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Antibodies to gangliosides and myelin proteins in Guillain-Barr syndrome.

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Antibodies to Ganghosides and Myelin
Proteins in Gdain-Barre Syndrome
Richard H. Quarles, PhD, Amjad A. Ilyas, PhD, and Hugh J. Willison, MRCP, PhD
An earlier investigation from our laboratory (Ilyas AA, Willison HJ, Quarles RH,et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol 1988;23:440-447) demonstrating the presence of high levels of antiganglioside antibodies in the sera of 5 of 26 patients with Guillain-Barre Syndrome (GBS) but not in control sera is
summarized. The ganglioside antigens varied among the 5 patients with positive findings, and the antiganglioside
antibodies decreased concurrently with clinical improvement in those patients for whom longitudinal samples were
available for analysis. The results are discussed in the context of antibodies to acidic glycolipids in other types of
neuropathy and other studies on antiglycolipid antibodies in GBS. Data showing the occurrence of lower levels of
antibodies to P2 protein, Po glycoprotein, and myelin-associated glycoprotein in some of the GBS patients are also
summarized. The findings from our laboratory combined with the results of others make it unlikely that antiganglioside antibodies have a notable pathogenic effect in most patients with GBS, but the possibility remains that they
are of pathogenic importance in some patients with the highest antibody titers.
Quarles RH, Ilyas AA, Willison HJ. Antibodies to gangliosides and myelin proteins in
Guillain-Barre syndrome. Ann Neurol 1990;27(suppl):S48-S52
The demonstration that monoclonal IgM antibodies
reacting with myelin-associated glycoprotein (MAG)
in patients with neuropathy also react with sulfate3-glucuronyl paragloboside (SGPG) 11-31 led to a
search for antiglycolipid antibodies in other patients
with paraproteinemic neuropathies. A high proportion
of the [MAG, SGPG1-negative IgM monoclonal antibodies in these patients react with various ganglioside
antigens {4}. Overall, about 80% of the IgM monoclonal antibodies tested in patients with neuropathy
react with an acidic glycolipid antigen, either SGPG
(and MAG) or a ganglioside. In view of the high frequency with which antibodies to acidic glycolipids
were detected in the paraproteinemic neuropathies,
sera from patients with inflammatory polyneuropathies
and other neurological diseases were tested for antibodies to acidic glycolipids [ 5 , 61. This article emphasizes data showing high levels of antibodies to gangliosides in the sera of some patients with Guillain-Barre
syndrome (GBS) 151 and also summarizes results demonstrating lower levels of antibodies to peripheral nervous system (PNS) myelin proteins in some of the
same group of patients [7}.
Antibodies to Gangliosides in GBS
A panel of sera from patients with the following diagnoses was obtained from the Department of Neurology at Johns Hopkins University: GBS, 26 patients;
From the Section on Myelin and Brain Development, Laboratory of
Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Instirutes of Health, Bethesda, MD.
S48
chronic inflammatory polyneuropathy (CIP), 19 patients; and other neurological diseases (OND), 19 patients. There were also 10 control sera from normal
individuals. In view of the high frequency of antibodies
to acidic glycolipids in the paraproteinemic neuropathies, as just described, the OND group did not
include patients with a detectable monoclonal IgM
protein. Sera were tested for antibodies to acidic
glycolipid antigens by an overlay procedure after fractionating the whole ganglioside fraction from human
peripheral nerve by thin-layer chromatography (TLC).
Findings for a serum were considered positive when
specific staining of one or more glycolipid bands was
obtained with a 1: 100 or greater dilution of the patient’s serum using a peroxidase-labeled second antibody. Quantitation of antibody levels for longitudinal
studies was accomplished by enzyme-linked immunosorbent assay (ELISA). The procedures used in our
laboratory to test for antiglycolipid antibodies in patients with neuropathy by TLC-overlay and ELISA
have been described in detail in a previous report [8].
Antibodies to gangliosides were detected in 5 of the
26 patients with GBS by the TLC overlay procedure,
but not in any of the O N D group or normal controls
151. The frequency of detection of antiganglioside antibodies in GBS was significantly greater than in the
combined OND and normal control samples ( p <
0.01). Antibodies to gangliosides were also detected in
Address correspondence to Dr Quarles, Park Bldg, Room 425,
NINDS, NIH, Bethesda, MD 20892.
1
2
3
1
4
A
Fig I. Binding of serum IgG fmm a patient with GuilLainBarn' syndrome to gangliosides of the human pevipheral nervous
system. Chromatogram was made of the gangliosidefractions
fmm human brain and nerve using an aluminum-backedthinLayer plate o f silica gel. (A)Resorcinol-stained gangliosides. Lane
1 contained known standard gangliosides G M l and GDla (2
pg sialic acid each). Lane 2 contained the gangliosidefraction
from human brain (about 15 pg sialic acid). Lanes 3 and 4
contained intact and neuriminidase-treated gangliosidefractions
from human nerve (about 15 pg sialic acid), respectively. (B)
Autoradiogramof the chromatogram after averlaying with the
patient's serum (diluted 1 :500),followed by radioiodinated goat
antihuman IgG (y-chain specifc). The patient's antibodies
reacted with two neuraminidase-sensitive components in the hum a n nerve gangliosidefraction. They were identijied as WMI
ganglioside and its higher homolog, with an additional lactosaminyl group, by use o f appropriate known standzrds. (Reproduced with permissionfrom Ilyas and colleagues is).)
3 of 19 patients with CIP; all 3 of these patients fell
into a clinical subgroup with multifocal motor neuropathy {6]. The antibodies in the 3 patients with CIP all
reacted with GM 1 ganglioside, although their fine
specificities differed as revealed by their pattern of
cross reactivity with other ganghosides [9). The occurrence of anti-GM1 ganglioside antibodies in patients
with motor nerve syndromes is currently an active area
of research in a number of laboratories. It was the
subject of a recent workshop held in Chicago in April
1989 [lo], and is covered in the article by Latov in this
supplement.
2
3
4
B
Among the 5 patients with GBS who had antibodies
to gangliosides, 1 had IgG reacting with LMI, the major ganglioside of human peripheral nerve myelin, and
a second component moving more slowly by chromatography (Fig 1).These two reactive gangliosides share
the same terminal carbohydrate structure (NeuAca23Galpl-4GlcNAcpl-3Galpl-). The antibody titer in
this patient fell five-fold over a 6-week period concurrent with clinical improvement. Two of the patients
with GBS had IgG that reacted with GDlb ganglioside, and antibody titers in both declined dramatically
as they improved clinically (Fig 2). The other 2 patients
had IgM antibodies that reacted with G D l a and GTlb
gangliosides, which share the same terminal sugar
configuration (NeuAca2-3Galp 1-3GalNAcP I-). Longitudinal serum samples from these 2 patients were
not available for correlation with clinical status.
Although high levels of antiganglioside antibodies
were found in 5 of the patients with GBS, the ganglioside antigens differed among the patients. It may be
noteworthy that despite the variability in specificity,
anti-GM 1 ganglioside antibodies, which have been associated with motor nerve syndromes, were not detected in any of the patients with GBS. When longitudinal samples were available from the patients with
positive findings, titers were highest in the earliest sera
collected, decreasing with clinical improvement. Because these patients underwent plasma exchanges, a
fall in antibody levels would be expected whether or
Quarles et al: Antibodies to Gangliosides in GBS S49
"\
I
85
DAYSAFTER ONSET OF SYMPTOMS
B
A
Fig 2. Longitudinalstudy of levels of antibodies to G D l b ganglioside in 2 patients with Guilkain-Bawi syndrome. Each well
of the enzyme-linked immunosorbent assay plate contained G D l b
standard (10 ng sialic acid), and the assay was done with
1 :100 dilutions of the patients' sera. (A, B) Optical densities
obtained in the assay plotted as a function of days after the onset
of neurological symptomsfor the 2 patients. The arrows indicate
days on which the patients underwent plasmpheresis.Antibody
levels decreased substantially in the .?-week period following onset
of disease, and they did not go back up many weeks after cessation of pkampheresis. (Reproducedwith permission from llyas
and colleagues {S).)
not they were related to the disease. Nevertheless,
their early presence suggests that they were not caused
by a secondary immune response to the neuropathy
itself. Also, the finding that antibody levels did not rise
again after cessation of plasmapheresis indicates that
they were not naturally occurring autoantibodies produced continuously in these individuals. Rather, the
antibodies appear to be associated with the early stages
of the demyelinating disease, consistent with having a
pathogenic effect.
Antibodies to PNS Myelin Proteins in GBS
The same GBS sera tested for antibodies to gangliosides were also tested by ELISA for antibodies to several PNS myelin proteins: PO glycoprotein, P2 protein,
and MAG [7]. Sera from the patients with CIP, from a
larger OND group (31 patients), and from 30 normal
controls were also analyzed. The results of the ELISAs
were expressed as the ratio of the optical density obtained for the patient's serum to the mean optical density obtained for a panel of normal control sera assayed
in the same experiment. A positive result was defined
as an optical density value more than two standard
deviations above the mean of the normal controls. The
positive sera were then tested for reactivity by Western blotting against the proteins of whole peripheral
S50 Annals of Neurology
Antibodies t o Myelin Proteins in Neurological Diseases"
Disease
GBS
CIP
OND
~
No. of
Patients
P2
Protein
26
3
3
3
20
31
PO
Glycoprotein
MAG
3
1
0
1
3
3
~~~
Values are the number of patients in each category with significantly
elevated antibodies to the myelin protein. A positive result is an
ELISA optical density greater than two standard deviations above
the mean optical density for normal controls analyzed in the same
experiment.
GBS = Guillain-BarrC syndrome; CIP = chronic inflammatory
polyneuropathy; OND = other neurological disease; MAG = myelin-associated glycoprotein.
nerve. An optical density ratio greater than 2 usually
was statistically significant, and sera with ratios greater
than 3 usually stained the protein on Western blots.
The Table summarizes the results. Among the 27
GBS sera, there were 7 positive results. The highest
optical density ratios (patiendmean control value) were
obtained for P2 protein in 3 of the patients with GBS
and ranged from 4 to 12. Reactivity to P2 protein was
readily demonstrated in these patients by Western
blotting (not shown). Substantially elevated antibodies
to P2 were also detected in some patients with CIP
and OND, and the frequencies of their occurrence in
patients with GBS and the other groups were similar.
Optical density ratios for the other diseases were not
above 4, however, except for one positive CIP serum
with a ratio of 8. In 1 of the patients with GBS who
had a positive P2 reaction, longitudinal samples were
available; levels of anti-P2 antibodies fell sharply in the
3 weeks following disease onset. The positive optical
density ratios obtained for PO glycoprotein and MAG
were not above 4 for any of the patients with GBS or
Supplement to Volume 27, 1990
other diseases. One patient with GBS had antibodies
to both PO and MAG. None of the patients with GBS
who had antibodies to myelin proteins were the same
as those with antiganglioside antibodies.
Importance of Elevated Antibodies in Patients
with GBS
Overall, antibodies to ganghosides or to a myelin protein were detected in 11 of the 26 patients with GBS
tested. Clearly, these studies have not identified a single “GBS antigen,” but it is possible that antibodies to
different components play a role in the pathogenesis of
the disease in different patients. Immune responses to
different antigens among patients could be related to
different antecedent factors in the disease-for example, the involvement of a variety of infectious
agents possessing immunogens that cross react with
different tissue components. It should be noted that
the levels of antibodies to gangliosides in the patients
described here appear much higher than those to the
myelin protein antigens. Thus, when the ELISA data
obtained for the ganglioside antibodies are expressed
as the ratios of the patients’ sera to the mean control
value, the ratios are in the range of 20 to 50, compared
with ratios from 2 to 12 for the protein antigens. The
ELISA data indicate that the strengths of antibody
binding to gangliosides in the early serum samples
from the 5 patients with GBS who had positive reactions, as reported by Ilyas and colleagues { 5 ] , are comparable to those in patients with paraproteinemic
neuropathies {4] and to those in patients with antiGM1 antibodies and multifocal motor neuropathy {6,
91. In contrast, our results on antibodiep to myelin
proteins suggest that relatively weak immune reactivity
to myelin proteins occurs in some patients with GBS, a
concept also gleaned from the variable data on immune responses to myelin proteins in the literature
{I 1-16]. The anti-ganglioside antibodies present in
higher levels may be better candidates for playing a
meaningful pathogenic role in some patients with GBS.
The potential importance of antiganglioside antibodies in GBS was rendered less clear by the report of
Svennerholm and Fredman (pp S36-S40), demonstrating antiganglioside antibodies in a higher proportion of patients with GBS but also in a high proportion
of controls without demyelinating disease. These investigators also used a TLC overlay procedure for detecting antibodies. A possible explanation for the discrepancy in the frequency of positive reactions is that
the procedure used by Svennerholm and Fredman was
capable of detecting lower levels of antibodies than
that used by Ilyas and co-workers { 5 ] , although a reason for such a difference in sensitivity was not obvious
when the findings were originally presented. If such a
difference in sensitivity does exist, it may be that Ilyas
and colleagues { 5 ] were selectively picking up the pa-
tients with GBS who had the highest titers of antiganglioside antibodies and, therefore, those in whom the
antibodies were more likely to be of pathogenic importance. This could be analogous to the situation with
anti-GM1 antibodies, in which individuals with very
high titers often have motor nerve syndromes, whereas
lower levels of anti-GM 1 antibodies frequently occur
in subjects without motor nerve disease (see {lo] and
article by Latov in this supplement, pp S41-S43). The
apparent discrepancy between the findings of Ilyas and
associates 151 on GBS and those reported by Svennerholm and Fredman in this supplement emphasizes
the need to quantify antibody levels and to establish
reliable means for comparing results among laboratories. This should be facilitated by establishment of a
panel sponsored by the National Institute for Neurological Disorders and Stroke to distribute known hightiter, standardizing sera and high-purity glycolipid antigens.
Nevertheless, the low frequency with which Ilyas
and co-workers 151 found high levels of antiganglioside
antibodies in GBS, combined with the relatively high
frequency with which Svennerholm and Fredman were
able to detect antiganglioside antibodies in controls,
make it unlikely that antibodies to gangliosides are a
common pathogenic factor in this disease. The antibodies to a Forssman-like, neutral glycolipid antigen of
myelin found in all patients with GBS tested and correlating with demyelinating activity of the sera in tissue
culture {17] may be more likely candidates to mediate
a common pathogenic effect in this disease. It may be
that the demyelinating process in all patients with GBS
is initiated by the anti-Forssman antibodies or some
other unknown common mechanism, but could be exacerbated in some patients by high levels of antibodies
to another antigen such as a ganglioside. In this regard,
it is of interest that the patient with antibodies to the
major LM1 ganglioside of PNS myelin described by
Ilyas and colleagues { 5 ] had a very severe and rapidly
progressing form of GBS (G. McKhann, personal
communication, 1989). It must be stressed, however,
that evidence for a pathogenic role for high levels of
antiganglioside antibodies in some patients with GBS
is only circumstantial at present. More research on the
pathological effects of antiganglioside antibodies in
animal models or tissue culture systems is an important
priority for the future, as is the need to standardize
procedures among laboratories for detecting and quantifying antibodies to potential antigens.
References
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neuropathy related to paraproteinemia binds to a carbohydrate
determinant in the myelin-associated glycoprotein and to a ganghoside. Proc Natl Acad Sci USA 1984;81:1225-1229
2. Chou DK, Ilyas AA, Evans JE, et al. Structure of sulfated
Quarles et al: Antibodies to Gangliosides in GBS
S51
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