Antibodies induced by ganglioside-mimicking Campylobacter jejuni lipooligosaccharides recognise epitopes at the nodes of Ranvier

Journal of Neuroimmunology 165 (2005) 179 – 185 www.elsevier.com/locate/jneuroim

Antibodies induced by ganglioside-mimicking Campylobacter jejuni lipooligosaccharides recognise epitopes at the nodes of Ranvier Anthony P. Moran*, Heidi Annuk, Martina M. Prendergast Department of Microbiology, National University of Ireland, Galway, Ireland Received 11 January 2005; accepted 20 April 2005

Abstract Molecular mimicry of gangliosides by Campylobacter jejuni lipooligosaccharides (LOSs) in the induction of anti-ganglioside antibodies has been hypothesised to contribute to GBS development. Rabbits were immunised with ganglioside-mimicking C. jejuni LOSs and antiLOS responses were analysed using passive haemagglutination, and anti-ganglioside responses by enzyme-linked immunosorbent assay and thin-layer chromatography with immunostaining. High titres of anti-LOS antibodies were demonstrated in rabbit antisera that were crossreactive with a panel of gangliosides. Non-ganglioside-mimicking C. jejuni HS:3 LOS induced a strong anti-LOS response, but no antiganglioside antibodies. Control rabbit antisera had no anti-LOS or -ganglioside responses. Moreover, IgG from a patient treated with parenteral gangliosides, who exhibited Guillain – Barre´ syndrome, had antibodies reactive with C. jejuni LOS. Biotinylated IgG fractions from the rabbit and the patient sera recognised epitopes at the nodes of Ranvier in sectioned human nerves, whereas fractions from controls did not. This study demonstrates that immunisation with ganglioside-mimicking C. jejuni LOS triggers the production of cross-reactive antiganglioside antibodies that recognise epitopes at the nodes of Ranvier. D 2005 Elsevier B.V. All rights reserved. Keywords: Campylobacter jejuni; Lipooligosaccharide; Guillain – Barre´ syndrome; Nodes of Ranvier; Anti-ganglioside antibodies

1. Introduction Guillain – Barre´ syndrome (GBS) is a post-infectious, paralytic neuropathy and occurs 10–14 days after a variety of antecedent bacterial and viral infections, particularly Campylobacter jejuni (Prendergast and Moran, 2000). Distinct subtypes of the disorder are recognised with acute inflammatory demyelinating polyradiculoneuropathy (AIDP) accounting for approximately 90% of GBS cases and acute motor axonal neuropathy (AMAN) occurring in up to 10% of patients with GBS (Hughes, 2004). The AIDP variant is characterised by an infiltration of endoneural vessels by lymphocytes and a macrophage-mediated demyelination of both sensory and motor nerves in a segmental fashion with deposition of complement activation products on Schwann-cell surface * Corresponding author. Tel.: +353 91 524411x3163; fax: +353 91 525700. E-mail address: [email protected] (A.P. Moran). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.04.013

membranes (Ho et al., 1998). AMAN is a form of GBS in which the motor axons appear to be the target of immune attack and is characterised by Wallerian-like degeneration without significant demyelination and with little or no lymphocytic infiltration (Griffin et al., 1996). Elevated antibody titres against a range of gangliosides, especially GM1 ganglioside, have been reported in different proportions (up to 82%) of GBS patients (reviewed in Prendergast and Moran, 2000). Moreover, the levels of these antibodies has been correlated with the severity of GBS, particularly AMAN (Prendergast and Moran, 2000). Furthermore, Illa et al. (1995) demonstrated that purified anti-GM1 antibodies from patients who exhibited AMAN after immunisation with a ganglioside preparation recognised epitopes at the nodes of Ranvier and at the presynaptic nerve terminals of motor end plates from human nerve biopsies. Accumulations of these antibodies at the nodes of Ranvier can cause disruption of Na+ and K+ channels and, thus, interfere with nerve conduction. Therefore, a causal link between C. jejuni infection, the presence of anti-ganglioside

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antibodies and development of GBS is considered likely (Moran et al., 1996; Prendergast and Moran, 2000; Moran and Prendergast, 2001). However, the induction of anti-C. jejuni antibodies in GBS and their precise involvement in disease development has not been established. Since the observation that only certain strains of C. jejuni are associated with GBS development, interest in the molecular basis for C. jejuni serotyping has been renewed. Traditionally, it had been considered that structural differences in high-molecular-weight lipopolysaccharide (LPS) of the bacterium accounted for the serospecificity of a strain (for review, see Moran and Penner, 1999). Proposals that something other than LPS such as exopolysaccharides (Kosunen et al., 1984), or capsular polysaccharides (Chart et al., 1996; Karlyshev et al., 2000) could account for strain serospecificity have emerged, but more recent evidence shows that both a low-molecular-weight type LPS, also called lipooligosaccharide (LOS), and extracellular polysaccharides can account for serotyping reactions (Moran et al., 2001). Importantly, a hypothesis of molecular mimicry has been proposed whereby anti-ganglioside antibodies are induced by LOSs from certain C. jejuni serotypes that contain ganglioside-mimicking epitopes (Yuki et al., 1993; Aspinall et al., 1994a; Schwerer et al., 1995; Moran et al., 1996; Prendergast et al., 1998; Neisser et al., 2000; Prendergast and Moran, 2000). Structures identical to the terminal oligosaccharide components of gangliosides have been demonstrated in the LOSs of all the C. jejuni GBS-associated strains isolated to date (for review, see Prendergast and Moran, 2000). Furthermore, these striking examples of molecular mimicry have prompted a search for reactivity of serum antibodies in GBS patients against C. jejuni LOSs. Several studies have demonstrated the cross-reactivity of anti-ganglioside antibodies with LOS from GBS-associated C. jejuni isolates and, thus, have verified the presence of ganglioside-like epitopes in C. jejuni LOS (Schwerer et al., 1995; Prendergast et al., 1998, 1999; Goodyear et al., 1999; Ang et al., 2000; Neisser et al., 2000; Prendergast and Moran, 2000). The presence of anti-ganglioside antibodies in C. jejuniassociated GBS sera, the cross-reactivity of the antibodies with C. jejuni LOS, the ability to induce anti-ganglioside antibodies with LOS, and demonstrations of antibody binding to neural targets provides cumulative evidence that GBS development is linked to an aberrant response to C. jejuni LOS (Prendergast and Moran, 2000; Moran and Prendergast, 2001). However, important evidence that these antibodies recognise and bind to ganglioside-expressing neural targets is lacking. Therefore, in this study, we used well-defined ganglioside-mimicking C. jejuni LOSs to elicit the production of cross-reactive anti-LOS antibodies, which included strains isolated from GBS patients. The reactivity of the raised antisera was examined using enzyme-linked immunosorbent assay (ELISA), passive haemagglutination (PHA), and thin-layer chromatography (TLC) with immunostaining. Importantly, and unique to this study, we asked whether the anti-ganglioside anti-

bodies induced by C. jejuni LOS could recognise and bind to GM1 epitopes at the nodes of Ranvier. In addition, the findings were compared to the binding characteristics of human IgG anti-GM1 antibodies, purified from a patient who developed GBS subsequent to the administration of parenteral gangliosides. This serum was used rather than serum from a C. jejuni-associated GBS patient since it was highly characterised for its IgG anti-GM1 antibody reactivity and patient data was available (Schwerer et al., 1994; Neisser et al., 2000), thereby representing a good comparative control, in contrast to a post-infectious GBS serum whose associated infection would have other influences on serum characteristics.

2. Materials and methods 2.1. Bacterial strains and growth conditions C. jejuni strains of serotype HS:2 (NCTC 11168), HS:3 (ATCC 43431), HS:4 (ATCC 43432), HS:19 (ATCC 43436), and HS:41 (16971.94GSH, GBS isolate) were routinely grown on blood agar (Columbia Agar Base [Oxoid, London, England] with 10% unlysed horse blood) under microaerobic conditions at 37 -C for 48 h. Biomass was harvested from agar plates as described previously (Moran et al., 1991). 2.2. LOS isolation and characterisation Biomass was subjected to the hot phenol – water extraction procedure as described previously (Westphal and Jann, 1965; Moran et al., 1991). Subsequently, the crude LOS from the water phase of extracts was purified by enzymatic treatments with RNase A, DNase II, and proteinase K (Sigma Chemical Co., St. Louis, MO), and ultracentrifuged as described elsewhere (Moran et al., 1991). Purity of C. jejuni LOSs was examined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) using a stacking gel of 5% acrylamide and a separation gel of 15% acrylamide containing 3.2 M urea (BDH Laboratory Supplies, Poole, England) (Prendergast et al., 1998). After SDS-PAGE, the gels were visualised by silver-staining (Tsai and Frasch, 1982) or examined for contamination by proteins using Coomassie blue staining. In addition, potential contamination by proteins and nucleic acids was assessed spectrophotometrically by scanning a sample of LOS (0.1 mg/ml ) between 200 –400 nm. The presence of GM1 epitopes on LOSs of C. jejuni HS:2, HS:4, HS:19, and HS:41 was demonstrated serologically using TLC with immunostaining (see below), and confirmed previous chemical structural studies (Aspinall et al., 1993a,b, 1994b; Prendergast et al., 1998; Prendergast and Moran, 2000). The LOS of C. jejuni HS:3 was used as a control as it is not sialylated, hence, does not mimic gangliosides (Aspinall et al., 1995), and is the only well characterised LOS of C. jejuni lacking such mimicry that is available.

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2.3. Immunisations Six New Zealand white adult rabbits were immunised subcutaneously (Kosunen et al., 1980). Five animals were immunised with the respective C. jejuni LOS mixed with complete Freund’s adjuvant (DIFCO Laboratories, Detroit, MI), followed four weeks later with a series of booster injections administered 3– 4 days apart (0.25, 0.5, 1.0, and 2.0 ml). As a control, another rabbit received injections with adjuvant only. Serum samples were taken at day 50, and animals were bled at day 57. As an additional control, before immunisation pre-immune sera were collected using sodium citrate as anticoagulant. Also, to ensure that the antibody responses did not result from an inter-current infection with C. jejuni, stool cultures for C. jejuni were performed on each animal prior to immunisation, and when the animals were bled (Ang et al., 2000). 2.4. Serological analysis Titres of anti-LOS antibodies in rabbit antisera were determined using PHA (Penner and Hennessy, 1980). Solutions of LOS (usually 30– 50 Ag/ml) were used to sensitise an equal volume of 1% suspension of sheep erythrocytes in PBS at 37 -C for 1 h. Sensitised erythrocytes were centrifuged (2000  g, 10 min), washed three times in PBS and used as a 0.5% suspension. Aliquots of 25 Al of antigen-sensitised erythrocytes were dispensed into Ushaped wells of microtitration plates containing 25 Al of two-fold dilutions of rabbit antisera. The plates were shaken and incubated at 37 -C for 1 h, stored overnight at 4 -C, and examined for agglutination of erythrocytes. The highest dilution of antisera showing agglutination was taken as the titre. The lowest dilution of the antisera used was 1 : 40 and the absence of agglutination at this dilution was considered a negative reaction. Reactivity of gangliosides (Sigma) and LOS with sera was assayed using TLC with immunostaining (Schwerer et al., 1995; Prendergast et al., 1998). Briefly, gangliosides or LOS (both 1 Ag aliquots) were analysed by TLC on precoated silica gel 60 glass plates (Merck, Darmstadt, Germany) with solvent systems consisting of chloroform-methanol-0.22% CaCl2&2H2O (50 : 45 : 10) or n-propanol-water-25% NH4OH (60 : 30 : 10), respectively. Developed TLC plates were dried for 30 min in a vacuum desiccator, fixed in 0.2% polyisobutylmethacrylate (Aldrich, Steinheim, Germany) in nhexane (Merck) for 1.5 min, and dried as before. Non-specific binding was reduced by submerging the plates for 1 h in a solution of PBS containing 0.3% gelatin (gelatin-PBS). Subsequently, lanes were overlaid with rabbit antisera, GBS patient serum, and polyclonal antisera to gangliosides (Matreya Inc., Pleasant Gap. PA) diluted 1 : 100 in gelatinPBS. Plates were incubated at 4 -C overnight, washed three times with cold PBS, overlaid with goat anti-rabbit IgGhorseradish peroxidase conjugate (Sigma), goat anti-human IgG-horseradish peroxidase conjugate (Bio-Rad, Richmond,

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CA), or goat anti-mouse IgG-horseradish peroxidase conjugate (Sigma), respectively, diluted 1 : 500 in gelatin-PBS, and incubated at room temperature for 1 h with gentle rocking. After incubation, the plates were washed with cold PBS, and the immunoreactants were visualised by use of an HRP development system (Bio-Rad). In addition, anti-GM1 antibodies in rabbit antisera were quantitated by ELISA as described by Kusunoki et al. (2003) with modifications. Briefly, each microtitre plate well was coated with 0.2 Ag of GM1 ganglioside overnight at 4 -C, followed by blocking with 1% bovine serum albumin in PBS at room temperature for 2 h with subsequent washing three times with PBS. Serum samples were serially diluted commencing at 1 : 100, and 100 Al of these were added to the wells with subsequent overnight incubation at 4 -C. After washing, peroxidase-conjugated anti-rabbit IgG (Sigma, 1 : 500) was added and incubated at room temperature for 2 h. Wells were washed and colour reaction was obtained with orthophenylenediamine dihydrochloride, after which the reaction was stopped by the addition of 3 M H2SO4. The absorbance values at 492 nm were corrected by subtracting the optical densities (ODs) obtained for each well without antigen. Anti-ganglioside antibody titre was the highest serum dilution at which the OD492 was 0.1 or greater. 2.5. Patient A patient who developed a slowly progressive axonal form of pure motor polyneuropathy after treatment with mixed gangliosides from bovine brain is the patient described elsewhere (Schwerer et al., 1994). Titres of IgG anti-GM1 antibody measured by ELISA two years after the administration of gangliosides, were extremely high at 1 : 200,000, as compared to 1 : 200 in normal controls (Neisser et al., 2000). Importantly, ELISA screening for diagnostic antibodies against C. jejuni was performed using acid-glycine extract as antigen as described previously (Kosunen et al., 1983), but no diagnostic anti-Campylobacter antibodies were detected in the patient’s serum indicating the absence of a previous C. jejuni infection (Neisser et al., 2000). Furthermore, the ganglioside reactivity of GBS patient serum, and control serum from an uninfected volunteer was examined using TLC with immunostaining (Prendergast et al., 1998; Neisser et al., 2000). 2.6. Biotinylation experiments The IgG fractions from rabbit antisera, GBS patient serum, polyclonal anti-GM1 antibodies (Matreya Inc.), and sera from controls (rabbit pre-immune serum, serum from adjuvant control and anti-Escherichia coli B4:0111 LPS antiserum) were purified using protein G-sepharose chromatography (Pharmacia, Uppsala, Sweden). The purified IgG fractions were biotinylated using a mixture of 1 mg in 1 ml of 0.1 M sodium bicarbonate (NaHCO3) solution, pH 8.4, with 0.2 ml of N-hydroxysuccinimidyl 6-(biotinamido)

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Table 1 The reactivity of rabbit antisera with homologous C. jejuni LOS in PHA and ganglioside GM1 in ELISA Rabbit antiserum

Anti-HS:2 antiserum Anti-HS:3 antiserum Anti-HS:4 antiserum Anti-HS:19 antiserum Anti-HS:41 antiserum

rough-form LPS (also called LOS), composed of lipid A and core oligosaccharide moieties. The absence of protein bands in the gels was confirmed by staining with Coomassie blue. The LOS preparations were essentially free from proteins (< 0.1%) and nucleic acids (< 0.1%) when assessed spectrophotometrically.

The titres of reaction C. jejuni LOS

GM1 ganglioside

1 : 5120 1 : 2560 1 : 10,240 1 : 10,240 1 : 20,480

1 : 5120 0 1 : 20,480 1 : 40,960 1 : 40,960

3.2. Anti-C. jejuni LOS antibodies All rabbits immunised with C. jejuni LOS showed a strong humoral response to homologous LOS. In general, IgG responses to LOS were seen after 57 days, as detected by PHA, with serum titres ranging from 1 : 2560– 1 : 20,480 (Table 1). As shown in Table 2, antisera from rabbits immunised with C. jejuni HS:2, HS:4, HS:19 and HS:41 LOSs reacted with GM1 ganglioside and asialo-GM1 glycolipid in TLC with immunostaining. In addition, antisera raised against HS:4 and HS:19 LOSs also reacted with GD1b ganglioside. Similar results were obtained with respective IgG fractions. With respect to anti-HS:4 and anti-HS:19 antisera, reactions against GD1a ganglioside were observed only with whole serum. This was also true for anti-GM2 responses in anti-HS:41 antiserum. Furthermore, titres of IgG anti-GM1 antibodies in the antisera as determined by ELISA were 1 : 5120– 1 : 40,960 (Table 1). Moreover, sera and IgG fractions from animals that were immunised with C. jejuni HS:3 LOS did not contain anti-ganglioside antibodies. In preimmune sera and samples from adjuvant control, anti-LOS or anti-ganglioside antibodies were undetectable. Stool cultures for C. jejuni performed before immunisation and also when animals were sacrificed, were negative, ensuring that the animals had no preceding C. jejuni infection.

hexanoate (Vector Laboratories Inc., Burlingame, CA) in dimethylsulfoxide (DMSO, Sigma) at 50 mg/ml (Illa et al., 1995). The solution was incubated at room temperature for 2 h and dialyzed against five changes of PBS. For immunohistological studies, the biotinylated IgG was applied to 5-Am-thick fresh-frozen sections of normal human peripheral motor nerves obtained during diagnostic nerve biopsies and prepared as described previously (Illa et al., 1995). Following serial sections were used for each IgG: unfixed, fixed in acetone, and fixed in chloroform:methanol (50 : 50 vol/vol). The sections were washed in Tris buffer, pH 7.6, blocked with albumin and normal goat serum, and incubated at 4 -C overnight with biotinylated IgG (10 Ag / ml). Immunostaining was visualised with peroxidase-conjugated avidin – biotin (Sigma) and diaminobenzidine (Sigma). In addition, anti-C. jejuni HS:2, HS:4, HS: 19 and HS:41 LOS antisera were pre-absorbed with GM1 ganglioside (200 Ag) as described previously (Prendergast et al., 1998), IgG fractions prepared as described above, and subsequently tested in immunohistochemistry.

3. Results

3.3. Patient serum

3.1. LOS characterisation

The patient in the present study, who developed GBS after treatment with parenteral gangliosides (Schwerer et al., 1994) was seronegative for diagnostic anti-C. jejuni antibodies, and had high titres of IgG anti-GM1 antibody (1 : 200,000) compared to controls (1 : 200) two years after the administration of gangliosides (Schwerer et al., 1994;

Consistent with earlier studies (Aspinall et al., 1994a; Prendergast et al., 1998; Prendergast and Moran, 2000), all strains exhibited a pattern of low-molecular-weight bands in silver stained gels (data not shown), corresponding to

Table 2 The reactivity of rabbit antisera and patient serum with gangliosides and asialo-GM1 glycolipid in TLC with immunostaining Rabbit antiserum/Human serum

Strength of reaction of anti-C. jejuni antibodies with gangliosides and asialo-GM1 glycolipida GM1

Anti-HS:2 antiserum Anti-HS:3 antiserum Anti-HS:4 antiserum Anti-HS:19 antiserum Anti-HS:41 antiserum GBS patient serumb,c a b c

Asialo-GM1

GM2

GD1a

GD1b

Whole serum

IgG

Whole serum

IgG

Whole serum

IgG

Whole serum

IgG

Whole serum

IgG

+++ – +++ +++ +++ ++++

+++ – +++ +++ +++ ++++

++ – + ++ + +

++ – + ++ + +

– – – – (+) –

– – – – – –

– – (+) (+) – –

– – – – – –

– – + + – +

– – + + – +

++++, very strong reaction; +++, strong reaction; ++, moderate reaction; +, weak reaction; (+), barely visible reaction; – , no reaction. Patient corresponds to that described by Schwerer et al. (1994) and Neisser et al. (2000). The patient serum and IgG fraction reacted with C. jejuni HS:2, HS:4, HS:19 and HS:41 LOSs in a similar manner to that observed by Neisser et al. (2000).

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Neisser et al., 2000). We showed using TLC with immunostaining that in addition to a reaction with GM1 ganglioside, the serum and IgG fraction of this patient also recognised asialo-GM1 glycolipid and GD1b ganglioside (Table 2). Furthermore, commercial anti-GM1-specific antibodies cross-reacted with C. jejuni HS:2, HS:4, HS:19 and HS:41 LOSs in TLC, consistent with previous observations (Aspinall et al., 1993a,b, 1994b; Schwerer et al., 1995; Prendergast et al., 1998; Prendergast and Moran, 2000), as did the patient serum (Neisser et al., 2000) and IgG fraction (not shown). Thus, the reaction of anti-ganglioside antibodies with C. jejuni LOSs was a result of shared epitopes between gangliosides and C. jejuni LOS, as the patient had no indication of a previous infection with C. jejuni (Neisser et al., 2000). Purified IgG from an uninfected volunteer was negative for anti-ganglioside and anti-C. jejuni antibodies. 3.4. Immunohistochemistry When biotinylated IgG fractions of sera from rabbits immunised with C. jejuni HS:2, HS:4, HS:19 and HS:41 LOSs, each containing high titre anti-GM1 antibodies, were

A

B

C

D

Fig. 1. Longitudinal section of a unfixed normal human nerve immunolocalised with IgG fraction of (A) rabbit antiserum from the animal immunised with C. jejuni O:41 LOS, (B) pre-immune rabbit serum, (C) anti-C. jejuni HS:3 LOS rabbit antiserum and (D) serum from an uninfected human not treated with gangliosides. The biotinylated IgG fraction (10 Ag/ ml) of the anti-C. jejuni O:41 LOS antiserum recognises epitopes at the node of Ranvier as indicated by the red-brown staining and arrow.

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applied to normal human nerve, a characteristic immunostaining pattern corresponding to the nodes of Ranvier was demonstrated (see Fig. 1). A similar reactive pattern was observed for the IgG fractions of the serum from the GBS patient and of the polyclonal anti-GM1 antibodies. However, no such staining pattern was observed for the preimmune sera, serum from the adjuvant control, anti-E. coli LPS antiserum, antiserum from the rabbits injected with C. jejuni HS:3 LOS, and serum from an uninfected human volunteer. As shown in Fig. 1, the presence of staining with biotinylated anti-HS:41 antiserum at the nodal gap of certain axons was demonstrated which extended to the surface of the paranodal myelin sheath. The immunolocalisation of IgG was observed only on the sections that were unfixed (Fig. 1) or treated with acetone. The staining disappeared after sections were delipidated with chloroform:methanol. Furthermore, when anti-C. jejuni HS:2, HS:4, HS: 19 and HS:41 LOS antisera were pre-absorbed with GM1 ganglioside reactivity disappeared.

4. Discussion It is well-established that GBS patient sera contain antiC. jejuni LOS antibodies and anti-ganglioside antibodies, and that anti-ganglioside antibodies cross-react with C. jejuni LOS (for reviews, see Prendergast and Moran, 2000; Moran and Prendergast, 2001; Moran et al., 2002). We have demonstrated in the present study an IgG response to gangliosides following immunisation of rabbits with wellcharacterised GM1-containing C. jejuni LOSs. Previously, immunohistochemical studies have localised GM1-like epitopes at the nodes of Ranvier, nodal axolemma and paranodal Schwann cell, by mapping the distribution of ligand and monoclonal antibody binding in peripheral nerve tissue (O’Hanlon et al., 1996, 1998; Sheikh et al., 1998). Also, the ability of anti-GM1 antibodies to bind to the surface of peripheral nerve structures, to nodes of Ranvier, and to neuromuscular junctions has been demonstrated. Moreover, incubation of isolated nerve preparations in vitro with human and rabbit anti-GM1 antibodies has produced acute conduction block of myelinated nerve fibres (Prendergast and Moran, 2000). The present study is the first to clearly demonstrate the binding of C. jejuni LOS-induced anti-GM1 antibodies from rabbits to sites at the nodes of Ranvier, in an identical manner to that of anti-GM1 antibodies from humans. Recently, Yuki et al. (2004) reported an animal model whereby upon sensitisation with C. jejuni LOS rabbits developed anti-GM1 antibody, flaccid limb weakness and pathological changes in peripheral nerves. However, long-term immunisation over a 12-month period was required with aggressive repeated immunisation with keyhole limpet haemocyanin adjuvant compared to the less aggressive immunisation protocol used in the present study. Also, to fully reproduce C. jejuni-associated GBS an animal model that allows colonisation by C. jejuni is

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required in addition to LOS-induction of neuropathy (Moran et al., 2002). Nevertheless, in the present absence of a C. jejuni-induced GBS animal model of disease our finding is an important step in clarifying the role of C. jejuni infection in GBS pathogenesis. Similar to the present study, the principle of eliciting antiganglioside antibodies with C. jejuni LOS has been demonstrated in rabbits using C. jejuni GM1-like LOSs (Ang et al., 2000), but also in rodents using GM1-like and GT1a/GD3-like C. jejuni LOSs (Goodyear et al., 1999; Willison and O’Hanlon, 1999). Using an immunisation protocol different to the one used in the present study, Ang et al. (2000) elicited both anti-GM1 IgM and IgG antibodies in rabbits using two GM1-like C. jejuni LOSs, one from a GBS strain (serotype HS:19), and one from an enteritis patient (serotype HS:2). Interestingly, both these immunoglobulin classes appear in the ganglioside antibody repertoires of GBS patients. However, little data is available on the potential differences in the specificities of IgG and IgM antibodies for neural targets. In a study by Goodyear et al. (1999), anti-GQ1b, -GT1a, or -GD3 antibodies induced after immunisation of mice with GT1a or GD3-like C. jejuni LOSs were shown to bind ganglioside-rich sites at the motor nerve terminals of sciatic and phrenic nerves. Consistent with our findings, Illa et al. (1995) demonstrated that human anti-GM1 antibodies purified from ganglioside-treated GBS patients were specific for GM1 sites at the nodes of Ranvier. However, that study did not address the issue of C. jejuni infection in the development of anti-ganglioside antibodies. Furthermore, the source of the nerve tissue used in pathogenesis studies should be a consideration, since sural nerves, as purely sensory nerves, are most often used, whereas GBS patients most often develop pure motor dysfunction. In this and the other cited studies (Goodyear et al., 1999; Ang et al., 2000) none of the immunised animals developed neuropathy. This failure to induce neuropathy by sensitisation with C. jejuni LOS may be due to short duration of the experiments or depend in part on the immunisation procedure used. Supporting such a view, Yuki et al. (2004) used repeated aggressive immunisation of long duration with C. jejuni LOS to achieve neuropathy-like symptoms in rabbits. Whether the less severe immunisation protocol employed in this study in a longer term experiment could induce neuropathy is the object of future studies. Nevertheless, there is evidence that accumulations of antiganglioside antibodies at motor nerve terminals and sites at the nodes of Ranvier (Illa et al., 1995) can cause disruption of Na+ and K+ channels in human tissue and, thus, interfere with nerve conduction (Takigawa et al., 1995; Arasaki et al., 1998). Incubation of isolated nerve preparations in vitro with human and rabbit anti-ganglioside antibodies has produced acute conduction block of myelinated nerve fibres, an effect that appears to be complement dependent (Goodyear et al., 1999; Willison and O’Hanlon, 1999). It should be noted that complement-deposition in rabbits and other

animals may not occur in a similar manner to that in humans, and may help explain the difficulty of inducing neuropathy development in such animals. However, it has been shown that IgG anti-GM1 antibodies can reversibly block the voltage gated Na+channels of nerve cells causing a decrease of the excitatory Na+ current (Willison and O’Hanlon, 1999). Using C. jejuni LOS-induced antiganglioside antibodies in a mouse hemidiaphragm model and in a macro-patch clamp electrode study, both pre- and post-synaptic complement-mediated conduction block with extensive deposits of IgM and C3c at nerve terminals has been demonstrated, an effect disputed by others (reviewed in Willison and O’Hanlon, 1999). Thus, unequivocal proof of the pathogenic potential of LOS-induced anti-ganglioside antibodies has yet to be provided, but importantly this study provides important evidence that anti-ganglioside antibodies can be induced by C. jejuni LOS and that these antibodies are specific for sites at the nodes of Ranvier. Furthermore, there are grounds to extend this study to investigate the minor specificity of C. jejuni LOS antibodies for cell types within the nodes of Ranvier and other neural sites. Acknowledgments The financial support of the Irish Health Research Board is gratefully acknowledged. We thank A. J. Lastovica (Cape Town, South Africa) for providing the C. jejuni O:41 strain, gratefully acknowledge B. Schwerer (Vienna, Austria) for providing the human serum, and thank T. Kosunen (Helsinki, Finland) for help with production of rabbit antisera. References Ang, C.W., Endtz, H.P., Jacobs, B.C., Laman, J.D., de Klerk, M.A., van der Meche´, F.G.A., van Doorn, P.A., 2000. Campylobacter jejuni lipopolysaccharides from Guillain – Barre´ syndrome patients induce IgG anti-GM1 antibodies in rabbits. J. Neuroimmunol. 104, 133 – 138. Arasaki, K., Kusunoki, S., Kudo, M., Tamaki, M., 1998. The pattern of antiganglioside antibody reactivities producing myelinated nerve conduction block in vitro. J. Neurol. Sci. 161, 163 – 168. Aspinall, G.O., McDonald, A.G., Raju, T.S., Pang, H., Moran, A.P., Penner, J.L., 1993a. Chemical structure of the core regions of Campylobacter jejuni serotypes O:1, O:4 O:23 and O:36 lipopolysaccharides. Eur. J. Biochem. 213, 1017 – 1027. Aspinall, G.O., McDonald, A., Raju, T.S., Pang, H., Kurjanczyk, L.A., Penner, J.L., Moran, A.P., 1993b. Chemical structure of the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide. Eur. J. Biochem. 213, 1029 – 1037. Aspinall, G.O., Fujimoto, S., McDonald, A.G., Pang, H., Kurjanczyk, L.A., Penner, J.L., 1994a. Lipopolysaccharide from Campylobacter jejuni associated with Guillain – Barre´ syndrome patients mimic human gangliosides in structure. Infect. Immun. 62, 2122 – 2125. Aspinall, G.O., McDonald, A.G., Pang, H., 1994b. Lipopolysaccharides of Campylobacter jejuni serotype O:19 structures of O antigen chains from the serostrain and two bacterial isolates from patients with the Guillain – Barre´ syndrome. Biochemistry 33, 250 – 255. Aspinall, G.O., Lynch, C.M., Pang, H., Shaver, R.T., Moran, A.P., 1995. Chemical structures of the core region of Campylobacter jejuni O:3

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