Intrathecal Anti-GalC Antibodies in Bickerstaff Brain Stem Encephalitis

We recently reported on a 9-year-old boy with ophthalmoplegia, ataxia, left-sided hemiplegia, and eventually coma diagnosed as Bickerstaff brain stem encephalitis (BBE) following Mycoplasma pneumoniae infection.[1] The etiologic diagnosis in this case was confirmed by the detection of an intrathecal synthesis of M. pneumoniae–specific antibodies.[2] Because we could not detect bacterial DNA in the cerebrospinal fluid (CSF) of the patient at disease onset,[1] we suggested a postinfectious immune-mediated process that manifested after clearance of the bacteria from the central nervous system (CNS).

We here aimed to identify a potential myelin target of cross-reactive antibodies elicited by M. pneumoniae in this BBE case.

For the analysis, the same serum and CSF samples used for the analysis of M. pneumoniae–specific antibodies taken at onset of neurologic symptoms and stored at −80°C were investigated.

IgM and IgG antibodies to GM1, GM2, GD1a, GD1b, GQ1b, AGM1, and galactocerebroside (GalC) (all from Sigma-Aldrich, Zwijndrecht, the Netherlands) were measured as described previously.[3] [4] To determine anti-GalC antibodies, half-area 96-wells plates (Costar, Corning B.V. Life Sciences, Amsterdam, the Netherlands) were coated with 450 pmol of glycolipid per well. All sera were diluted 100-fold. The optical densities (ODs) from uncoated (only ethanol containing wells) were subtracted from the glycolipid-coated wells. Cutoff values were either predefined (i.e., a background-subtracted OD of 0.2 for IgG and 0.3 for IgM) or obtained by measuring 30 healthy control sera (mean OD plus three times the standard deviation). Positive samples were titrated using twofold serial dilution series starting at a 1:100 dilution. The titer was defined as the reciprocal of the highest dilution that resulted in an OD higher than the cutoff value.

Since patients with Guillain–Barré syndrome (GBS) may also produce antibodies to complexes of two glycolipids instead of a single glycolipid,[5] we additionally tested antibodies to glycolipid complexes as described previously,[5] with the modification that 225 pmol/well was used for GalC and 75 pmol/well for other glycolipids in half-area plates.

Our analysis demonstrated the isolated presence of anti-GalC IgM and IgG antibodies in both CSF and serum ([Fig. 1]). In serum, the binding activity of IgG antibodies to GalC was inhibited by the presence of GM1, GD1a, GD1b, or GQ1b and attenuated by the presence of GM2 and asialo-GM1. The presence of serum antibodies against N-methyl-d-aspartate receptor, voltage-gated potassium channel complex, and aquaporin-4 had previously been excluded.

Fig. 1 Antibody profile against various glycolipids in a patient with Bickerstaff brain stem encephalitis following Mycoplasma pneumoniae infection. Both IgM and IgG antibodies against single glycolipids (x – Neg) and glycolipid complexes (x – y) in cerebrospinal fluid and serum were determined by enzyme-linked immunosorbent assay. Abbreviations: AGM1, asialo-GM1; GalC, galactocerebroside.

To our knowledge, this is the first case of BBE with the detection of anti-GalC antibodies.

BBE is closely related to GBS, forming a continuous spectrum.[6] [7] This is supported by immunological findings in which antibodies against the myelin glycolipid GQ1b were strongly associated with BBE.[8] Anti-GQ1b antibodies were also found in the serum of the two previously reported BBE cases associated with M. pneumoniae infection.[9] [10]

In GBS, antibodies against M. pneumoniae infection have been found to cross-react with the myelin glycolipid GalC.[11] [12] GalC is a major glycolipid antigen in the myelin of both the peripheral and CNS, and accounts for 32% of the CNS myelin lipid content.[13] Anti-GalC antibodies caused demyelinating neuropathy in rabbits immunized with GalC[14] and have also been associated with demyelination in GBS,[12] encephalitis,[15] and encephalomyelitis.[16] Anti-GalC antibodies were not tested in the other two BBE cases associated with M. pneumoniae infection.[9] [10]

Because we detected an intrathecal antibody synthesis of M. pneumoniae–specific antibodies in this case and because it has been demonstrated that anti-GalC antibodies cross-react with M. pneumoniae,[11] [12] there is evidence that these anti-GalC antibodies were induced by M. pneumoniae. In fact, we detected anti-GalC antibodies in CSF and serum, although the mere presence of anti-GalC in CSF may have a pivotal role for the pathogenesis of BBE. However, the mechanisms of M. pneumoniae–driven antibody responses within the CNS are unclear. It is also possible that during inflammation the blood–brain barrier (BBB) can become permeable, which would thereby enable serum antibodies to cross the BBB and cause disease.

In conclusion, the presence of antibodies to GalC (in absence of antibodies to GQ1b) in this patient may suggest that these antibodies are involved in the development of BBE. Their hypothesized role in the pathogenesis may provide a basis for immunomodulatory treatment in BBE associated with M. pneumoniae infection.

• References

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