanisms byWhich Anti-GangliosideAntibodies Paralyse the PNS

Anti-ganglioside antibodies could potentially bind

any ganglioside-containing membranes, provided that

they can gain access and binding is not subject to steric

inhibition locally in the membrane. One site for antibody

action we discovered and have worked on extensively

is the ganglioside-rich, pre-synaptic component of the

neuromuscular junction (NMJ), lying outside the BNB

where its membranes are readily accessible to circulat-

ing antibodies. Other important proximal axonal and glial

sites of injury exist, especially exposed axolemma at

nodes of Ranvier and paranodal myelin, as has been

well demonstrated in a wide range of human studies

described above and in animal studies (Kusunoki et al.,

1996; Yuki et al., 2001; Sheikh et al., 2004).

One reason why our studies initially focused on

the NMJ was because of the longheld appreciation of

1.2

0.6

HS:4

HS:4 GD1a

1.4GD1a GD1a

GM1

Blank

GalNAcT+/+

GalNAcT–/–

0.7

0Pre 1st 2nd 3rd

Pre 1st 2nd 3rd Pre 1st 2nd 3rd

Pre 1st 2nd 3rd0

1.2Opt

ical

den

sity

1.4

0.7

*

0

Immunisation number

0.6

0

Figure 2. Anti-ganglioside IgG antibody responses inGalNAc transferase (GalNAcT)þ/þ and GalNAcT–/– mice fol-lowing intraperitoneal immunisation with HS:4 lipo-oligosac-charides (bearing a GD1a epitope, left-hand panels) or GD1a/ovalbumin liposomes (right-hand panels). Mice were immu-nised at 2-weekly intervals and bled 4 days after each immu-nisation. Elevated anti-GD1a antibody titres are evident inGalNAcT–/– mice (deficient in GD1a) by the second or thirdimmunisation, compared with non-responding GalNAcTþ/þ

mice (*p < 0.005). Modified from Bowes et al. (2002).

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

99

the clinical resemblance between MFS and botulism

(Marvaud et al., 2002). Botulinum toxins bind the nerve

terminal in part by using gangliosides as ectoacceptors

(Montecucco et al., 1988; Kitamura et al., 1999;

Bullens et al., 2002), prior to their cytosolic uptake

and proteolytic action on proteins of the release

machinery (Schiavo et al., 2000). It thus seemed logical

that anti-ganglioside antibodies might also act at the

NMJ to induce paralysis, albeit through a different

mechanism. Emerging clinical electrophysiological evi-

dence suggests that nerve terminal dysfunction occurs

in both MFS and GBS (Uncini and Lugaresi, 1999;

Wirguin et al., 2002; Kuwabara et al., 2003; Spaans

et al., 2003; Lo et al., 2004). However, it is also impor-

tant to recognise the immunohistological observations

of Chiba and colleagues that have clearly demonstrated

anti-GQ1b antibodies binding to extra-ocular nerve

nodes of Ranvier and it may well be that multiple neural

sites for antibody attack exist that may differ both

between syndromes and individuals (Chiba et al., 1993).

AMechanistic Pathway for NerveTerminalAxonal Injury Culminating in SynapticNecrosis

We first demonstrated using in vitro mouse hemi-

diaphragm preparations that anti-GQ1b antibodies asso-

ciated with MFS bind the motor nerve terminal where

they locally activate complement (Roberts et al., 1994;

Plomp et al., 1999; Halstead et al., 2004). A schematic

diagram of the pathological processes occurring in this

model that summarises an extensive series of experi-

ments is shown in Figure 4. Following antibody binding

with local complement fixation, an uncontrolled influx of

calcium into the nerve terminal through membrane

attack complex (MAC) complement pores then provokes

spontaneous exocytosis, accompanied by calpain-

mediated structural degradation of the terminal axonal

cytoskeleton and calcium-mediated mitochondrial injury,

with resultant paralysis (O’Hanlon et al., 2001; 2003).

MAC formation is essential because C6-deficient condi-

tions abolish the effect (Halstead et al., 2004). Features

of this lesion are similar to those induced by the pore-

forming toxin, a-latrotoxin (O’Hanlon et al., 2002). We

have used the term ‘synaptic necrosis’ to describe these

events in distinction from other forms of synapse elim-

ination including synaptosis (Gillingwater and

Ribchester, 2003). To illustrate this process, Figure 5

shows synaptic necrosis occurring at nerve terminals in

Thy1-CFP/YFP transgenic mice that express fluorescent

protein in the cytosolic compartment of the whole motor

axon including the nerve terminal (collaboration, R.

Ribchester) and Figure 6 shows the electrophysiological

events occurring concomitantly with this structural dis-

integration (collaboration, J. Plomp). There is evidence

that anti-GM1, anti-GD1a, and anti-GalNAcGD1a antibo-

dies may also act in part at the NMJ (Roberts et al., 1995;

Taguchi et al., 2004a; Goodfellow et al., 2005), as

described below and seen in Figures 6 and 7.

The extent to which anti-ganglioside antibody-

mediated nerve terminal degeneration extends proxi-

mally up the axon remains uncertain and may be influ-

enced by the ganglioside specificity of the anti-

ganglioside antibodies and the integrity of the BNB

and local regulators of complement activation. The

pathophysiological pathways underlying synaptic and

axonal degeneration may be distinct, as suggested by

studies in the slow Wld (Wlds) mutant mouse (Mack

et al., 2001; Gillingwater and Ribchester, 2003). When

applying anti-GQ1b antibodies to this MFS model, the

nerve terminal of Wlds mice is not protected from

MAC-mediated injury, undergoing degeneration to the

same extent as seen in WT mice (Willison et al.,

unpublished data), however, any influence of Wlds in

protecting the more proximal axon from injury is

unknown.

GM1

GD1a

GQ1b C4 C2

C3

C5b

MA

C

Ca2+

MAC

–CD59

CR1

Crry

DAF

Complement components and regulators

Soluble oligosaccharidefragments as inhibitors

C6,7,8,9

C5

C3 convertase

C5 convertase

C1

From patientGM1-sepharose column

To patient

Immunoadsorption columns

Axon

Axon/synapse

necrosis

Figure 3. A schematic diagram of potential therapeutic tar-gets and strategies based on inhibition or removal of anti-ganglioside antibodies, inhibition of steps in complementactivation, or enhancement of complement regulation.

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

100

Anti-Disialylated GangliosideAntibodiesTarget the Perisynaptic Schwann Cell

An interesting neuropathogenic mechanism that

has not been explored in human disease is the extent

to which perisynaptic Schwann cell (pSC) injury might

contribute to distal motor axonal degeneration.

Similarly to the axonal element of the nerve terminal,

the pSC lies outside the BNB where it is fully exposed

Ca2+

α-Latrotoxin

Ca2+

Calcium-mediatedmitochondrial dysfunction

Ab

Lipid Raft

Synapticvesicle

C1q

Ca2+

Ca2+

Calpain-mediatedcytoskeleton disintegration

MACVGCC

NF+

VGCC

Complement classicalpathway activation

Figure 4. A schematic diagram of the events occurring at nerve terminals in experimental nerve-muscle preparations exposed toanti-ganglioside antibodies in the presence of complement. Exocytosis is normally initiated by highly regulated calcium entry throughvoltage-gated calcium channels that activate the adjacent SNARE complex and initiate synaptic vesicle fusion with the pre-synapticmembrane. When membrane attack complex (MAC) pores are deposited in the pre-synaptic membrane, unregulated calcium entrytriggers massive, uncontrolled exocytosis and calcium-/calpain-mediated intra-terminal injury, including cytoskeletal degradation andmitochondrial death. The pore-forming toxin, a-latrotoxin, is believed to act in part through a similar mechanism, allowing unregulatedcalcium influx with electrophysiological and morphological sequelae similar to those resulting from MAC pores.

A B

Figure 5. Neuromuscular junctions in triangularis sternae muscle preparations from B6.Cg-Tg(Thy1-CFP) green fluorescent micewere exposed in vitro to anti-GQ1b monoclonal antibody and the nuclear marker, ethidium dimer (EthD-1). Images were collectedbefore (A) and 7 min after (B) the addition of human serum as a source of complement. (A) Normally appearing green fluorescence isvisible in axon terminals overlying the post-synaptic membrane (stained for bungarotoxin, blue) and EthD-1 is excluded (and thereforenot seen in this image) from perisynaptic Schwann cell (pSC) nuclei, as normal. (B) Green fluorescence has disappeared throughleakage from the axon terminal undergoing acute synaptic necrosis. Concomitantly, EthD-1, which is normally excluded from theintracellular compartment, has been taken up into pSC nuclei (that now appear red) because of pSC plasma membrane injury.

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

101

to the extracellular fluid environment. Using anti-

ganglioside antibodies with different specificities for

GQ1b, GT1a, and GD3, and for GM1 and GD1a, we have

shown that anti-ganglioside antibodies can (a) destroy

the nerve terminal (i.e., synaptic necrosis), (b) kill the

pSC that envelops the pre-synaptic region, or (c)

destroy both nerve terminal and pSC (Halstead et al.,

2004; Halstead et al., 2005). Model schemes of this

injury are shown in Figure 7, and an electron micro-

graph of selective pSC damage induced by an anti-

disialosyl antibody is shown in Figure 8. pSCs support

the underlying motor nerve terminal, both in the steady

state and during development and regeneration (Kong

et al., 1998; Auld and Robitaille, 2003; Love et al.,

2003; Reddy et al., 2003; Halstead et al., 2004; Liu

et al., 2004). The selective ablation of pSCs at mam-

malian NMJs has not been previously achieved and

their role in human disease not considered. We have

recently been able to segregate injury to these two

compartments. Interestingly, pSC ablation induced no

acute electrophysiological or morphological changes to

the underlying nerve terminal, suggesting that at mam-

malian NMJs, acute pSC injury or ablation has no major

deleterious influence on synapse function. However, it

is expected that the absence of pSCs may well have a

deleterious effect on the longer-term integrity of the

underlying synapse, and this area deserves further

attention. Information on ganglioside distribution in dif-

ferent membranes and cell types at the human NMJ is

sparse, although this is known to be a ganglioside- and

glycan-rich area (Martin, 2003). One might expect to

see variations in ganglioside distribution that correlate

with clinically affected sites (e.g., cranial nerves, poly-

sialylated gangliosides; limb nerves, ‘a series’ ganglio-

sides) (Ogawa-Goto et al., 1992; Chiba et al., 1997;

Ogawa-Goto and Abe, 1998; Gong et al., 2002), and

this is an area that deserves further study.

The Role of Complement Regulators andTherapeutic Inhibitors in Attenuating NerveInjury

An important body of evidence has proven that anti-

ganglioside antibodies exert their paralytic effects

directly (Buchwald et al., 1998; 2001; Ortiz et al., 2001;

Taguchi et al., 2004a; 2004b). Nevertheless, tissue-

bound antibody of the appropriate class should automa-

tically fix complement that would therefore exacerbate

injury over and above any direct actions of antibody

(Koski et al., 1987). It is for this reason that many of our

recent studies have focused on complement-dependent

injury and begun to investigate complement inhibitors

and regulators as therapeutic targets, as outlined in

Figure 3. Complement activation is regulated by an

equally complex inhibitory process, mediated by human

and mouse decay-accelerating factor (DAF), CD59, com-

plement receptor 1 (CR1), and mouse CR1-related gene

y (Crry) (Morgan and Harris, 2003; Turnberg and Botto,

2003; Mizuno and Morgan, 2004). CD59 inhibits the

formation of MAC and DAF accelerates the decay of

C3/C5 convertases (Lukacik et al., 2004; Mizuno and

Morgan, 2004; Harris et al., 2005). Thus, we recently

discovered that CD59-deficient mice are excessively

vulnerable to anti-ganglioside antibody-dependent com-

plement-mediated injury (Halstead et al., 2004). An

example of this effect is shown in Figure 9. These find-

ings agree with data from experimental myasthenia

Before

EP

P a

mpl

itude

(m

V)

Complementaddition

40

B

A

30

20

10

00

10 m

V

20 ms

5Time (min)

10 15

Afteranti-GD1a

mAb

WT1 mv

100 ms

t = 0t = 5t = 10

t = 11

t = 12

t = 13

GD3s–/–

Figure 6. Microelectrode recordings from GD3s–/– and wild-type (WT) mouse ex vivo hemidiaphragm preparations exposedto anti-GD1a antibodies raised in GalNAcT–/– mice immunisedwith Campylobacter jejuni lipo-oligosaccharides. (A) Acute expo-sure of nerve terminals to anti-GD1a antibody causes a massiveincrease in miniature endplate potential (MEPP) frequency at theGD3s–/– neuromuscular junction (NMJs), with no effect at WTNMJs. The anti-GD1a antibody effect is entirely dependent onthe presence of a source of complement. (B) An example ofneurotransmission failure at GD3s–/– NMJ induced by anti-GD1aantibody plus complement. The phrenic nerve of a GD3s–/–

diaphragm nerve-muscle preparation was stimulated onceevery 30 s over a 13-min monitoring period, in the presence ofm-conotoxin-GIIIB to prevent muscle action potentials. Endplatepotentials (EPPs) were recorded at an NMJ where MEPP fre-quency was very high (>100/s, visible at the baseline of traces).EPPs were normalised to �75 mV and superimposed. The EPPdecreased with time after complement addition and becameblocked. This did not occur at WT or GalNAcT–/– nerve terminalsand did not occur without the anti-GD1a monoclonal antibody(experiments conducted by J. Plomp, modified fromGoodfellowet al., 2005).

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

102

gravis (Lin et al., 2002; Kaminski et al., 2004) and experi-

mental allergic encephalomyelitis (Mead et al., 2004).

It is very clear that complement activation with MAC

formation drives neural membrane injury in anti-GQ1b-

treated mouse tissue (Halstead et al., 2004). It is also

well established that MAC is present in human GBS

biopsy material (Lu et al., 2000; Putzu et al., 2000;

Wanschitz et al., 2003). It would thus appear likely that

blocking MAC formation locally should prevent MAC-

dependent tissue injury, even if anti-ganglioside antibody

is deposited in the membrane. One therapy we have

used to investigate this is the complement inhibitor,

APT070 (collaboration, R. Smith, Inflazyme). APT070

contains the C3/C5 convertase-inhibiting region of CR1

and a membrane-localising peptide that allows it to inhi-

bit complement accumulation (Linton et al., 2000; Smith

and Smith, 2001; Pratt et al., 2003). Using APT070, we

can completely abrogate MAC formation and acute tis-

sue injury by pre-treatment of anti-ganglioside antibody-

immunised mice both ex vivo and in vivo, as shown in

Figure 10 (Halstead et al., unpublished data). The thera-

peutic window for APT070 intervention in passive and

active immunisation models of GBS remains unknown,

but clearly, it is a possible intervention in human neuro-

pathies in which complement deposits are implicated.

The Influence of Ganglioside Density onNerveVulnerability toAnti-GangliosideAntibodyAttack

One of the most interesting enigmas surrounding

GBS is the regional pattern of clinical involvement and

the way that this links in with anti-ganglioside antibody

specificities. Clearly, this regional involvement can

potentially be accounted for at many different levels.

The most striking example of this is MFS, in which

anti-GQ1b and anti-GT1a antibodies appear to target

the oculomotor and bulbar nerves because GQ1b and

PerisynapticSchwann cell

Normal NMJ Injured NT; reactive pSC

Injured pSC; normal NTInjured NT and pSC

Nerve terminal

A B

C D

Muscle surface

Key

Vesicles

Antibody

MAC pores

Mitochondria

Neurofilaments

Figure 7. (A) Schematic representation of the normal neuromuscular junction (NMJ) and (B–D) different patterns of NMJ injuryoccurring on exposure to complement-fixing anti-ganglioside antibodies. At NMJs in which the nerve terminal (NT) alone is injured(e.g., by anti-GD1a antibodies), the terminal axon disintegrates and the perisynaptic Schwann cell (pSC) becomes reactive,extending processes that interdigitate between the axonal fragments and envelope the remaining terminal with wraps ofSchwann cell membranes. If the pSC is concomitantly injured by complement deposits along with the NT (e.g., by anti-disialosylantibodies), the pSC reactive response does not take place, and the pSC undergoes rapid necrotic disintegration. Antibodies thatinduce acute pSC death alone appear to spare the nerve terminal in the short term, both morphologically and functionally.

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

103

GT1a are enriched in those sites. By corollary, because

AMAN or AIDP occurring in the absence of anti-GQ1b

antibody spares the oculomotor nerves, the relevant

antigens should be missing or at a low enough density

to escape targeting by antibody. In considering this,

the term density is solely intended here to refer to the

total amount of membrane ganglioside, independent of

any effect of raft compartmentalisation that might con-

centrate antigen in discrete regions (Herreros et al.,

2001; McKerracher, 2002; Guan, 2004).

In this area, GalNAcT–/– and GD3s–/– mice have told

us a lot about the extent to which ganglioside density

in neural membranes influences the degree of anti-

ganglioside antibody-mediated neural injury. Firstly, it is

necessary to recall that glycosyltransferase KO mice

develop precursor substrate build-up above the enzyme

block and direct excess gangliosides down other avail-

able routes in the biosynthetic pathway (Sandhoff and

Kolter, 2003). Thus, GalNAcT–/– mice contain only GM3

and GD3, but in excess amounts, whereas GD3s–/– mice

lack all ‘b series’ gangliosides and overexpress the ‘a

series’ products, GM1 and GD1a (Kawai et al., 2001;

Okada et al., 2002). We have discovered in two models

(passive immunisation of anti-GD3 antibody into

GalNAcT–/– and anti-GD1a antibody into GD3s–/– mice)

that this enhancement of ganglioside levels confers sen-

sitivity to development of disease compared with the

relatively insensitive WT mice that express ‘normal’

levels of ganglioside (Bullens et al., 2002; Goodfellow

et al., 2005). This is particularly well illustrated in the

GD3s–/– mouse exposed to anti-GD1a antibody, as

shown pictorially in Figure 11. What is now required is

an extensive regional evaluation of the human PNS to

map ganglioside density in key sites, including axonal,

glial, and nerve terminal membranes, most easily con-

ducted by quantitative immunofluorescence in order to

preserve gross anatomical information that is largely lost

when using preparative biochemical techniques.

Treatment of GBS by Removal orNeutralisation of Anti-GangliosideAntibodies

As stated earlier, GBS is unlikely to ever be pre-

ventable; thus, optimising acute therapy is paramount.

We have considered a number of therapeutic interven-

tions that would be suitable for use at clinical presenta-

tion in what we believe to be antibody-mediated forms

pSCpSC

MNMN

NT

NT

Figure 8. Mouse monoclonal anti-disialosyl antibodies segregate into groups according to whether they injure the nerveterminal, the perisynaptic Schwann cell (pSC), or both, following passive exposure of mouse neuromuscular junctions toantibody in the presence of complement. In this low-magnification electron micrograph, an antibody that kills the pSC andspares the axon is shown. A motor axon is wrapped by the last myelinating Schwann cell just proximally to the nerve terminals(NTs) that form synaptic contact with a muscle fibre. The NTs have a normal morphology, with electron-dense mitochondriaand tightly packed synaptic vesicles. Two pSCs sitting on either side of the terminal axon appear severely damaged withswollen and electron lucent cytoplasm, damaged organelles, nuclear membrane blebbing, and perinuclear bodies (arrow-heads), all indicative of necrotic cell death. Two myonuclei (MN) beneath the post-synaptic membrane projecting into this planeof section are of normal appearance. By immunofluorescence, these pSCs would be laden with anti-disialosyl antibody andcomplement products. Scale bar ¼ 5 mm (Halstead et al., unpublished data).

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

104

of GBS, as outlined in Figure 3. Treatment could be

aimed at antibody neutralisation or removal or at sup-

pression of antibody effector function (e.g., inhibition

of complement activation) and key downstream

inflammatory pathways (e.g., chemotaxis and cellular

extravasation). In consideration of the former, anti-

ganglioside antibodies could be (a) removed from the

circulation by immunoadsorption plasmaphoresis, (b)

neutralised with soluble oligosaccharides/glycans, pep-

tide mimics, or anti-idiotypic IVIg, or (c) deleted through

selective plasma cell/B-cell ablation (e.g., by using ricin–

antigen complexes) (Tanemura et al., 2002).

In an approach aimed at antibody neutralisation or

removal, we have identified the minimum epitope

requirements for murine monoclonal antibody and

human anti-GQ1b, anti-GM1, and anti-GD1a antibody

binding and have chemically or enzymatically synthe-

sised these epitopes and related candidate analogues

in collaborations (D. Bundle, Edmonton; A. Bernardi and

S. Sonnino, Milan; N. Bovin and O. Galanina, Moscow)

(Andersen et al., 2004; Willison et al., 2004). Some of

these structures are shown in Figure 1. By using such

approaches, we have established that all mouse anti-

GQ1b monoclonal antibodies and >50% human MFS

sera bind NeuNAc (a2–8)NeuNAc(a2–3)Gal- (disialyl-

galactose, DSG), the terminal trisaccharide of GQ1b,

GT1a, and GD3. Anti-GM1 monoclonal antibodies

either require Gal(b1–3)GalNAc or whole GM1 penta-

saccharide (100 nM GM1 for >90% inhibition). By

using DSG- or GM1-sepharose immunoabsorption col-

umns, we can readily deplete anti-GQ1b and anti-GM1

antibody from MFS and GBS sera.

In a collaborative study with K. Nillson (Glycorex,

Lund), who manufactures glycan immunoadsorption

columns in human use for ABO-mismatched transplan-

tation (Tyden et al., 2003; Rydberg et al., 2005), and

with E. Samain (Grenoble), who is able to synthesise

gram quantities of GM1 pentasaccharide from glycosyl-

transferase-transfected Escherichia coli (Antoine et al.,

2003), we are now taking this proposal towards clinical

80

A

B C

60

40

CD59+/+, anti-GQ1b antibody

CD59+/+, PBS

CD59–/–, PBS

CD59–/–, anti-GQ1b antibody

20Sig

nal (

% B

Tx

area

)

0IgM

BTxNF BTxNF

BTxMAC

MAC

Figure 9. Complement regulator intact (CD59þ/þ) and deficient (CD59–/–) mice were passively immunised for 20 h with anti-GQ1bimmunoglobulin (Ig)M monoclonal antibody plus human serum as a heterologous source of complement. Diaphragm neuromuscularjunctions (NMJs) were analysed for deposits of anti-GQ1b IgM and membrane attack complex (MAC) and for neurofilament (NF)degradation as an index of neuronal injury. Whereas IgM deposits were equal, MAC deposits were increased nearly twofold in CD59–/–

mice compared with CD59þ/þ wild-type mice (p < 0.001). NF signals over the NMJ were greatly reduced in CD59–/– compared withCD59þ/þ mice, in relation to their phosphate-buffered saline (PBS)-treated controls (p < 0.001). (A) Deposits of MAC over bungarotoxin(BTx)-labelled NMJs in CD59–/– mice. (B) Normally appearing synaptic and pre-synaptic NF as a measure of axonal integrity in a CD59–/–

mouse exposed to PBS, compared with the CD59–/– mouse passively immunised with anti-GQ1b antibody plus complement, in whichthe nerve terminal NF signal is lost (C). Scale bar ¼ 20 mm. Modified from Halstead et al. (2004).

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

105

use. Substitution of blood group glyco-antigens for

ganglio-series glycans is relatively straightforward, and

prototype columns are currently being constructed. We

are also testing the inhibitory activity of soluble oligo-

saccharides in ex vivo physiological preparations and in

mouse models in vivo. However, the pharmacokinetics

and glycan metabolism aspects of such an approach in

humans are likely to present greater complexities than

extra-corporeal immunoadsorption, and we thus favour

the former therapeutic strategy.

ConclusionOur advances in understanding the immuno-

pathological pathways underlying GBS have come a

very long way in a short space of time and opened

up a wealth of potential therapeutic avenues.

Whether the identification of further ganglioside

and glycolipid antigens will lead to new areas for

enquiry remains to be seen. Progress is most

especially needed in the search for the elusive AIDP

antigen(s), and this is the most pressing research

goal in the GBS field. If further advances in identifying

novel glycan antigens are not fruitful, the tables

may turn back to the myelin proteins or other

Schwann cell antigens in the quest for major disease

targets, either as T- or B-cell antigens, and many

preliminary studies suggest that such searches

may be fruitful (Pestronk et al., 1998; Gabriel et al.,

2000).

A B

C D

BTx s100 MAC NF/BTx

Figure 10. Neuromuscular junctions (NMJs) in triangularis sternae muscle preparations from C57/Bl6 mice exposed in vitro to anti-GQ1b monoclonal antibody plus human serum as a source of complement, in the absence (A, B) or presence (C, D) of thecomplement inhibitor APT070. Post-synaptic membranes are stained with bungarotoxin (BTx). In the absence of APT070 (A, B),membrane attack complex (MAC) (A, green) is formed that disrupts the nerve terminal and overlying perisynaptic Schwann cell (pSC)membrane integrity, resulting in loss of neurofilament (NF) (B, green) and S100 (A, blue) staining, respectively. In the presence ofAPT070, no MAC is formed and NMJ architecture is preserved with normal NF architecture (D, green) and intact S100-positive pSCs(C, blue) overlying the nerve terminal. Scale bar ¼ 20 mm (Halstead et al., unpublished data).

Willison Journal of the Peripheral Nervous System 10:94–112 (2005)

106

An important consequence of identifying antibo-

dies as the main pathogenic mediators of GBS (or

not) is that such knowledge should direct therapeutic

approaches towards blockade of antibody-mediated

effector pathways, such as complement inhibition or

B-cell suppression. Because an ever-growing array of

‘pathway-specific’ immunotherapies becomes increas-

ingly available for human use, this issue has a true

urgency. Therapeutic progress in the GBS field is

especially confounded by the fact that large trials are

complex and time consuming to organise and execute;

this reinforces the need to make rational choices for

novel immunotherapy testing, informed by basic stu-

dies that reasonably reflect the pathogenesis of the

human disease. Such an approach is essential to pre-

vent the GBS field from following false trails or floun-

dering in the therapeutic doldrums well into the 21st

century, especially as other autoimmune diseases

make significant headway by the use of contemporary

treatments.

AcknowledgementsThe research work described here was supported

by grants from the Wellcome Trust, National Institutes

of Health, Guillain-Barre Syndrome Support Group UK,

and Guillain-Barre Syndrome Foundation International.

The dedicated effort of all the graduate students and

scientific staff in my laboratory over the last decade

who conducted this work is gratefully recognised.

Amongst many external collaborators, particular men-

tion is due to my long-standing collaborator, Dr. Jaap

Plomp, Leiden University, whose electrophysiological

expertise has constantly stimulated and advanced our

work.

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