Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinemia also react with sulfatide

Journal of Neuroimmunology, 37 (1992) 85-92 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

85

JNI 02139

Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinemia also react with sulfatide Amjad A. Ilyas a, Stuart D. Cook a, Marinos C. Dalakas b and Francis A. Mithen c a Department of Neurosciences, Unwersity of Medicine and Dentistry of New Jersey - New Jersey Medical School, Newark, NJ 07103, USA, b NINDS, National Institutes of Health, Bethesda, MD 20892, USA, and c John Cochran VA Medical Center and Department of Neurology, St. Louis University, St. Louis, MO 63110, USA

(Received 29 August 1991) (Revised, received 11 October 1991) (Accepted 11 October 1991)

Key words: Myelin-associated glycoprotein (MAG); Anti-MAG lgM paraprotein; Polyneuropathy; Paraproteinemia; Sulfatide

Summary Sera from five of 11 patients with neuropathy associated with IgM paraproteinemia (NAIgMPP) and reactivity against myelin-associated glycoprotein (MAG) also had elevated levels of IgM against sulfatide. Although three patients had anti-sulfatide IgM titers of ~< 1 : 1000, two patients had titers of >_-1 : 50,000. Absorption of patient serum with sulfatide revealed that anti-MAG IgM paraproteins in two patients with high titer anti-sulfatide IgM crossreacted with sulfatide. Our study is the first to show that some anti-MAG IgM paraproteins crossreact with sulfatide, a major acidic glycolipid of myelin. Moreover, some patients with NAIgMPP have polyclonal anti-sulfatide IgM in addition to anti-MAG IgM paraproteins. Therefore, sulfatide may be a target antigen in some patients with NAIgMPP.

Introduction IgM paraproteins in more than 50% of patients with neuropathy associated with IgM paraproteinemia (NAIgMPP) react with carbohydrate antigenic determinants shared by myelin-associated glycoprotein (MAG) (Quarles, 1989), sulfated glucuronyl glycolipids (SGGLs) (Ilyas et al.,

Correspondence to: A.A. llyas, Ph.D., Department of Neurosciences, New Jersey Medical School, University of Medicine and Dentistry of N J, 185 S. Orange Avenue, Newark, NJ 07103, USA. Tel. (201) 456-4509; Fax (201) 456-5059.

1984b, 1986a, b, 1990; Quarles et al., 1986) and a number of other glycoproteins including neural cell adhesion molecule (NCAM) and Po glycoprotein (Nobile-Orazio et al., 1984; Noronha et al., 1986; O'Shannessy et al., 1986; Bollensen et al., 1988). The two major SGGLs have been characterized as sulfoglucuronyl paragioboside (SGPG) and sulfoglucuronyl lactosaminyl paragloboside (SGLPG) (Chou et al., 1985, 1986; Ariga et al., 1987). In some patients with NAIgMPP IgM paraproteins do not react with MAG and the SGGLs but do react with various gangliosides or chondroitin sulfate (Sherman et al., 1983; Ilyas et al., 1985a, b, 1988a, b; Quarles et al., 1986; Miy-

8;6

atani el al., 1987; Ycc ct al., 1989). Sulfatide is a major acidic glycolipid of peripheral nerve myelin (Svennerho[m and Frcdman, 1990). In the current study, we tested sera from patients with N A I g M P P containing anti-MAG IgM paraproteins for reactivity with sulfatide.

Materials and methods

Silica gel 6(I aluminum-backed thin-laycr chromatography (TLC) plates (E. Merk) and Unisil wcrc purchased from Brinkmann Instruments (Westbury, NY, USA) and Clarkson Chemical Co. (Williamsport, PA, USA), respectively. Vibrio cholerae ncuraminidasc was obtained from Calbiochem-Bering (San Diego, CA, USA). Bovine brain ganglioside standards and sulfatide were purchascd from Sigma Chemical Co. (St. lmuis, MO, USA). Pcroxidasc-conjugated goat anti-human lgM (p,-chain specific), anti-human IgG (3'chain specific), rabbit anti-human kappa light chains and lambda light chains were purchased from Accurate Chemical and Scientific Corporation (Westbury, NY, USA).

Patient sera Sera wcrc obtained from 11 patients with demyelinating neuropathy associated with IgM paraproteincmia and 21 normal controls. One patient (C) had IgM lambda and ten patients had lgM kappa monoclonal proteins. All sera were obtained from volunteers under research protocols approved by the Human Studics Committee or Institutional Review Board at all affiliatcd institutions in accordance with regulations mandated by the D e p a r t m e n t of Hcalth and H u m a n Services or the D e p a r t m e n t of Veterans Affairs.

Rad) (Laemmli, 19711). After clcctrophorcsis, proteins wcrc transferred electrophorctically to a nitrocellulose shect in an clcctrophoretic transfer cell (Mini Trans-Blot, Bio-Rad) (Towbin et al., 1979). The blot was first blocked with 3% bovine serum albumin (BSA) in Tris-buffercd saline (containing 0.9% NaCI, 10 mM Tris-HCl, ptl 7.4) at 50°C for 2 h and then incubated with patient serum (1:51) dilution)in 3% BSA in Tris-buffcrcd saline at room tcmpcraturc overnight, followed by 1:20.0011 dilutions of pcroxidase-conjugated goat anti-human IgM or lgG antibody for 5 h at room tcmperature. Peroxidase activity was detected using 3,3'-diaminobenzidinc (Bio-Rad, Richmond, CA, USA) as substrate.

Isolation of acidic glycolipid fractions and SGPG purification Dog sciatic nerves wcrc purchased from Pcl Frcez Biologicals (Rogcrs, AR, USA). Dog sciatic nerves wcrc used to isolate acidic glycolipid fractions because they contain substantial amounts of S G G L s (llyas et al., 1986a). Ganglioside fractions were isolated by diethylaminocthyl (DEAE)-Sephadex A 25 column chromatography, alkali treatment and Unisil chromatography (Ledeen et al., 1973). S G P G was purificd from acidic glycolipid fractions (llyas et al., 1986a) and quantitated by the micro-orcinol method (Balazs ct al., 1971). I)csulfatcd SGPG, known as glucuronyl paragloboside (GPG), thc methyl ester of G P G ( M c G P G ) , and sulloglucosyl paragloboside (SGIcPG), a derivative of S G P G in which glucuronic acid has bccn rcduced to glucosc, wcrc prepared (llyas et al., 1986a, 1990). Ganglioside fractions were digested with Vibrio cholerae neuraminidasc (llyas ct al., 1984a).

Binding of antibodies to glycolipids Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting Human brain myelin was prepared (Norton and Poduslo, 1973). The lyophilized myelin samplcs were delipidated and the insoluble protein was solubilized in a sample buffer (2% ( w / v ) SDS, 10% glycerol, 5% 2-mcrcaptoethanol) in a boiling watcr bath for 5 min. S D S - P A G E was carried out in 12.5% polyacrylamide gels using a slab elcctrophoretic cell ( M i n i - P R O T E I N 11, Bio-

Sera were assayed for antibodics to S G P G and sulfatide by enzyme-linked immunosorbent assay (EI,ISA) and TLC-immunostaining. The ELISA procedure was as described (llyas et al., 1984b) with slight modifications. The plates were incubated at 4°C instead of room temperature and the amounts of antigen indicated below wcre used. 200 ng sulfatide or 100 ng of S G P G in 20 p-I of methanol was added to each ELISA well and evaporated to dryness in air at room temperature.

87

Sera were tested at various dilutions beginning at 1 :I(X). The absorbance at 490 nm of each well was read on EL 309 Microplate Autoreader. Absorbance values were corrected by subtracting optical densities obtained in wells without antigen. Serum was considered positive if its absorbancc value was 3 standard deviations (SD) above the mean absorbance value for normal sera. The serum titer is defined as the highest dilution that exhibited reactivity. Thin-layer chromatography was performed on silica gel 60 aluminum-backed TLC plates developed in c h l o r o f o r m / m e t h a n o l / 0 . 2 % KCI, 5 0 : 4 0 : 1 0 (by volume) (Ilyas et al., t984b). For dog acidic glycolipid fractions, 5 /zg sialic acid was loaded per TLC lane. For sulfatide, 10/zg of lipid was used per TLC lane. After chromatography, the plates were air-dried and dipped in a solution of 0.1% polyisobutylmethacrylate (Polysciences) in hexane. Then the plates were overlayed with test sera diluted 1:500 in Tris-HClsaline at pH 7.4 containing 1% bovine serum albumin and incubated for 4 h at 4°C. After washing with cold phosphate-buffered saline (PBS), pH 7.4, thc plates were overlayed with either peroxidase-conjugated goat anti-human lgM (/z-chain specific) or peroxidase-conjugated goat anti-human IgG (y-chain specific) overnight at 4°C and washed with cold PBS as above. The plates were developed with o-phenylenediamine as substratc (Ilyas et al., 1990).

serum at various dilutions was added to the gel, mixed vigorously and incubated at 4°C overnight. The mixture was then diluted with 10 ml cold PBS and centrifuged at 1200 rpm for 10 min to remove the beads. Sera were also absorbed with unconjugated octyI-Sepharose beads as a negative control.

Results Antibodies" to M A G IgM paraproteins from two of 11 patients with NAIgMPP in the current study had previously been shown to react with MAG (llyas et al., 1984b; O'Shannessy et al., 1986). lgM from the other nine patients were also shown to react with MAG from human brain myelin on Western blots. Use of peroxidase-conjugated anti-human lambda and kappa chains suggested that it was the IgM paraprotein that was reacting with MAG. Fig. 1 shows binding of lgM on immunoblots with MAG from five patients with NAIgMPP. None of the healthy controls exhibited IgM binding to MAG on immunoblots, lgG from patients and healthy

...~:. o ~

.'::.!~..!::.

MAG Absorption of patient serum Sera from some patients with NAIgMPP were absorbed with purified sulfatide and SGPG using glycolipid-conjugated octyl-Sepharose CL-4B beads (Pharmacia, Piscataway, N J, USA) (Hirabayashi et al., 1983). Conjugates of sulfatide or SGPG and octyl-Sepharose CL-4B were prepared as follows. 1 mg of sulfatide or 0.5 mg of SGPG was dissolved in 4 ml of methanol/water, 1:1 (v/v) containing 0.1 M KCI. The glycolipid suspension was warmed at 70°C in a water bath, poured into the same volume of an octyl-Sepharose gel suspension, mixed vigorously, and then left at room temperature for 30 min with occasional shaking. The gel was then washed completely with PBS to remove the organic solvent, salts, and unadsorbed glycolipid. 500 ~1 of test

m

,,a~.

.:.r • .::.. :i

:.

I!! ;~::z.~: ::

.:i ~ .~.i

1

,:

:'..

2

3

4

5

Fig. 1. Binding of lgM paraproteins from five patients with NAIgMPP to MAG. Western blots of protein from human CNS myelin separated by SDS-PAGE (60 /.tg protein per lane) and immunostained with 1 : 50 dilution of test serum and peroxidase-conjugated anti-human lgM (tz chain specific).

88

TABLE 1 STRU(SYURES OF GLYCOSPHINGOLIPIDS Sulfatide SGPG SGLPG SGIcPG GPG McGPG

SO,-3GaI,(31 - l ( ' e r S O 4 - 3 G I c A / 3 1 - 3 G a l / 3 1 - 4 G I c N A c / 3 1 - 3 G a l / 3 1 - 4 G l c [ : l 1 - 1Cer SO4-3GIcA/3(1-3Gal/31-4GIcNAc)231-3Gal/31-4Gic/31- ICcr SO4-3Glc 3 1 - 3 G a l 3 1 - 4 G l c N A c / 3 1 - 3 G a l / 3 1 - 4 G i c / 3 1 - 1Cer G I c A / 3 1 - 3 G a l / 3 1 - 4 G l c N A c / 3 1 - 3 G a l / 3 1 - 4GIc,B l - 1Cer M e G I c A / 3 1 - 3 G a l / 3 1 - 4 G l c N A c 3 1 - 3 G a l / 3 1 - 4 G I c 31 - 1Cer

A b b r e v i a t i o n s : S G P G , sulfoglucuronyl p a r a g l o b o s i d e ; S G I , P G , sulfoglucuronyl lactosaminyl p a r a g l o b o s i d e ; SGIcPG, sulfoglucosyl p a r a g l o b o s i d e ; G P G , glucuronyl p a r a g l o b o s i d e ; M e G P G , m e t h y l e s t e r of G P G : Cer, c e r a m i d e ( N - a c y l s p h i n g o s i n e ) .

controls did munoblots.

not

react

with

MAG

on

A

im-

B

C

t

Antibodies to sulfoglucuronyl glycolipids SGPG and S G L P G (Table 1) share antigenic

determinants with MAG and have been shown to crossreact with anti-MAG IgM paraproteins from all previously examined patients with NAIgMPP (llyas et al., 1984b, 1986a, 1990; O'Shannessy et al., 1986). As expected, all 11 anti-MAG lgM paraproteins reacted strongly with both SGPG and SGLPG on TLC. None of thc 11 patients with NAIgMPP exhibited anti-SGPG lgG binding. Two healthy controls also exhibited weak binding to SGPG and SGLPG on TLC. Fig. 2

GM1 GDla j ~ GDlb GTlb 1

~G

~"

PG

2

1

2

1

Fig. 2. B i n d i n g of s e r u m l g M from two p a t i e n t s with N A I g M P P to sulfoglucuronyl glycolipids on TLC. L a n e s I c o n t a i n e d bovine brain gangliosides. L a n e s 2 c o n t a i n e d dog sciatic nerve acidic glycolipid fractions (5 p,g sialic acid). P a n e l A was s t a i n e d with orcinol. P a n e l s B and C were i m m u n o s t a i n e d with 1:500 dilutions of test s e r u m a n d p e r o x i d a s e - c o n j u g a t e d a n t i - h u m a n IgM ( # chain specific). Test sera w e r e o b t a i n e d from p a t i e n t A ( p a n e l B ) and B (panel C).

TABLE 2 SUMMARY Pa-

OF BINDING OF lgM FROM PATIENTS WITH NAIgMPP TO MAG AND GLYCOLIPIDS

Age/sex

tients

Clinical

MAG "

Sulfatide b

SGPG b

syndrome

A B C D

80F 53F 61F 69M

SM-D SM-D SM-D SM-D

+ + + +

E F-K

54M *

SM-D SM-D

+ +

>

: 50,000 : 100,000 : I(R)0 : 8(X) : 400 ( l g G ) : 2(X)

> > > >

1 : 100,000 1:100,000 1 : 64,000 1 : 64,000

> 1 : 64,000 > 1 : 64,000

Reactivity with M A G was d e m o n s t r a t e d by W e s t e r n blotting. b H i g h e s t s e r u m d i l u t i o n s that s h o w e d reactivity. ~ Reactivity t e s t e d by E L I S A . ( + ) B i n d i n g to the antigen. ( - ) No binding. ( * ) Five p a t i e n t s were male, one was female. M e a n age = 63 ( 5 3 - 7 3 ) years. n.d. = not done. SM-D = sensorimotor demyelinating neuropathy.

S G P G derivatives c GPG

SGIcPG

+ -

+ -

n.d.

+ n.d.

MeGPG

n.d.

89 1.8 1.6

//0""0~0

1.4

~

1.0

Z w

0.8.

~

0.6.

~

0.4-

0.0

%.

_

10 -1

1

'0

_

2

10

T_

3

10

4

,_

10

,

5

10-6

SERUM DILUTION

Fig. 3. Determinations of titers of anti-sulfatide IgM antibodies in five patients with NAIgMPP. Patient A, open circles; patient B, open squares; patient C, closed circles; patient D, closed squares; patient E, open diamonds.

shows binding of anti-MAG IgM paraproteins from patients A and B with SGPG and SGLPG on thin-layer chromatograms. IgM from both patients also bound weakly to a third antigen migrating between G D l b and G T l b (Fig. 2, arrow). The slowest migrating glycolipid antigen has been observed before (Ilyas et al., 1990) and its chromatographic mobility suggests that it is a higher analog of SGLPG. Determination of antibody titers using purified SGPG revealed that all 11 anti-MAG IgM paraproteins gave anti-SGPG IgM titers of more than 1:64,000 (Table 2). Two healthy controls gave anti-SGPG IgM titers of 1:100 and 1:400 and one gave anti-SGPG IgG titer of 1 : 200. Antibodies to sulfatide Five of 11 patients with NAIgMPP also had elevated levels of anti-sulfatide IgM antibodies (Fig. 3). One of the five patients with elevated levels of anti-sulfatide IgM also had elevated levels of anti-sulfatide IgG (patient D) (Table 2). One healthy control had an anti-sulfatide IgM titer of 1 : 100 and one had an anti-sulfatide IgG titer of 1 : 200. Patients A and B Patients A and B had anti-sulfatide IgM titers of > 1 : 50,000 as well as anti-SGPG IgM titers of

more than 1:50,000 (Table 2). This observation suggests that the a n t i - M A G / S G P G IgM paraproteins in patients A and B crossreacted with sulfatide. In order to determine a n t i - M A G / SGPG IgM paraprotein crossreactivity with sulfatide, patient sera were absorbed with sulfatide and SGPG-conjugated octyl-Sepharose beads. Absorption of the serum from these patients with suifatide-conjugated beads reduced the reactivity of IgM against both SGPG and sulfatide by more than 90%. Absorption of the serum from both patients with SGPG-conjugated beads also reduced the reactivity of IgM against SGPG and sulfatide by 74-82%. These observations support the suggestion that a n t i - M A G / S G P G IgM paraprotein was binding to both SGPG and sulfatide. Sulfatide-absorbed sera from both patients also did not react with MAG on immunoblots. Further, use of peroxidase-conjugated anti-human kappa and lambda light chains revealed that anti-SGPG and anti-sulfatide IgM in both patients had kappa light chains. Binding of IgM paraproteins from patients A and B to sulfatide was confirmed by TLC-immunostaining (Fig. 4). Patients A and B had sensorimotor demyelinating neuropathy (Table 2) and thus were clinically indistinguishable from other neuropathy pa-

B

A

C

SULF ..ir':~ • : ,:,,:, ::,

•

: A, : , ,

!;):,:..'.. ~':,, :,::, ,,, ,, , , ,

..'~:': ~,~.:i. • .;i " .

GM1 ' . . . . ':~ '~!: ':~ : GDlaGDlb~ ~: :~:~,,:;..~.~,:::..: .. G Tlb ~ . ~ i ; ~ ~;~; ~'

.~/.~,~ ~ ~ ..

"'~:~:~' : ~ " :": :

( ~ ~ ; ~~,, :-:':~i :~: !:~:~i:?

1

Z

3

4

Fig. 4. Thin-layer chromatograms showing the binding of IgM from two patients with N A I g M P P to sulfatide. Lane 1 contained bovine brain ganglioside standards. Lanes 2 - 4 contained 10 ~ g of purified bovine brain sulfatide. The plates were developed with c h l o r o f o r m / m e t h a n o l / 0 . 2 % KCI (50:40: 10; v / v / v ) . Panel A was stained with orcinol. Panels B and C were immunostained with 1:500 dilutions of test serum obtained from patient A and B, respectively and peroxidase-conjugated anti-human IgM ( ~ chain specific).

90

tients with anti-MAG IgM paraprotcins not reactive with sulfatidc. l'atients C - E Patients C - E had lower titcrs of anti-sulfatidc lgM (~< 1: 10{10) than anti-SGPG lgM ( > 1:64,(X)0) (Tablc 2). Absorption of scra from these three paticnts with sulfatide-conjugatcd beads complctcly abolishcd lgM reactivity against sulfatide but not against SGPG, an observation suggesting that sulfatidc and SGPG were recognized by different populations of IgM antibodies. Usc of peroxidase-conjugatcd anti-human light chain antibodies revealed that sulfatidc was rccognizcd by polyclonal lgM antibodies in all thrcc paticnts. Patients F - K IgM and lgG antibodies from six patients ( F K) cxhibitcd no reactivity with purified sulfatidc in EI,ISA (Table 2). Binding to derit'atil'es of" SGt'G Our data suggested that anti-MAG IgM paraproteins from patients A and B crossreacted with SGPG and sulfatide. In order to characterizc furthcr thc fine specificities of IgM paraproteins from these two patients, sera were tested for reactivity with derivatives of SGPG. The failure of lgM antibodies from patients A and B to rcact in ELISA experiments with desulfated derivatives of SGPG, GPG or M c G P G (Tablc 2) suggcsts that the sulfate group was a critical part of the cpitope for both lgM paraproteins. Although lgM paraprotein from patient B bound to sulfoglueosyl paragloboside (SGIcPG), IgM from patient A did not (Table 2).

Discussion

Previous studics have shown that anti-MAG IgM paraproteins from all patients with NAIgMPP also rcact with SGPG, SGLPG and some low M~ glycoproteins of the peripheral nervous system (PNS) (llyas et al., 1986a, 1990; O'Shannessy et al., 1986; Quarles el al., 1986). The current study is the first to demonstrate that antiM A G / S G P G lgM paraprotcins from some patients with NAIgMPP crossreact with sulfatide, a

major acidic glycolipid of human PNS myelin (Svcnncrholm and Frcdman. 199(I). Moreover. we confirmed the report by Pestronk ct al. (1991) that somc patients with anti-MAG IgM paraproreins and peripheral neuropathy also have antisulfatide lgM or IgG antibodies. Heterogcncity in the fine specificitics of antiMAG lgM paraprotcins from patients with NAIgMPP had bccn shown previously using purificd SGPG and its modified dcrivativcs (llyas ct al.. 1986a, 199(I). For cxamplc, some anti-MAG igM paraproteins can react with GPG, a desulfated dcrivativc of SGPG, while other paraproteins require a sulfatc group for binding to SGPG (llyas ct al., 1986a, 1990). Thus. the binding of two lgM anti-MAG paraprotcins with sulfatidc in the current study is not surprising. Although lgM paraproteins from patients A and B had similar reactivity to MAG, SGPG, SGLPG and sulfatidc, they exhibited a subtle diffcrcncc in fine specificity. IgM paraprotein from patient B reacted with SGIcPG, a dcrivativc of SGPG in which glucuronic acid had bccn rcduced to glucose. while lgM from patient A did not react. Thus, lgM from patient A was capable of binding to 3-sulfogalactosyl (SO~-3-Gal) and 3-sulfoglucuronyl (SO4-3-GIcA) moietics but not to 3sulfoglucosyl (SO4-3-Glc) moicty. In contrast, IgM from patient B had broader reactivity in that it bound to sulfoglucuronyl, sulfogalactosyl and sulfoglucosyl moictics in SGPG, sulfatide and SGIcPG, respectively (Tablc 2). It should bc noted, however, that the absolute requirement for sulfatc of somc anti-MAG paraprotcins for binding to SGPG does not necessarily mean that the a n t i - M A G / S G P G antibodies will crossrcact with sulfatide. For example, a mouse monoclonal antibody, HNK-1, binds to MAG and othcr related glyeoconjugates and also requires sulfate for binding to SGPG, but does not erossreact with sulfatide (Ilyas et al., 1990). The present study providcs additional support for prcvious dcmonstrations that anti-MAG lgM paraproteins from patients with NAIgMPP differ in their fine specificitics (llyas et al., 1986a, 199(1). Anti-sulfatide lgM and lgG antibodies have been reported in some patients with GuillainBarrd syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (C1DP) as well as

91

in some healthy and disease controls (Fredman et ai., 1991; llyas et al., 1991). High titer anti-sulfatide and anti-MAG antibodies have also been reported in some patients with sensory or sensorimotor peripheral neuropathies (Pestronk et al., 1991). In the latter study, one of five patients with neuropathy and anti-MAG IgM paraprotein also had IgM to sulfatide but it is not clear from the report if anti-MAG IgM paraprotein from this patient crossrcacted with sulfatide. The role of anti-MAG/SGPG paraproteins in the pathogenesis of neuropathy remains unclear (Ouarles, 1989). The strongest evidence for the possible pathogenic role for anti-MAG/SGPG IgM paraproteins has come from the production of segmental demyelination in chickens by systemic transfusion of anti-MAG IgM paraproteins (Tatum, 1989). Anti-MAG IgM paraprotein was found to localize in the outer lamellae of myelin with widening of the intermediate line, an observation characteristic of paraproteinemic neuropathy (Vital ct al., 1989). Moreover, injections of serum from NAIgMPP patients with antiM A G / S G P G paraproteins and complement into feline peripheral nerve produced extensive myelin destruction within 2-9 days of injection (Hays et al., 1987; Willison et al., 1988). Although sensitization of rabbits with purified SGPG produced peripheral nerve dysfunction in conjuction with the development of elevated levels of anti-SGPG antibodies (Kohriyama et al., 1988), similar nerve dysfunction was not observed in rats sensitized with SGPG (Maeda ct al., 1991). In conclusion, two of 11 anti-MAG IgM paraproteins from patients with NAIgMPP have been shown to crossreact strongly with sulfatide, a major acidic glycolipid of human peripheral nerve myelin. Thus, in addition to MAG, SGPG, SGLPG and a number of other glycoproteins, suifatide must also be considered as a potential target antigen to be involved in the pathogenesis of neuropathy in some patients with NAIgMPP. Moreover, determination of effects, if any, of suifatide sensitization in experimental animals and the effects of anti-MAG/sulfatide antibodies in intraneural injection models and in myelinatcd PNS cell cultures might be of use in understanding the pathogenesis of neuropathy in some patients with NAIgMPP.

Acknowledgements This work was supported in part by National Multiple Sclerosis Society Grants RG 2064-A-1 (A.A.I.) and RGI636-A-I (F.A.M.), and by the Department of Veterans Affairs (F.A.M.). We thank Z.W. Chen for expert technical assistance.

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