Journal of Neuroimmunology, 38 (1992) 221-228
221
© 1992 ElsevierSciencePublishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02184
Influence of gangliosides on experimental allergic neuritis * H. Wieth61ter, M. Schabet, A. Stevens, A. Melrns, N. Sommer and M. Weller Department of Neurology, University of Tiibingen, Tiibingen, FRG
(Received 18 October 1991) (Revised, received8 January1992) (Accepted 23 January1992)
Key words: Experimentalallergicneuritis; Ganglioside;Antibody;Guillain-Barr6syndrome
Summary The development of myelin-induced experimental allergic neuritis (EAN) in Lewis rats can be depressed and delayed by adding a ganglioside mixture (GM1, GDla, GDlb, GTlb) to the immunization compound; however, gangliosides may enhance the induction of adjuvant arthritis. Antibodies against multiple gangliosides are produced in rats after immunization with gangliosides after addition of myelin, but only low titers can be detected in animals immunized with myelin and complete Freund's adjuvant alone. We conclude that this antibody production is not the result of peripheral nerve inflammation but depends rather from external applied gangliosides.
Introduction The ganglioside mixture of bovine brain gangliosides (GM1, GDla, GDlb, GTlb) has been widely used in trials of the treatment of peripheral neuropathies (for example Hallett et al., 1987). Polyclonal antibodies against gangliosides have been described in various neurological diseases and in some, especially peripheral neuropathies, they are considered to play a pathogenetic role (Svennerholm and Fredman, 1990). Significant amounts of ganglioside antibodies were detected in patients with postinfectious GBS
Correspondence to: H. Wieth61ter,Departmentof Neurology, University of Tiibingen, Hoppe-Seyler-Str. 3, D 7400 Tfibingen, FRG. * This studywas supported by the DFG (Wi 770/2-3)
(Ilyas et al., 1988; Quarles et al., 1990; Weller et al., submitted), which has been interpreted as pathogenetically irrelevant concomitant reactions, preferably in cases with previous campylobacter infections (Yuki et al., 1990). Experimental allergic neuritis (EAN) is a well-known animal model of the human acute Guillain-Barr6 syndrome with great similarities in the clinical, electrophysiological and histological findings and has been helpful in elucidating pathophysiological and therapeutical aspects of the human disease. In this study, the influence of gangliosides on the conventionally induced EAN was investigated by (a) modifying the clinical course of the disease by adding gangliosides to the immunization mixture and (b) measuring serum levels of anti-ganglioside antibodies in animals immunized with or without gangliosides.
222 Materials and methods
Clinical score
Animals Female inbred Lewis rats weighing approximately 190 g were obtained from Zentralinstitut ffir Versuchstierzucht (Hannover, FRG). They were housed in pairs in Macrolon cages in a temperature-controlled room. Food pellets and tap water were available ad libitum. All experiments were performed according to the legal rights of animal care.
Clinical disease was ranked from 0 to 10 according to King et al. (1983): 0 = normal, 1 = less lively, 2 = impaired righting/limp tail, 3 = absent righting, 4 = ataxic gait, abnormal position, 5 = mild paraparesis, 6 = moderate paraparesis, 7 = severe paraparesis, 8 = tetraparesis, 9 = moribund, 10 = death. Scoring was performed by two observers independently. Body weight was recorded daily.
Electrophysiological testing Immunization procedure Myelin was prepared from homogenized bovine dorsal roots according to Schott et al. (1988), emulsified with sterile saline to a concentration of 20 m g / m l or 4 m g / m l respectively. Myelin was mixed 1 : 1 with Freund's complete adjuvant containing 20 m g / m l of Mycobacterium tuberculosis (Difco, Detroit, MI). The ganglioside mixture (GM1, 21%; G D l a , 40%; G D l b , 16%; G T l b , 19%) (kindly supplied by Fidia, Italy) was added to a final concentration of 10 mg gangliosides per 0.1 ml emulsion. A total of 0.1 ml of emulsion was injected intradermally at the neck and proximal to the front paws of each animal. Animals were divided into groups according to the following protocol (Table 1).
Anaesthesia Immunization, electrophysiological recordings and blood sampling required anaesthesia by a mixture of 63 m g / k g ketanest (Ketavet R, Parke Davis) and 3 m g / k g xylazin (Rompun R, Bayer), given intraperitoneally. TABLE 1 IMMUNIZATION PROCEDURE IN THE FIVE DIFFERENT GROUPS OF ANIMALS CFA, 0.1 ml CFA (containing 2 mg mycobact./animal); gangl., 10 mg gangliosides/animal; myelin, isolated myelin from bovine dorsal root Group Animalnumber A B C D E
811-814 815-818 819-822 823-825 826-828
CFA CFA CFA CFA CFA
gangl. gangl. 0.2 mg myelin gangl. 1.0 mg myelin 1.0 mg myelin 0.2 mg myelin
Electrophysiological measurement was performed on days 8, 22, 34, 49 after immunization. The tibial nerve was stimulated at the ankle by a pair of subcutaneous monopolar needle electrodes. A 0.5-ms rectangular pulse was delivered by a T6nnies D A II R unit. Spinal somatosensory evoked potentials (SEP) were recorded from fine needle electrodes placed between the spinous processes of L 5 and L 6 and of L~ and L 2. The responses of 128 stimuli ( 3 / s ) were averaged. The latencies of spinal somatosensory evoked potentials were measured as described earlier (Wieth61ter and Htilser, 1985; Wieth61ter, 1989).
Histology Animals were killed with an overdose of anaesthetics. Tissues from spinal cord, lumbar and sacral nerve roots, sciatic nerves and soleus muscles were removed, fixed for 24 h in periodate-lysine-paraformaldehyde (PLP), embedded in paraffin, and cut to sections of 6 p.m. Inflammatory infiltrates were systematically scanned in hematoxylin and eosin stained sections. In addition immunohistochemical techniques visualized monocytic cells by ED1 monoclonal antibody (Stevens et al., 1989) using the avidin-biotin (ABC) peroxidase method (Hsu et al., 1981). Cellular infiltration was assessed semiquantitatively: - -- no infiltration; + = scanty infiltrate, not more than one cell layer thick surrounding a few vessels, or isolated cells scattered in the parenchyma; + + = scanty infiltrates around numerous vessels, or more than one cell layer thick around a few vessels, or diffusely scattered cells in the parenchyma; + + + = multilayered inflammatory infiltrates surrounding numerous ves-
223 Clinical score Igrade]
sels or a diffuse dense infiltration of the parenchyma.
Ganglioside antibodies Serum was taken from one of the tail veins on day 8, 22, 34, 49 after immunization for ELISA (IgG and IgM antibodies against GM1, GDla, GDlb, GTlb, AGM1).
3 2 1 0
ELISA 96-well microtiter plates were coated overnight at room temperature with purified gangliosides (150 ng/well) dissolved in PBS (phosphatebuffered saline). Plates were washed three times with PBS containing 0.05% Tween 20, rinsed four times with PBS, and blocked by PBS with 3% BSA (bovine serum albumin) for 1 h. Serum samples diluted in PBS containing 1% BSA and 0.5% Tween 20 were added to the plates and incubated at 37°C for 2 h. After washing, HRP(horseradish-peroxidase)-conjugated goat antibody to rat IgG or IgM was added and the plates were incubated for 2 h. The plates were then washed and 0.1 ml substrate (OPD Cromogen) was added. Reaction was stopped 10 min later by adding 4 N H2SO 4. Optical density was measured at 492 nm using an automatic ELISA reader. Absorptions greater than the negative control plus four standard deviations were considered positive (+); levels greater than negative control plus two standard deviation were described as equivocal ( + / - ) .
J''''J'J'' Imm.
-
,,,b,,,,
8
21
34
49
Days after Imm. GROUP
A
-e-
GROUP
B
~
GROUP
E
Fig. 2. Mean clinical score of the groups A, B and E in the course of disease. Imm. = Immunization.
Statistics Student's t-test and ANOVA were applied for statistical analysis.
Results
An early and reliable sign for the development of EAN is loss of body weight, seen at the beginning of the disease. Animals of all groups showed a certain delay in gaining body weight with intermittent weight loss in group B and D between day 16 and 21, respectively (Fig. 1). Animals in all groups except A developed clinical signs of EAN. In group A we recorded only some animals being less lively than normal. Animals in groups B and E showed a similar clinical course with respect to the grading (Fig. 2). Since Clinical score [gradel
Body weight I%1
7 r~7
130
• D ~ 0.04
120
110 3 2
100
1 90
Imm.
8
21
34
49
Days after Imm. --
GROUP A
-e-
GROUP B
-B-
GROUP D
~
GROUP E
0
,,,,h~,,, Imm.
8
21
34
49
Days after Imm. ~
GROUP C
Fig. 1. Mean body weight of each group of animals calculated in percent of original body weight. Imm. = Immunization.
GROUP
A
~
GROUP
C
~
GROUP
D
Fig. 3. Mean clinical score of the groups A, C and D in the course of disease. Imm. = Immunization.
224 all animals immunized with gangliosides develo p e d typical signs of adjuvant arthritis mainly in the ankle joints, their clinical grading d e p e n d e d rather on arthritic gait disturbance, than on E A N (in g r o u p B also histologically no signs of neuritis could be detected). By contrast, only one rat in group E showed a slight t e m p o r a r y arthritis. T h e most severe disease was seen in group D on day 20 after immunization. In g r o u p C clinical signs started 5 days later and peaked on day 21 with a plateau and slow recovery thereafter (Fig. 3). The difference between the two groups was statistically significant (on day 16, P < 0.04; on day 20 to 23, P < 0.1). Electrophysiologically we f o u n d near normal values for Ls/6 and L l / 2 median SEP-latencies in g r o u p A, B and E at all timepoints, except a minimal increase on day 34 in group B. A characteristic delay was found in group C and D with an initial p e a k on day 21 in group D (statistically significant m o r e in D, P < 0.02) followed by a continuous recovery and a peak on day 34 in group C with far better recovery regarding L1/2 latencies. SEP-latencies at L5/6 were delayed in group C and D from day 21 to 49 to the same extent (Table 2). Monocytic infiltrates detected by immunohistochemical staining with ED1 are summarized in Table 3. In the groups A and B no cellular
infiltration could be detected, wereas in group E we found scanty and in groups C and D intensive infiltration particularly proximally in the sciatic nerve and less also in the nerve roots. Spinal cord and muscles were free of any infiltration. The extent of demyelination c o r r e s p o n d e d well to the grade of infiltration, but could not be quantified. Ganglioside antibodies taken on day 49 after immunization were mostly positive for the IgM class in animals from groups A and B, and partly in group C. Antibodies against G D l a a p p e a r e d before G T l b , G D l b , GM1 and A G M 1 when tested at different times and reached their highest levels on day 22 after immunization (data not shown here). N o antibodies could be detected in groups D and E. I g G antibodies were only seen in some animals of groups A and B and seem to result from a cross-reaction to I g M antibodies (Table 4). A summary of the antibody levels at day 34 for IgM a n t i - G D l a and G T l b is given by Fig. 4. A n t i b o d y titers in the different groups are quantified by the factor of standard deviation above normal controls.
Discussion Highly purified gangliosides from bovine brain are t h o u g h t to be rather weakly immunogenic. It
TABLE 2 MEAN LATENCY (MS) OF SEP RECORDED FROM Ls/6 AND ANIMALS (STANDARD DEVIATION IN BRACKETS) Group
Latency L5/6 on day
L1/2 ON SEVERAL DAYS OF EACH GROUP OF
8
21
34
49
Latency Lt/2 on day 8 21
34
49
A
2.18 (0.04)
2.18 (0.04)
2.23 (0.08)
2.18 (0.04)
3.20 (0.04)
3.23 (0.04)
3.20 (0.07)
3.15 (0.05)
B
2.18 (0.03)
2.18 (0.08)
2.40 (0.17)
2.20 (0.07)
3.20 (0.03)
3.23 (0.04)
3.23 (0.08)
3.20 (0.01)
C
2.18 (0.04)
2.53 (0.15)
2.73 (0.36)
2.50 (0.24)
3.20 (0.02)
3.70 * (0.27)
4.3 (0.91)
3.63 (0.31)
D
2.18 (0.03)
2.73 (0.33)
2.57 (0.45)
2.70 (0.37)
3.20 (0.03)
5.20 * (1.31)
4.47 (0.99)
3.80 (0.65)
E
2.18 (0.04)
2.23 (0.05)
2.23 (O.l 2)
2.23 (0.09)
3.30 (0.01)
3.30 (0.01)
3.27 (0.19)
3.30 (0.14)
* P < 0.02.
225 TABLE 3
cobacteria tuberculosis applied in divided doses
MONOCYTIC INFILTRATES IN SPINAL CORD, NERVE ROOTS, SCIATIC NERVE AND MUSCLE, DETECTED BY IMMUNOHISTOCHEMICAL STAINING WITH ED1
intradermally at multiple sites. The antibodies were mainly of type IgM and mostly directed against G D l a , followed by G T l b , G D l b and GM1 (data not shown). Only three animals developed I g G antibodies that reacted against all five gangliosides tested. IgM antibodies to AGM1, regarded as more immunogenic were only positive in one animal. The highest titers of antibodies were produced ( 4 / 4 ) in animals immunized with ganglioside plus low-dose myelin (group B), less frequently and of lower titers in group A (immunized without myelin) and group C (with high dose myelin). In E A N animals immunized without additional gangliosides (groups D and E) no antibodies were found on day 49 and low titers on day 22 (data not shown) and 34 (Fig. 4, demonstrated for IgM). Antibody production, though possible with gangliosides alone, is more often seen after immunization with a mixture of gangliosides plus minor amounts of myelin and declines with high myelin concentration (1.0 nag/animal). Looking at the clinical course of single animals we found no correlation between antibody production and severity or onset and duration of the disease. There has been a longstanding discussion concerning the 'ganglioside syndrome', an EAN-like syndrome, which was induced in few susceptible strains of rabbits after repeated injections of brain gangliosides admixed with methylated bovine serum albumin and CFA (Nagai et al., 1976). Later these results could not be confirmed. However, another effect was demonstrated: the induction of a weak E A N by P2 (as immunogen) could be clearly enhanced by addition of brain gangliosides. This effect was explained as a help to impart a "special conformation for the Pz protein molecule by locating or exposing the proper neuritogenic determinant of P2 molecules" (Nagai et al., 1980). Recently Raine and Ponzin (1991) immunized Lewis rats with a purified ganglioside mixture plus the P2 protein resulting in E A N with a significant lower level of disease severity than E A N in animals inoculated with 1'2 alone (Ponzin et al., 1991). Using dorsal root myelin for the induction of E A N in rats, we could demonstrate a protecting effect of gangliosides when they were added to
Group
Animal
Spinal
Nerve
Sciatic
no.
cord
roots
nerve
811 812 813 814
. . .
. . .
(+) . . .
. . .
B
815 816 817 818
. . . .
. . . .
. . . .
. . . .
C
819
.
.
.
.
820 821 822
-
D
823 824 825
E
826 827 828
A
Muscle
-
-
(+) + +
++ ++ ++
-
-
+ + -
++ ++ (+)
-
-
(+) + -
(+) + (+)
-
- , n o infiltration; + , s c a n t y infiltrate, n o t m o r e t h a n o n e cell. l a y e r t h i c k surr o u n d i n g a few vessels, o r i s o l a t e d cells s c a t t e r e d in the parenchyma; + + , s c a n t y i n f i l t r a t e s a r o u n d n u m e r o u s vessels, o r m o r e t h a n o n e cell l a y e r t h i c k a r o u n d a few vessels, o r diffusely s c a t t e r e d cells in t h e p a r e n c h y m a ; + + + , m u l t i l a y e r e d i n f l a m m a t o r y i n f i l t r a t e s s u r r o u n d i n g num e r o u s vessels o r a d i f f u s e d e n s e i n f i l t r a t i o n of the parenchyma.
has been demonstrated repeatedly that it is necessary to prepare special immunogenic mixtures like a complex of gangliosides and extraneous protein to obtain anti-ganglioside antibodies in animals (Doherty and Walsh, 1987) and that a mixture with CFA alone is not sufficient. Most often, methylated bovine serum albumin is used as conjugate and multiple injection sites with booster injections are described as mandatory (Schwerer et al., 1986; Shimada and Iwata, 1987). We demonstrate here that a significant amount of anti-ganglioside antibodies are produced in rats after a single immunization with a ganglioside mixture of GM1, G D l a , G D l b , and G T l b emulsified in CFA enhanced with additional My-
226 TABLE 4 ANTI-GANGLIOSIDE Group
A
Animal
IgG
number
GMI
811 812 813 814
B
C
D
E
ANTIBODIES MEASURED
.
IgM GDla .
. . +
BY E L I S A IN E A C H A N I M A L
GDlb
.
. .
.
.
. .
. .
+ .
+
815
.
816 817
+ .
818
+
819 820 821 822
. . . .
. . . .
. . . .
. . . .
. . . .
823 824 825
. . .
. . .
. . .
. . .
. . .
826 827 828
. . .
. . .
. . .
. . .
. . .
+ .
.
.
.
+
.
+
+
AGM1
.
. . +
GTlb
GDla
-
+
-
+
m
Jr-
_
-
+
+
+
--
+
+
+
+
+
--
+
- -
-}-
_
GDlb
GTlb
AGM1
+
+
+ -
+
+
+
--
+
+
+
+
+
+ +
+ +
---
+
+
- -
!
+
+
-
+
.
+
GM1
__
w
m
m
_
m
m
+ , A n t i b o d y level g r e a t e r t h a n n e g a t i v e control plus f o u r s t a n d a r d deviations; + / - , A n t i b o d y level g r e a t e r t h a n n e g a t i v e c o n t r o l plus two s t a n d a r d deviations; - , A n t i b o d y level e q u a l to o r l o w e r t h a n n e g a t i v e c o n t r o l plus two s t a n d a r d deviations.
the immunization mixture. Development of EAN was clearly depressed and delayed in rats of groups B and C (ganglioside groups) (C versus D, P < 0.04) but otherwise the induction of adjuvant
arthritis was enhanced. Usually adjuvant arthritis (experimental allergic arthritis) is induced in rats by immunization with CFA without additional protein. It can be speculated that gangliosides are
IgM ANTI-GTlb
IgM ANTI-GDla
Antibody tiler [x SDI
Antibody tiLer [x SD]
10
i
10
811-814
816"818
819-822
Groups of animals m
Fig.
4,
GTlb
823-826
826-828
:' II II,I .I.I ,,,.._ :,,,
811-814
818-818
819"822
823-826
828-828
Groups of animals m
GDla
A n t i b o d y tiLer of I g M a n t i - G T l b a n d G D l a on day 34. T h e tiLer is c a l c u l a t e d as a f a c t o r o f s t a n d a r d d e v i a t i o n a b o v e n o r m a l controls.
227
able to protect myelin from exposure in the mixture. The protecting effect of gangliosides for EAN can better be proved by electrophysiology. The most severe and earliest delay in nerve conduction properties, documented by SEP latencies, is seen in group D. A milder delay later, after immunization, occurred in group C. In the groups B and E the SEP latencies were only mildly delayed, in group E more than in B. Another approach was chosen by Ledeen et al. (1990) who administered ganglioside mixture (20 m g / k g i.m.) from day 6 to 21 after induction of EAN in rats. They described a markedly attenuated clinical course of the disease with significant reduction and delay of mortality. Long-term observation of the disease development and recovery in their study was difficult. Some studies have shown elevated anti-ganglioside titers in patients with acute Guillain-Barr6 syndrome (Quarles et al., 1990) and investigators speculated that antibodies are of pathogenic importance in some patients with very high titers. Walsh et al. (1991) detected antibodies that reacted with gangliosides GM1 a n d / o r G D l b but not GM2, G D l a and GTlb in 15% of GBS patients but no control patients. They found a correlation between the presence of antibodies and the patients' prognosis in terms of disability at 3 and 12 months after presentation. They regarded the antibodies as a good correlation with the ongoing pathology and the degree of axonal damage. Patients with higher incidence of antibodies had a higher incidence of previous Campylobacter infections. In our own studies we detected elevated antibodies titers in 35% of GBS patients most often against GM1. We found no correlation with the severity of the disease (Wieth61ter et al., 1991). Further investigations with application of gangliosides in the early stages of disease to mimic clinical condition are necessary to give an idea of the potential clinical usefulness and harmless use in polyradiculoneuritis. References Doherty, P. and Walsh, F.S. (1987) Ganglioside GM1 antibodies and B-cholera toxin bind specifically to embryonic
chick dorsal root ganglion neurons but do not modulate neurite regeneration. J. Neurochem. 48, 1237-1244. Hallett, M., Flood, T., Slater, N. and Dambrosia, J. (1987) Trial of ganglioside therapy for diabetic neuropathy. Muscle Nerve 10, 822-825. Hsu, S.M., Raine, L. and Fanger, H. (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580. Ilyas, A.A., Willison, H.J., Quarles, R.H., Jungalwala, F.B, Cornblath, D.R., Trapp, B.D., Griffin, D.E. and Griffin J.W. (1988) Serum antibodies to gangliosides in GuillainBarr6 syndrome. Ann. Neurol. 23, 440-447. King, R.H.M., Craggs, R.I., Gross, M.L.P., Tompkins, C. and Thomas, P.K. (1983) Suppression of experimental allergic neuritis by Cyclosporin-A. Acta Neuropathol. 59, 262-268. Ledeen, R.W., Oderfeld-Nowak, B., Brosnan, C.F. and Cervone, A. (1990) Gangliosides offer partial protection in experimental allergic neuritis. Ann. Neurol. 27 (Suppl.) 69-74. Nagai, Y., Momoi, T., Saito, M., Mitsuzawa, E. and Ohtani, S. (1976) Ganglioside syndrome, a new autoimmune neurologic disorder, experimentally induced with brain gangliosides. Neurosci. Lett. 2, 107-111. Nagai, Y., Sakakabira, K. and Uchida, T. (1980) Immunomodulatory roles of gangliosides in EAE and EAN. In: A. Boese (Ed.), Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous System. Verlag Chemie, Weinheim. Ponzin, D., Menegus, A.M., Kirschner, G., Nunzi, M.G., Fiori, M.G. and Raine, C.S. (1991) Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system. Ann. Neurol. 30, 678-685. Quarles, R.H., Ilyas, A.A. and Willison, H.J. (1990) Antibodies to gangliosides and myelin proteins in Guillain-Barr6 Syndrome. Ann. Neurol. 27 (Suppl.), 48-52. Raine, C.S. and Ponzin, D. (1991) Comparative effects of gangliosides and galactocerebrosides on the expression of autoimmune demyelination in the PNS. Gangliosides: the Pharmacology of Neuronal Plasticity, Fidia Research Foundation, Rome. Schott, K., Jenner, A., Wieth61ter, H., Schabet, M. and Stevens, A. (1988) Demyelination of sciatic nerve in acute experimental allergic neuritis of the rat. A gel electrophoresis investigation. J. Neuroimmunol. 20, 53-61. Schwerer, B., Lassmann, H. and Bernheimer, H. (1986) Ganglioside GM1, a molecular target for immunological and toxic attacks: similarity of neuropathological lesions induced by ganglioside-antiserum and cholera toxin. Acta Neuropathol. 72, 55-61. Shimada, S. and Iwata, D. (1987) Preparation of monoclonal antibodies against a glycolipid asialo GM1. Microbiol. Immunol. 31,923-933. Stevens, A., Schabet, M., Schott, K., Wieth61ter, H. (1989) Role of endoneural cells in experimental allergic neuritis and characterisation of a resident phagocytic cell. Acta Neuropathol. 77, 412-419.
228 Svennerholm, L, and Fredman, P. (1990) Antibody detection in Guillain-Barr6 Syndrome. Ann. Neurol. 27 (Suppl.) 36-40. Walsh, F.S., Cronin, M., Koblar, S., Doherty, P., Wirier, J., Leon, A. and Hughes, R.A.C. (1991) Characterisation of Ganglioside antibodies in sera of patients with GuillainBarr~ Syndrome. Gangliosides: the Pharmacology of Neuronal Plasticity, Poster, Rome. Wieth61ter, H. (1989) Verlaufstbrmen der experimentell-allergischen Neuritis - - Elektrophysiologische Untersuchungen und Behandlung durch Plasmaseparation. Springer, Berlin and New York.
Wieth61ter, H. and Hiilser, P.J. (1985) Spinal somatosensory evoked potentials in the rat after stimulation of the tibial nerve. ]. Exp. Neurol. 89, 24-31. Wieth61ter, H., Weller, M., Stevens, A , Sommer, N., Kappler, B., Dichgans, J. (1991) Diagnostic value of multiple ganglioside antibody determination. Gangliosides: the Pharmacology of Neuronal Plasticity, Poster, Rome. Yuki, N., Yoshino, H., Sato, S. and Miyatake, T. (1990) Acute axonal polyneuropathy associated with anti-GMl antibodies following Carnpylobacter enteritis. Neurology 40 (12), 1900-1902.
221
© 1992 ElsevierSciencePublishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02184
Influence of gangliosides on experimental allergic neuritis * H. Wieth61ter, M. Schabet, A. Stevens, A. Melrns, N. Sommer and M. Weller Department of Neurology, University of Tiibingen, Tiibingen, FRG
(Received 18 October 1991) (Revised, received8 January1992) (Accepted 23 January1992)
Key words: Experimentalallergicneuritis; Ganglioside;Antibody;Guillain-Barr6syndrome
Summary The development of myelin-induced experimental allergic neuritis (EAN) in Lewis rats can be depressed and delayed by adding a ganglioside mixture (GM1, GDla, GDlb, GTlb) to the immunization compound; however, gangliosides may enhance the induction of adjuvant arthritis. Antibodies against multiple gangliosides are produced in rats after immunization with gangliosides after addition of myelin, but only low titers can be detected in animals immunized with myelin and complete Freund's adjuvant alone. We conclude that this antibody production is not the result of peripheral nerve inflammation but depends rather from external applied gangliosides.
Introduction The ganglioside mixture of bovine brain gangliosides (GM1, GDla, GDlb, GTlb) has been widely used in trials of the treatment of peripheral neuropathies (for example Hallett et al., 1987). Polyclonal antibodies against gangliosides have been described in various neurological diseases and in some, especially peripheral neuropathies, they are considered to play a pathogenetic role (Svennerholm and Fredman, 1990). Significant amounts of ganglioside antibodies were detected in patients with postinfectious GBS
Correspondence to: H. Wieth61ter,Departmentof Neurology, University of Tiibingen, Hoppe-Seyler-Str. 3, D 7400 Tfibingen, FRG. * This studywas supported by the DFG (Wi 770/2-3)
(Ilyas et al., 1988; Quarles et al., 1990; Weller et al., submitted), which has been interpreted as pathogenetically irrelevant concomitant reactions, preferably in cases with previous campylobacter infections (Yuki et al., 1990). Experimental allergic neuritis (EAN) is a well-known animal model of the human acute Guillain-Barr6 syndrome with great similarities in the clinical, electrophysiological and histological findings and has been helpful in elucidating pathophysiological and therapeutical aspects of the human disease. In this study, the influence of gangliosides on the conventionally induced EAN was investigated by (a) modifying the clinical course of the disease by adding gangliosides to the immunization mixture and (b) measuring serum levels of anti-ganglioside antibodies in animals immunized with or without gangliosides.
222 Materials and methods
Clinical score
Animals Female inbred Lewis rats weighing approximately 190 g were obtained from Zentralinstitut ffir Versuchstierzucht (Hannover, FRG). They were housed in pairs in Macrolon cages in a temperature-controlled room. Food pellets and tap water were available ad libitum. All experiments were performed according to the legal rights of animal care.
Clinical disease was ranked from 0 to 10 according to King et al. (1983): 0 = normal, 1 = less lively, 2 = impaired righting/limp tail, 3 = absent righting, 4 = ataxic gait, abnormal position, 5 = mild paraparesis, 6 = moderate paraparesis, 7 = severe paraparesis, 8 = tetraparesis, 9 = moribund, 10 = death. Scoring was performed by two observers independently. Body weight was recorded daily.
Electrophysiological testing Immunization procedure Myelin was prepared from homogenized bovine dorsal roots according to Schott et al. (1988), emulsified with sterile saline to a concentration of 20 m g / m l or 4 m g / m l respectively. Myelin was mixed 1 : 1 with Freund's complete adjuvant containing 20 m g / m l of Mycobacterium tuberculosis (Difco, Detroit, MI). The ganglioside mixture (GM1, 21%; G D l a , 40%; G D l b , 16%; G T l b , 19%) (kindly supplied by Fidia, Italy) was added to a final concentration of 10 mg gangliosides per 0.1 ml emulsion. A total of 0.1 ml of emulsion was injected intradermally at the neck and proximal to the front paws of each animal. Animals were divided into groups according to the following protocol (Table 1).
Anaesthesia Immunization, electrophysiological recordings and blood sampling required anaesthesia by a mixture of 63 m g / k g ketanest (Ketavet R, Parke Davis) and 3 m g / k g xylazin (Rompun R, Bayer), given intraperitoneally. TABLE 1 IMMUNIZATION PROCEDURE IN THE FIVE DIFFERENT GROUPS OF ANIMALS CFA, 0.1 ml CFA (containing 2 mg mycobact./animal); gangl., 10 mg gangliosides/animal; myelin, isolated myelin from bovine dorsal root Group Animalnumber A B C D E
811-814 815-818 819-822 823-825 826-828
CFA CFA CFA CFA CFA
gangl. gangl. 0.2 mg myelin gangl. 1.0 mg myelin 1.0 mg myelin 0.2 mg myelin
Electrophysiological measurement was performed on days 8, 22, 34, 49 after immunization. The tibial nerve was stimulated at the ankle by a pair of subcutaneous monopolar needle electrodes. A 0.5-ms rectangular pulse was delivered by a T6nnies D A II R unit. Spinal somatosensory evoked potentials (SEP) were recorded from fine needle electrodes placed between the spinous processes of L 5 and L 6 and of L~ and L 2. The responses of 128 stimuli ( 3 / s ) were averaged. The latencies of spinal somatosensory evoked potentials were measured as described earlier (Wieth61ter and Htilser, 1985; Wieth61ter, 1989).
Histology Animals were killed with an overdose of anaesthetics. Tissues from spinal cord, lumbar and sacral nerve roots, sciatic nerves and soleus muscles were removed, fixed for 24 h in periodate-lysine-paraformaldehyde (PLP), embedded in paraffin, and cut to sections of 6 p.m. Inflammatory infiltrates were systematically scanned in hematoxylin and eosin stained sections. In addition immunohistochemical techniques visualized monocytic cells by ED1 monoclonal antibody (Stevens et al., 1989) using the avidin-biotin (ABC) peroxidase method (Hsu et al., 1981). Cellular infiltration was assessed semiquantitatively: - -- no infiltration; + = scanty infiltrate, not more than one cell layer thick surrounding a few vessels, or isolated cells scattered in the parenchyma; + + = scanty infiltrates around numerous vessels, or more than one cell layer thick around a few vessels, or diffusely scattered cells in the parenchyma; + + + = multilayered inflammatory infiltrates surrounding numerous ves-
223 Clinical score Igrade]
sels or a diffuse dense infiltration of the parenchyma.
Ganglioside antibodies Serum was taken from one of the tail veins on day 8, 22, 34, 49 after immunization for ELISA (IgG and IgM antibodies against GM1, GDla, GDlb, GTlb, AGM1).
3 2 1 0
ELISA 96-well microtiter plates were coated overnight at room temperature with purified gangliosides (150 ng/well) dissolved in PBS (phosphatebuffered saline). Plates were washed three times with PBS containing 0.05% Tween 20, rinsed four times with PBS, and blocked by PBS with 3% BSA (bovine serum albumin) for 1 h. Serum samples diluted in PBS containing 1% BSA and 0.5% Tween 20 were added to the plates and incubated at 37°C for 2 h. After washing, HRP(horseradish-peroxidase)-conjugated goat antibody to rat IgG or IgM was added and the plates were incubated for 2 h. The plates were then washed and 0.1 ml substrate (OPD Cromogen) was added. Reaction was stopped 10 min later by adding 4 N H2SO 4. Optical density was measured at 492 nm using an automatic ELISA reader. Absorptions greater than the negative control plus four standard deviations were considered positive (+); levels greater than negative control plus two standard deviation were described as equivocal ( + / - ) .
J''''J'J'' Imm.
-
,,,b,,,,
8
21
34
49
Days after Imm. GROUP
A
-e-
GROUP
B
~
GROUP
E
Fig. 2. Mean clinical score of the groups A, B and E in the course of disease. Imm. = Immunization.
Statistics Student's t-test and ANOVA were applied for statistical analysis.
Results
An early and reliable sign for the development of EAN is loss of body weight, seen at the beginning of the disease. Animals of all groups showed a certain delay in gaining body weight with intermittent weight loss in group B and D between day 16 and 21, respectively (Fig. 1). Animals in all groups except A developed clinical signs of EAN. In group A we recorded only some animals being less lively than normal. Animals in groups B and E showed a similar clinical course with respect to the grading (Fig. 2). Since Clinical score [gradel
Body weight I%1
7 r~7
130
• D ~ 0.04
120
110 3 2
100
1 90
Imm.
8
21
34
49
Days after Imm. --
GROUP A
-e-
GROUP B
-B-
GROUP D
~
GROUP E
0
,,,,h~,,, Imm.
8
21
34
49
Days after Imm. ~
GROUP C
Fig. 1. Mean body weight of each group of animals calculated in percent of original body weight. Imm. = Immunization.
GROUP
A
~
GROUP
C
~
GROUP
D
Fig. 3. Mean clinical score of the groups A, C and D in the course of disease. Imm. = Immunization.
224 all animals immunized with gangliosides develo p e d typical signs of adjuvant arthritis mainly in the ankle joints, their clinical grading d e p e n d e d rather on arthritic gait disturbance, than on E A N (in g r o u p B also histologically no signs of neuritis could be detected). By contrast, only one rat in group E showed a slight t e m p o r a r y arthritis. T h e most severe disease was seen in group D on day 20 after immunization. In g r o u p C clinical signs started 5 days later and peaked on day 21 with a plateau and slow recovery thereafter (Fig. 3). The difference between the two groups was statistically significant (on day 16, P < 0.04; on day 20 to 23, P < 0.1). Electrophysiologically we f o u n d near normal values for Ls/6 and L l / 2 median SEP-latencies in g r o u p A, B and E at all timepoints, except a minimal increase on day 34 in group B. A characteristic delay was found in group C and D with an initial p e a k on day 21 in group D (statistically significant m o r e in D, P < 0.02) followed by a continuous recovery and a peak on day 34 in group C with far better recovery regarding L1/2 latencies. SEP-latencies at L5/6 were delayed in group C and D from day 21 to 49 to the same extent (Table 2). Monocytic infiltrates detected by immunohistochemical staining with ED1 are summarized in Table 3. In the groups A and B no cellular
infiltration could be detected, wereas in group E we found scanty and in groups C and D intensive infiltration particularly proximally in the sciatic nerve and less also in the nerve roots. Spinal cord and muscles were free of any infiltration. The extent of demyelination c o r r e s p o n d e d well to the grade of infiltration, but could not be quantified. Ganglioside antibodies taken on day 49 after immunization were mostly positive for the IgM class in animals from groups A and B, and partly in group C. Antibodies against G D l a a p p e a r e d before G T l b , G D l b , GM1 and A G M 1 when tested at different times and reached their highest levels on day 22 after immunization (data not shown here). N o antibodies could be detected in groups D and E. I g G antibodies were only seen in some animals of groups A and B and seem to result from a cross-reaction to I g M antibodies (Table 4). A summary of the antibody levels at day 34 for IgM a n t i - G D l a and G T l b is given by Fig. 4. A n t i b o d y titers in the different groups are quantified by the factor of standard deviation above normal controls.
Discussion Highly purified gangliosides from bovine brain are t h o u g h t to be rather weakly immunogenic. It
TABLE 2 MEAN LATENCY (MS) OF SEP RECORDED FROM Ls/6 AND ANIMALS (STANDARD DEVIATION IN BRACKETS) Group
Latency L5/6 on day
L1/2 ON SEVERAL DAYS OF EACH GROUP OF
8
21
34
49
Latency Lt/2 on day 8 21
34
49
A
2.18 (0.04)
2.18 (0.04)
2.23 (0.08)
2.18 (0.04)
3.20 (0.04)
3.23 (0.04)
3.20 (0.07)
3.15 (0.05)
B
2.18 (0.03)
2.18 (0.08)
2.40 (0.17)
2.20 (0.07)
3.20 (0.03)
3.23 (0.04)
3.23 (0.08)
3.20 (0.01)
C
2.18 (0.04)
2.53 (0.15)
2.73 (0.36)
2.50 (0.24)
3.20 (0.02)
3.70 * (0.27)
4.3 (0.91)
3.63 (0.31)
D
2.18 (0.03)
2.73 (0.33)
2.57 (0.45)
2.70 (0.37)
3.20 (0.03)
5.20 * (1.31)
4.47 (0.99)
3.80 (0.65)
E
2.18 (0.04)
2.23 (0.05)
2.23 (O.l 2)
2.23 (0.09)
3.30 (0.01)
3.30 (0.01)
3.27 (0.19)
3.30 (0.14)
* P < 0.02.
225 TABLE 3
cobacteria tuberculosis applied in divided doses
MONOCYTIC INFILTRATES IN SPINAL CORD, NERVE ROOTS, SCIATIC NERVE AND MUSCLE, DETECTED BY IMMUNOHISTOCHEMICAL STAINING WITH ED1
intradermally at multiple sites. The antibodies were mainly of type IgM and mostly directed against G D l a , followed by G T l b , G D l b and GM1 (data not shown). Only three animals developed I g G antibodies that reacted against all five gangliosides tested. IgM antibodies to AGM1, regarded as more immunogenic were only positive in one animal. The highest titers of antibodies were produced ( 4 / 4 ) in animals immunized with ganglioside plus low-dose myelin (group B), less frequently and of lower titers in group A (immunized without myelin) and group C (with high dose myelin). In E A N animals immunized without additional gangliosides (groups D and E) no antibodies were found on day 49 and low titers on day 22 (data not shown) and 34 (Fig. 4, demonstrated for IgM). Antibody production, though possible with gangliosides alone, is more often seen after immunization with a mixture of gangliosides plus minor amounts of myelin and declines with high myelin concentration (1.0 nag/animal). Looking at the clinical course of single animals we found no correlation between antibody production and severity or onset and duration of the disease. There has been a longstanding discussion concerning the 'ganglioside syndrome', an EAN-like syndrome, which was induced in few susceptible strains of rabbits after repeated injections of brain gangliosides admixed with methylated bovine serum albumin and CFA (Nagai et al., 1976). Later these results could not be confirmed. However, another effect was demonstrated: the induction of a weak E A N by P2 (as immunogen) could be clearly enhanced by addition of brain gangliosides. This effect was explained as a help to impart a "special conformation for the Pz protein molecule by locating or exposing the proper neuritogenic determinant of P2 molecules" (Nagai et al., 1980). Recently Raine and Ponzin (1991) immunized Lewis rats with a purified ganglioside mixture plus the P2 protein resulting in E A N with a significant lower level of disease severity than E A N in animals inoculated with 1'2 alone (Ponzin et al., 1991). Using dorsal root myelin for the induction of E A N in rats, we could demonstrate a protecting effect of gangliosides when they were added to
Group
Animal
Spinal
Nerve
Sciatic
no.
cord
roots
nerve
811 812 813 814
. . .
. . .
(+) . . .
. . .
B
815 816 817 818
. . . .
. . . .
. . . .
. . . .
C
819
.
.
.
.
820 821 822
-
D
823 824 825
E
826 827 828
A
Muscle
-
-
(+) + +
++ ++ ++
-
-
+ + -
++ ++ (+)
-
-
(+) + -
(+) + (+)
-
- , n o infiltration; + , s c a n t y infiltrate, n o t m o r e t h a n o n e cell. l a y e r t h i c k surr o u n d i n g a few vessels, o r i s o l a t e d cells s c a t t e r e d in the parenchyma; + + , s c a n t y i n f i l t r a t e s a r o u n d n u m e r o u s vessels, o r m o r e t h a n o n e cell l a y e r t h i c k a r o u n d a few vessels, o r diffusely s c a t t e r e d cells in t h e p a r e n c h y m a ; + + + , m u l t i l a y e r e d i n f l a m m a t o r y i n f i l t r a t e s s u r r o u n d i n g num e r o u s vessels o r a d i f f u s e d e n s e i n f i l t r a t i o n of the parenchyma.
has been demonstrated repeatedly that it is necessary to prepare special immunogenic mixtures like a complex of gangliosides and extraneous protein to obtain anti-ganglioside antibodies in animals (Doherty and Walsh, 1987) and that a mixture with CFA alone is not sufficient. Most often, methylated bovine serum albumin is used as conjugate and multiple injection sites with booster injections are described as mandatory (Schwerer et al., 1986; Shimada and Iwata, 1987). We demonstrate here that a significant amount of anti-ganglioside antibodies are produced in rats after a single immunization with a ganglioside mixture of GM1, G D l a , G D l b , and G T l b emulsified in CFA enhanced with additional My-
226 TABLE 4 ANTI-GANGLIOSIDE Group
A
Animal
IgG
number
GMI
811 812 813 814
B
C
D
E
ANTIBODIES MEASURED
.
IgM GDla .
. . +
BY E L I S A IN E A C H A N I M A L
GDlb
.
. .
.
.
. .
. .
+ .
+
815
.
816 817
+ .
818
+
819 820 821 822
. . . .
. . . .
. . . .
. . . .
. . . .
823 824 825
. . .
. . .
. . .
. . .
. . .
826 827 828
. . .
. . .
. . .
. . .
. . .
+ .
.
.
.
+
.
+
+
AGM1
.
. . +
GTlb
GDla
-
+
-
+
m
Jr-
_
-
+
+
+
--
+
+
+
+
+
--
+
- -
-}-
_
GDlb
GTlb
AGM1
+
+
+ -
+
+
+
--
+
+
+
+
+
+ +
+ +
---
+
+
- -
!
+
+
-
+
.
+
GM1
__
w
m
m
_
m
m
+ , A n t i b o d y level g r e a t e r t h a n n e g a t i v e control plus f o u r s t a n d a r d deviations; + / - , A n t i b o d y level g r e a t e r t h a n n e g a t i v e c o n t r o l plus two s t a n d a r d deviations; - , A n t i b o d y level e q u a l to o r l o w e r t h a n n e g a t i v e c o n t r o l plus two s t a n d a r d deviations.
the immunization mixture. Development of EAN was clearly depressed and delayed in rats of groups B and C (ganglioside groups) (C versus D, P < 0.04) but otherwise the induction of adjuvant
arthritis was enhanced. Usually adjuvant arthritis (experimental allergic arthritis) is induced in rats by immunization with CFA without additional protein. It can be speculated that gangliosides are
IgM ANTI-GTlb
IgM ANTI-GDla
Antibody tiler [x SDI
Antibody tiLer [x SD]
10
i
10
811-814
816"818
819-822
Groups of animals m
Fig.
4,
GTlb
823-826
826-828
:' II II,I .I.I ,,,.._ :,,,
811-814
818-818
819"822
823-826
828-828
Groups of animals m
GDla
A n t i b o d y tiLer of I g M a n t i - G T l b a n d G D l a on day 34. T h e tiLer is c a l c u l a t e d as a f a c t o r o f s t a n d a r d d e v i a t i o n a b o v e n o r m a l controls.
227
able to protect myelin from exposure in the mixture. The protecting effect of gangliosides for EAN can better be proved by electrophysiology. The most severe and earliest delay in nerve conduction properties, documented by SEP latencies, is seen in group D. A milder delay later, after immunization, occurred in group C. In the groups B and E the SEP latencies were only mildly delayed, in group E more than in B. Another approach was chosen by Ledeen et al. (1990) who administered ganglioside mixture (20 m g / k g i.m.) from day 6 to 21 after induction of EAN in rats. They described a markedly attenuated clinical course of the disease with significant reduction and delay of mortality. Long-term observation of the disease development and recovery in their study was difficult. Some studies have shown elevated anti-ganglioside titers in patients with acute Guillain-Barr6 syndrome (Quarles et al., 1990) and investigators speculated that antibodies are of pathogenic importance in some patients with very high titers. Walsh et al. (1991) detected antibodies that reacted with gangliosides GM1 a n d / o r G D l b but not GM2, G D l a and GTlb in 15% of GBS patients but no control patients. They found a correlation between the presence of antibodies and the patients' prognosis in terms of disability at 3 and 12 months after presentation. They regarded the antibodies as a good correlation with the ongoing pathology and the degree of axonal damage. Patients with higher incidence of antibodies had a higher incidence of previous Campylobacter infections. In our own studies we detected elevated antibodies titers in 35% of GBS patients most often against GM1. We found no correlation with the severity of the disease (Wieth61ter et al., 1991). Further investigations with application of gangliosides in the early stages of disease to mimic clinical condition are necessary to give an idea of the potential clinical usefulness and harmless use in polyradiculoneuritis. References Doherty, P. and Walsh, F.S. (1987) Ganglioside GM1 antibodies and B-cholera toxin bind specifically to embryonic
chick dorsal root ganglion neurons but do not modulate neurite regeneration. J. Neurochem. 48, 1237-1244. Hallett, M., Flood, T., Slater, N. and Dambrosia, J. (1987) Trial of ganglioside therapy for diabetic neuropathy. Muscle Nerve 10, 822-825. Hsu, S.M., Raine, L. and Fanger, H. (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580. Ilyas, A.A., Willison, H.J., Quarles, R.H., Jungalwala, F.B, Cornblath, D.R., Trapp, B.D., Griffin, D.E. and Griffin J.W. (1988) Serum antibodies to gangliosides in GuillainBarr6 syndrome. Ann. Neurol. 23, 440-447. King, R.H.M., Craggs, R.I., Gross, M.L.P., Tompkins, C. and Thomas, P.K. (1983) Suppression of experimental allergic neuritis by Cyclosporin-A. Acta Neuropathol. 59, 262-268. Ledeen, R.W., Oderfeld-Nowak, B., Brosnan, C.F. and Cervone, A. (1990) Gangliosides offer partial protection in experimental allergic neuritis. Ann. Neurol. 27 (Suppl.) 69-74. Nagai, Y., Momoi, T., Saito, M., Mitsuzawa, E. and Ohtani, S. (1976) Ganglioside syndrome, a new autoimmune neurologic disorder, experimentally induced with brain gangliosides. Neurosci. Lett. 2, 107-111. Nagai, Y., Sakakabira, K. and Uchida, T. (1980) Immunomodulatory roles of gangliosides in EAE and EAN. In: A. Boese (Ed.), Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous System. Verlag Chemie, Weinheim. Ponzin, D., Menegus, A.M., Kirschner, G., Nunzi, M.G., Fiori, M.G. and Raine, C.S. (1991) Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system. Ann. Neurol. 30, 678-685. Quarles, R.H., Ilyas, A.A. and Willison, H.J. (1990) Antibodies to gangliosides and myelin proteins in Guillain-Barr6 Syndrome. Ann. Neurol. 27 (Suppl.), 48-52. Raine, C.S. and Ponzin, D. (1991) Comparative effects of gangliosides and galactocerebrosides on the expression of autoimmune demyelination in the PNS. Gangliosides: the Pharmacology of Neuronal Plasticity, Fidia Research Foundation, Rome. Schott, K., Jenner, A., Wieth61ter, H., Schabet, M. and Stevens, A. (1988) Demyelination of sciatic nerve in acute experimental allergic neuritis of the rat. A gel electrophoresis investigation. J. Neuroimmunol. 20, 53-61. Schwerer, B., Lassmann, H. and Bernheimer, H. (1986) Ganglioside GM1, a molecular target for immunological and toxic attacks: similarity of neuropathological lesions induced by ganglioside-antiserum and cholera toxin. Acta Neuropathol. 72, 55-61. Shimada, S. and Iwata, D. (1987) Preparation of monoclonal antibodies against a glycolipid asialo GM1. Microbiol. Immunol. 31,923-933. Stevens, A., Schabet, M., Schott, K., Wieth61ter, H. (1989) Role of endoneural cells in experimental allergic neuritis and characterisation of a resident phagocytic cell. Acta Neuropathol. 77, 412-419.
228 Svennerholm, L, and Fredman, P. (1990) Antibody detection in Guillain-Barr6 Syndrome. Ann. Neurol. 27 (Suppl.) 36-40. Walsh, F.S., Cronin, M., Koblar, S., Doherty, P., Wirier, J., Leon, A. and Hughes, R.A.C. (1991) Characterisation of Ganglioside antibodies in sera of patients with GuillainBarr~ Syndrome. Gangliosides: the Pharmacology of Neuronal Plasticity, Poster, Rome. Wieth61ter, H. (1989) Verlaufstbrmen der experimentell-allergischen Neuritis - - Elektrophysiologische Untersuchungen und Behandlung durch Plasmaseparation. Springer, Berlin and New York.
Wieth61ter, H. and Hiilser, P.J. (1985) Spinal somatosensory evoked potentials in the rat after stimulation of the tibial nerve. ]. Exp. Neurol. 89, 24-31. Wieth61ter, H., Weller, M., Stevens, A , Sommer, N., Kappler, B., Dichgans, J. (1991) Diagnostic value of multiple ganglioside antibody determination. Gangliosides: the Pharmacology of Neuronal Plasticity, Poster, Rome. Yuki, N., Yoshino, H., Sato, S. and Miyatake, T. (1990) Acute axonal polyneuropathy associated with anti-GMl antibodies following Carnpylobacter enteritis. Neurology 40 (12), 1900-1902.
