C. Wim Ang

The crucial role of Campylobacter jejuni genes in anti-ganglioside antibody induction in Guillain-Barre syndrome

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

Clinical features and response to treatment in Guillain‐Barré syndrome associated with antibodies to GM1b ganglioside

GM1b is a minor ganglioside in human peripheral nerves. Serum anti‐GM1b antibodies frequently are present in patients with Guillain‐Barré syndrome (GBS). In this collaborative study, we investigated the antecedent infections, clinical features, and response to treatment of GBS patients with anti‐GM1b antibodies. Of 132 GBS patients who participated in the Dutch GBS trial that compared the effect of intravenous immunoglobulins and plasma exchange, 25 (19%) patients had anti‐GM1b antibodies. IgM antibodies were present in 14, IgG antibodies in 15, and both isotypes in 4 patients. The 25 patients with anti‐GM1b antibodies had a clinical pattern distinct from that of the other 107 GBS patients. They more often had an episode of gastrointestinal illness and frequently showed serological evidence of recent infection by Campylobacter jejuni. The anti‐GM1b–positive subgroup was marked by more rapidly progressive, more severe, and predominantly distal weakness. Cranial nerve involvement and sensory deficits were less common in the patients with anti‐GM1b antibodies. The presence of anti‐GM1b antibodies was associated with slower recovery. The clinical manifestations predominantly were associated with anti‐GM1b antibodies of the IgG isotype. Fourteen (56%) of the 25 patients with anti‐GM1b antibodies also had anti‐GM1 antibodies. The group of patients with both antibodies was clinically more homogeneous and had a more rapidly progressive, pure motor neuropathy. The subgroup of anti‐GM1b–positive GBS patients responded well to treatment with immunoglobulins but not to plasmapheresis. The distinctive clinical features of the patients with anti‐GM1b antibodies show that acute motor neuropathy represents a specific subgroup within GBS and that recognizing these patients may have consequences as to the choice of therapy. Ann Neurol 2000;47:314–321

Structural characterization of Campylobacter jejuni lipooligosaccharide outer cores associated with Guillain-Barré and Miller Fisher syndromes

ABSTRACT Molecular mimicry between lipooligosaccharides (LOS) of Campylobacter jejuni and gangliosides in peripheral nerves plays a crucial role in the pathogenesis of C. jejuni-related Guillain-Barré syndrome (GBS). We have analyzed the LOS outer core structures of 26 C. jejuni strains associated with GBS and its variant, Miller Fisher syndrome (MFS), by capillary electrophoresis coupled with electrospray ionization mass spectrometry. Sixteen out of 22 (73%) GBS-associated and all 4 (100%) MFS-associated strains expressed LOS with ganglioside mimics. GM1a was the most prevalent ganglioside mimic in GBS-associated strains (10/22, 45%), and in eight of these strains, GM1a was found in combination with GD1a mimics. All seven strains isolated from patients with ophthalmoplegia (GBS or MFS) expressed disialylated (GD3 or GD1c) mimics. Three out of 22 GBS-associated strains (14%) did not express sialylated ganglioside mimics because their LOS locus lacked the genes necessary for sialylation. Three other strains (14%) did not express ganglioside mimics because of frameshift mutations in either the cstII sialyltransferase gene or the cgtB galactosyltransferase gene. It is not possible to determine if these mutations were already present during C. jejuni infection. This is the first report in which mass spectrometry combined with DNA sequence data were used to infer the LOS outer core structures of a large number of neuropathy-associated C. jejuni strains. We conclude that molecular mimicry between gangliosides and C. jejuni LOS is the presumable pathogenic mechanism in most cases of C. jejuni-related GBS. However, our findings suggest that in some cases, other mechanisms may play a role. Further examination of the disease etiology in these patients is mandatory.

Diagnostic value of anti-GM1 ganglioside serology and validation of the INCAT-ELISA

The Inflammatory Neuropathy and Treatment (INCAT) group developed a standardized ELISA method for the detection of serum anti-GM1 antibodies. The diagnostic value of anti-GM1 antibodies determined by this method has not yet been established in large groups of patients. We assessed the reproducibility, sources of variation, optimal cut-off values and evaluated the diagnostic relevance of the INCAT-ELISA in various groups of patients and controls (N=1232). The coefficient of variance was 11.2% for IgM and 3.8% for IgG. High IgG titers were only found in Guillain-Barré syndrome (GBS) and other inflammatory polyneuropathies. High IgM titers were associated with GBS and multifocal motor neuropathy. Low IgM titers had no additional diagnostic value. The INCAT-ELISA is a reliable test with additional diagnostic value in specific clinical situations.

Sialylation of Campylobacter jejuni lipo-oligosaccharides is associated with severe gastro-enteritis and reactive arthritis

We used various genotyping methods to identify bacterial genetic markers for development of arthritic symptoms following Campylobacter enteritis. We genotyped a collection of population derived Campylobacter strains, with detailed information on clinical characteristics, including arthritic symptoms. Besides using whole genome screening methods, we focused on the lipo-oligosaccharide (LOS) gene locus in which marker genes for developing post-Campylobacter neurological disease are present. Patients with arthritic symptoms were more frequently infected with Campylobacter jejuni strains with a class A LOS locus. We also found that patients who were infected with a C. jejuni strain containing sialic acid-positive LOS (class A, B or C) more frequently had bloody diarrhoea and a longer duration of symptoms. Furthermore, the IgM antibody response against Campylobacter was stronger in patients with a sialic acid containing LOS. Ganglioside auto-antibodies were observed in a small number of patients following infection with a class C strain. We conclude that sialylation of C. jejuni LOS is not only a risk factor for development of post-infectious symptoms, but is also associated with increased severity of enteric disease.

Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, by horizontal exchange.

ABSTRACT Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, has been shown to be genetically closely related to the completely sequenced strain C. jejuni NCTC 11168 by various molecular typing and serotyping methods. However, we observed that the lipooligosaccharide (LOS) biosynthesis genes strongly diverged between GB11 and NCTC 11168. We sequenced the LOS biosynthesis locus of GB11 and found that it was nearly identical to the class A LOS locus from the C. jejuni HS:19 Penner serotype strain (ATCC 43446). Analysis of the DNA sequencing data showed that a horizontal exchange event involving at least 14.26 kb had occurred in the LOS biosynthesis locus of GB11 between galE (Cj1131c in NCTC 11168) and gmhA (Cj1149 in NCTC 11168). Mass spectrometry of the GB11 LOS showed that GB11 expressed an LOS outer core that mimicked the carbohydrate portion of the gangliosides GM1a and GD1a, similar to C. jejuni ATCC 43446. The serum from the GB11-infected patient was shown to react with the LOS from both GB11 and ATCC 43446 but not with that from NCTC 11168. These data indicate that the antiganglioside response in the GB11-infected patient was raised against the structures synthesized by the acquired class A LOS locus.

Origin of ganglioside complex antibodies in Guillain–Barré syndrome

The origin of antibodies to ganglioside complexes, as new immunotargets for Guillain-Barré syndrome (GBS), is unknown. This was investigated in 21 GBS patients from which Campylobacter jejuni was isolated. Two of these patients had serum IgG to the GM1/GD1a complex and two other patients had IgG to the GQ1b/GD1a complex. These pairs of patients were clinically distinct. These antibodies all cross-reacted to lipo-oligosaccharides (LOS) from the autologous C. jejuni strain. Previous mass spectrometry studies on these LOS showed the presence of oligosaccharides with a similar structure, further supporting the hypothesis that in these patients LOS induced the ganglioside complex antibodies.

Detection and prevalence of α-latrotoxin–like effects of serum from patients with Guillain–Barré syndrome

Anti‐GQ1b antibodies are associated with the Miller Fisher syndrome (MFS), a variant of the Guillain–Barré syndrome (GBS). In the ex vivo mouse diaphragm, anti‐GQ1b–positive MFS serum induces muscle fiber twitching, a temporary dramatic increase of spontaneous quantal acetylcholine release, and transmission blockade at neuromuscular junctions (NMJs). These effects resemble those of α‐latrotoxin (α‐LTx) and are induced by antibody‐mediated activation of complement. We developed an assay for detection of the α‐LTx–like effect, using muscle fiber twitching as indicator. We tested 89 serum samples from GBS, MFS, and control subjects, and studied correlations with clinical signs, anti‐ganglioside antibodies, micro‐electrode physiology, and complement deposition at NMJs. Twitching was observed with 76% of the MFS and 10% of the GBS samples. It was associated with ophthalmoplegia and anti‐GQ1b antibodies in patients, and with increased spontaneous acetylcholine release and C3c‐deposition at mouse NMJs. This study strongly suggests that antibodies to GQ1b (with cross‐reactivity to related gangliosides) are responsible for the α‐LTx–like activity. The twitching assay is an efficient test for detection of this effect, and allows for screening of large numbers of samples and modifying drugs.

PCR-Restriction Fragment Length Polymorphism Analysis of Campylobacter jejuni Genes Involved in Lipooligosaccharide Biosynthesis Identifies Putative Molecular Markers for Guillain-Barré Syndrome

ABSTRACT Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy.

Risk Factors Associated with Campylobacter jejuni Infections in Curaçao, Netherlands Antilles

ABSTRACT A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.