Delayed type skin response to myelin basic protein in chronic relapsing experimental allergic encephalomyelitis

Journal of Neuroimmunolofy, 5 (1983) 295-304

295

Elsevier JNI 00161

]:)e]ayed Type Skin Response to Myelh~ Basic Protein in Chronic Relapsing Experimental Allergic Encephalomyelitis Takeshi Tabira ~, Yasuto Itoyama 2, Yo~higo~o K'aroiwa 2, Chiharu Kubo 3 and Yoshitaka Nagai 4 I National Centerfor Nervous, Mental and Muscular D,sorders, 2620 Ogawa-Hisasbi- Machi, Kodaira, Tokyo 187; z Department of Neurolosy, Neurolo$icalInstituteand "~Department of Microbiolosy,Facultyof Medicine, Kyushu University,Fukuoka City 812; and 4 Department of Biochemistry,Facultyof Medicine, University.of Tokyo, Tokyo (Japan) (Received 19 October, 1982)

(Revised,received11 November,1982and 21 June, 1983) (Accepted11 July,1983)

Summary The skin response to myelin basic protein (MBP) was studied in chronic relapsing experimental allergic encephalomyelitis (EAE) using strain 13 guinea pigs. The delayed, type skin response showed a monophasic curve; it ~adually increau~ after immunization, reached maximum levels around 80 days post-immunization, and deoreased thereafter. Relapses were more frequent while it was at high lewis although it did not correlate directly in individuals with the clinical stage. The response was also high in MBP-immunized animals which had recovereA from acute EAE. Our results suggest that delayed type hypersensitivity to MBP is involved but is not sufficient by itself to cause relapsing EAE. Key words: Chronic relapsing experimental allergic encephalomyelitis - Guinea pigs Myelin basic protein - Skin response

~mpmulez~e: Dr. TakeghiTab~ra,NationalCenterfor Nervo~as,Mentaland MuscularDimcden 2620 Olawa.Hif,ashi-Maehi,Kodah'aCity,Tokyo187, Japan. 0165-5~/83/'$03.00 © 1983 ElsevierScience Publishers B.V.

296 Introduction Acute experimental allergic eneephalomyefitis (EAE) is produced in mature animals by inoculatic,n with myefin basic protein (MBP) in Fretmd's complete adjuvant (FCA). It is acute monophasic clinically and the pathogenefic mechani~;mis based c:1 T-cell media~.d hypersensitivity to MBP of central nervout; system (CN~,3) myelin (Paterson 1960; Ortiz-Ortiz and Weigle 1976; l~m-Nun et al. 1981). Delayed type h3q~ersensitivity(DTH) to MBP, expressed by skin test; to MBP (Shaw et al. 1965; Lsak and Zwe~man 1974), has been shown to correla~,ewith clinical activity. A ch~'onic form of EAE can be r,roduced by immmfizing juvenile strain 13 guinea pigs witi~ whole CNS antigens in FCA (Stone and Leraer 1965; Stone et al. 1969). It is characterized by wi~e-spread demyelination (Raine et al. 1974) and spontaneously remir,sio~is and exacerbations (Snyder et al. 1975; Wisniewski and Keith 1977). Its causativ~ antigens ac.d mode of immune reactions are not yet understood. Since imraune reactions to MBP have not been well studied in chronic relapsing EAE, we have exlmined the skin response to MBP in this type of EAE.

Materials and Metho~ One hundred and thirteen juvenile and 8 adult strain 13 guinea pigs were used in this experiment, 108 male and 13 female. Ninety-six juvenile animals at 2-4 weeks old (body weight 210 :t: 42 g) were immunized subcutaneously in the dorsum of each hind foot with 0.1 ml of an emulsion containing 0.05 ml of a 50~ suspension of isologous spinal cerd emulsified with 0.05 mi FCA incorporating 1 mg of killed dried Mycobacterium tuberculosis (Difco). Seventeen juvenile animals were similarly immunized in each hind foot with 0.1 ml of an emulsion containing 10 pg MBP and FCA with 1 mg M. tuberculosis. MBP was purified from bovine spinal cord as described previously (Nagai et al. 1978). Animals were weighed and evaluated clinically at least twice a week except for paralyzed animals on which closer examinations were performed. Clinical scores were as follows; poor righting reflex (0.5), absent righting reflex with mild paraparesis (1.0), moderate paraparesis (1.5), complete paraplegia (2.0), quadriplegia (3.0) and death (4.0). The disease activity was assessed in 50 consecutive males by counting the number of clinical bouts per a~timal in each 20 days after inm~unization. When the clinical course was progressive follow~ng a preceding level c,f paralysis which lasted for more than a week, it was counted as a new bout. Death was not classed as a boat since the cause of death was b.t~erogeneous. For pathological .qudies, v|oribund animals were perfused with glutaraldehyde, ~,nd the specimens were embedded in epoxy resin and examined histologicalilywith conventional light raicros~opy. Dead animals were fixed in 10~ formalin, embedded in paraffin and stain,~ with LuJtol fast blue-hematoxylin and emin. Skin tests were performed b,.f injecting intradermally with 50 ~tg MBP in 0.1 eal saline on a shaved area of the flmk. Our preliminary study u.~ng a small group of anim~l~ proved that erythema ~md induration were well con~ated at 24 arid 48 h

after chaUenge, Since induration wz~ so slight as to resider accurate measurement difficult, only the diameter of the erythema was measured. Measurements at 6 h were done i~ some animals. Groups of animals were tested on 0, 7,11, 20, 30, 40, 50, 60,83, 130~,180, 215, and 300 days post-innnnniT~tion (P.I.). Longitudinal studies were done in some animals, The area of induration and erythema was excised at 48 h in some animals tested on 50 ant2 13)days P.l. Theti~.ue was fixed in 10~ formalin, embedded in paraffin and stained wide. hem~ toxylin and cosin. Skin tests with 0.1 ml saline and 5 ~tg PPD in 0.1 ml saline were ~one in small groups of animals. The lymphopro~,iferative response t o Mi~P was also tested in small groups of animals. The method was modified from that described before (Ka~kow et al. 1981). Briefly, lymphocytes separated fror,~ 5 ml of blood by a FicoU-Com-ay method were suspended in the medium at a concentration of 5 × 105 ceils/ml in RPMI 1640 containing 10~ heat-inacti'~ated fetal calf serum, 5 x l0 -s M 2-mercaptoethanol, 100 /~g/ml streptomychl and 100 U / m l penicillin with or without 16.7/tg/ml of MBP. 0.2 ml of the cell suspension (1 × 105 cells/well) was cultured in a microplate for 4 days. During the last 18 h of incubation, [3H]thymidine (0.2 ~tCi/well) was added. Stimulation indices (S.I.) were obtained f~'om 8 normal strain 13 guinea pigs, and from 14 animals immunized with spinal cord in FCA and tested on 40 (n ~ 5), 80 (n = 5) and 120 days P.L (n -- 4).

Remits Seventy percent of animals immunized with spinal cord developed a chronic relapsing form of EAE, 24% of animals died during acute stage, and the remainder did not show a,.~y evidence of clinical illness. Eighty percent of animals which developed chronic relapsing EAE had acute transient paralysis 10-20 days after immunization. The first relapse appeared as early as 40 days P.I.; mean onset of the first relapse was 76 days P.I. "Ele disease activity (No. of bouts/animal/each 20 days after immunization) was hit~+hest(0.78) in the first period (0-19 days P.l.), and TABLE 1 DISEASEACTIVITYIN STRAIN13 GUINEA PIGS IMMUNIZEDWlTlt SPINALCORD IN FCA Days P.l, O19

2039

4059

6079

8099

100- 1 2 0 - 140- 1 6 0 - 180- 200119 139 159 179 199 219

:;0

42

37

37

35

31

26

21

20

19

13

39

2

10

22

17

20

13

$

9

7

4

0.37

0.31

II

of boots

No.

Disease activity"

0.78

0.05

0.27

0.59

0.49

0.65

0.50

0.38 0.45

• Diseaseactivityis expreuedby No. of bouts/animal/each 20 days after in~nunization.

298 lowest (0.05) in the second period (20-39 days P.L). It gradually increased to the 6th |~er~od (100-119 days P.I.) (0.65) and then decreased thereafter (Table 1)~ The m~.at time of death during the chronic stage was 155 days P.I, Histologically there ~rere inflanmtatory lesions without significant demyelination in the CNS of animals dyi~g du~ing acute stage. Animals with chronic r e l a p ~ g EAE slunved large demyelinating plaques in the Gpinal cord, optic n e r v e s a n d periventricular white matter of the cerebrum. A mixture of fresh and old lesions in each individual was charactmistic. Among the animals immunized wi~h MBP, 5 died of acute paralysis mid 9 developed transient acute paralysis. N~ne of the surviving animals showed a significant relapse or a demyelinating plaque. The mean sizes of the erythema (sum of the longest and shortest diameters) in animals having received spinal cord in FCA tested with saline on 130 days P.l., were 0.8 + 1.9 (SD) m m at 6 h, 0.6 + 1.3 m m at 24 h and 1.1 + 1.6 m m at 48 h. The si~; of the ery~;hema in the animals tested with PPD on 40 days P.I. was 16.8 4-1.0 m m ~t 6

!

!

•

o

o

~

.

I0o

ti o

~0 . . . .

t00 . . . .

,SO . . . .

=e0 Days~ Lwmmlm~l*~ Fig. 1. Skin responset~ MBP evaluatedet 2.4h ~ter chaJlenge.The ~ responsewas nqpttivein a~imals on ]1 days P.l., alth-.,ughmost had acute paralyuis.TI~. ~ respome increm~ up Io 13 days P.I. an~' thmt decreased. No direct correlationbetween t[~ ~;~ of er~themaand c"imical~ ~,nm~ in individtmls. However,relapseswere more frequent while it wu at high levd; O: male.guim~ ~ without paraly~; @: maleswith paralysis; 4: femaleswitlu~t pataly~; a: femalts with I0al~J~;. ~u~ ~ u m t m ~ ± l SD.

299 TABLE 2 COMPARISOI~ O F DELAYED SKIN RESIK)NSE ( ~ h) BETWEEN ANIMAt.S WITH A N D WITHOUT PARALYSIS Days P.l. 0

]I

20

8

10

50

60

83

105

130

180

210

0

6

2

5

3

2

3

n

0 Para~s~ + Mean s i ~ (nun) of (SD)

2

-

4.4

9.0

9.5

-

9.8

12.5

13.1

8.0

6.0

4.0

(-)

(2.0)

(2.6)

(2.1)

(-)

(2.7)

(2.1)

(2.0)

(5.2)

(1A)

(4.0)

1

0

8

6

4

2

0

5

4

2

-

10.6

12.3

12.5

14.0

-

12.5

9.5

5.5

(-)

(0.8)

(5.4)

(3.4)

(1.4)

(-)

(3.0)

(4.8)

(0)

n 5 ,Pm,'+~,s+s-,,: (Mean slze (nun) of

3.4

4.0

Etythem, (SD)

(2.3)

+' 95

,iL r - ~

(-)

t

I'

I0.~

12

,

,

II

10

I 4

il

;

,--,-,t 6.5

t 12

2"o

~o

,-,

io

;o

,~ 'Ii0..

~Xo

~;o

days a f t e r i m m u n i z a t i o n

Fis. 2. L o ~ t e d i n a l m~.y of delayed skin response to MBP in chronic rebpsinS EAE. ~ tats wq~ rep~t4xl mole than 3 tir~s in $ animals with re~ps~n s ~--~AF.Arrows and fik'm'~ show f i m ~ 5 ot" ~ and s~e of e~'ythema meuured at 24 h ~ t e r challense. The size of erythema is unreltted to+the ~ rose.

300

? ~,0

n 80

i~0 Days Days .~fter fn~r,tmi~.atton

Fi E. 3. Longitudinal study of delayed skin response (24 h) to MBP. The skin respo~,~: vadually increased up to 80-100 days P.I. and then de,;eased, inespectivc of clinical stage. Q: ~nimal~ wi,hout paralysis; 81: anim.'tls with paralysis.

h, 23.3 + 4.9 mm at 24 h and 15.8 + 9.3 mm at 48 h, in wlIdc:l the skin was heavily indurated and swollen. Skin tests in animals immunizeA with spinal cord in FCA measured at 6 h were as follows; mean sizes of erythema were 8.1 + 2.7 mm (20 d.), 17.0 ± 2.0 mm (50 d.), 15.3 + 1.3 mm (83 d,), 13.2 + 3.4 mm (!05 d.), 3.3 + 3.8 mm (130 d.), 2.3 + 3,7 mm (180 d.) and 4.4 + 4.6 mm (215 d.). No clinical correlation was seen with the size of the erythema. Skin tests measured at 24 h are illustrated in Fig. 1. Generally, the sizes ,of th~ erythema and indtlration were much smaller than those produced with PPD. The nlargin of erythema was sharp, and the skin was indurated sfightly at this stage. The diameter was 5.8 ± 2.6 mm in aninuds 7 days P.L, before the onset of acut~ paralysis. It decreased ;lightly to 4.4 ± 1.9 mm in aninuds 11 days P.I., when most o~ the animals had acute paralysis. Then, it gradually increased and reached a maxin mum (13.3 ± 1.7 ram) on 83 da):; P.L, and decreased i~radually to 4.6 ± 2.9 nan o~ 2]5 days P.l. Mos~. of the animals were rlinically in remission on 40 and 50 days P.I., but ~he size of the r,~acfion kept increasing. As shown in Fig. 1, no correlatiot,, bet~!~en the size of the erythema m~d clinical stage was seen in individuals. Wh~m th~

3oi

<

DJ~~e

IO

/ r

0.2

m

o

.

.

_

.

~

.

:do

.

.

.

.

i!

.

150

200

DAYS

AFTER

IIIRUIIIZAT~O~

Fig. 4. Delayed skin response and disease ~tctivity.The disease activity (bar graph) is expressed by 1~o.of bouts/animal/each 20 days P.I. During the chronic stage, relapses are more frequent whik. DTH to MBP is at high levels.The dissociation during the acute st~e is discussed in the text. e: skin response evaluated at 24 h; O: skin response evaluated at 48 h.

size of file reaction was compa~'ed between groups of animals with and without paralysis, it was relatively smaller in animals with paralysis, but the difference was not significant (Table 2). Figure 1 shows a longitudinal study evaluated at 24 h in 5 animals. The results of repeated tests using more animals are illustrated in F:g. 3. From these, it was obvious that ttte skin response was unrelated to the c "lmical sta4ge. The erythema evaluated at 48 ~t after challenge was relatively smaller; it increased gradually in size, r e ~ h e d m a x i m u m on 83 days after a small dip on 60 days, end decreased Ihereafter. Excised skin showed infiltration of lymphocytes to various degrees with a few polymorphonuclear le,kocytes. The time courses of the skin responses at 24 and 48 h are illustrated ~together with disease activity in Fig. 4. In animals immunized with MBP in FCA, skin tests were performed on 40 (n = 7) and 300 days P.I. (n - 2). The mean sizes of the erythema m the former animals were 15.3 4- 2.0 m m at 6 h, 13.1 4- 0.7 m m at 24 h and 10.5 4- I.:2 m m at 48 h. It was nil in the latter animals. The stimulation index of peripheral lymphocytes was 0.78 + 0.29 for normal adult controls (n -- 8). In animals immunized with spinal cord, the i n d i e s were 1.25 + 0.38 on 40 days P.l. (n - 5, P < 0.05), 1.09 + (3.20 on 80 d a y s (n = 5, P < 0.05) and 0.91 -t- 0.08 on 120 days P.l. (n =, 4, N.S.). I~. did not correlate with clinical state.

302

Discussio~

In the present study we have er,amined the skin response to MBP in animals with chronic relapsing EAE, The response measured at 24 and 48 h after ehalleage was associated with slight induration of the skin. Histological observation cor~firmed ~l~lt the reaction was of the delayed type hypersensitivity {DTH) form. The DTH was gradually increased up to 80 days after immunization and then decreased gradually. It did not correlate with clinical activity but clinical relapses were more frequent while it was at high levels during the chronic phase, We have noticed several differ~mces in the skin response to MBP between chronic relapsing and acute EAE. In acttte EAE in guinea pigs, delayed skin reactions to whole nervous antigens (Waksman 1959) or to MBP (Shaw et al. 1965) reached a maximum intensity shortly before the onset of neurologic sig,'~ and then decreased as the animals began to show clinical evidence of EAE. In our experiment using juvenile guinea pigs, the delayed skin reaction to MBP was still low when animals began to show clinical and histological evidence of acute EAE on 11 days P.L It! was kept increasing up to 83 days P.I., although animals recovered from the acute stage at about 40 days P.l. Furthennore in acute EAE, the size of the skin reactions correla,'ed well with the subsequent time of onset and histological severity of EAE (Shaw et al. 1965). In chronic relapsing EAE, we saw no correlation between size of erythema and clinical activity in individual animals. However, when the skin response was considered in its entire course, it seemed to he a monophasic phenomenon; viz. it gradually increased, reached a maximum around 80 days P.I., and then gradually decreased. It is interesting to note that c"hnical relapses were more frequent while the skin reactivity was at high levels during the chronic phase. The relapse rate decreased gradually according to declining skin reactivity. These phenomena implicate that DTH to MBP has some causative role in chronic relapsing EAE. The decline of the skin reactivity seems to be related to decrease of antigen remaining in the local immunizing loci (Tabira et aL 1982). The role of MBP in chronic relapsing EAE is not yet known. We have confirmed that MBP alone in FCA is insufficient in causing chronic relapsing EAE (Keith and McDermott 1980; WisniewsH et al. 1980). Administration of MBP in incomplete adjuvant after inoculation is kr~own to suppress relapsing EAE (Raine et al. 1978). Pretreatment with a synthetk pol.~,-ptide, Cop 1, was claimed to he effective i~n few animals (Keith et al. 1979). MBP with myelin lipids produced signifwa~tt demyelination irrespective of whetl,.er animals relapsed or not (Madrid et al. 198~). "i o ,'late, chronic relapsiitg EAE has not been induced with significant demye~tation without MBP. Thus, MBP probably has an important role. in causing relapsing EAE. Our data on the stin tesponse have provided additional evidence that ldBP is involved in chronic ~'elapsing EAE as an antigen. Two dissociations were observed between skin response and clinical ~activi~.y. First. at an early stage on day 11 P.l., animals showed evidence of acute EkE clinically and pathologically, but the skin response was low. This phenomenon m i ~ t be explained by the observ~ttion that newborn or young guinea pigs can develop DTH: but do not manifest it i~ the skin (Freund 1927; Salvin et al. 1962). S,~co,idlv,

303

a reversal was seen after recovery from acute EAE. M o s t of the a v ~ a a h were clinically in remission, yet, D T H t o M B P was high. C o m p a r i ~ m of these d a t a m a y n o t b e a p p r o p r i a t e d u e to the difference in a m o u n t of M B P useA in i m m u n i z a t i o n . However, !t similar p h e n o m e n o n was found in M B P - i m m u n i z e d a n i m a l s which ) ~ d recovered f r o m acu~.e EAE. These animals showed highly elevated D T H t o M B P b u t never relapsed. Thi~ m i g h t sugges t that in o r d e r to develop chronic r e l a p s i n g E A E a n i m m u n e r e s p o n ~ to a second myelir, a n t i g e a is necessary, especially in the h u m o r a l response, a s suggested previously ( l ~ t e a n d T r a u g o t t 1982). Alternatively, D T H ~'.o non-encephalitoger:ic regions o f M B P could explain the dissociation ( H ~ J ~ m 1976). F u r t h e r studies are necessary to find a factor related directly to a relapse o f the disease.

References Ben-Nun, A., H, Wekerle and I.R. Cohen, The rapid isolation c)f cionable antigen-specific T-lymphocyte lines capable of mediating autoimmune encephalomyefitis, Europ. J. Immanol., 11 (1981) 195-199. Frennd, J., The influence of age on the skin sensitiveness of tuberculous guinea pigs, J. lmmunol., 13 (1927) 285-288. Hashim, G.A., R.D. Sherpe, E.F. Carvalho and L.E. Stevens, Suppression and reversal of allergic encephalomyelitis in guinea pigs with a non-encephalitogenic analogue of the tryptophan region of the myelin basic protein, J. lmmunol., 116 (1976) 126-130. Kasckow, J.W., T. Tabira, Y. ltoyama, Y. Kuroiwa, A. Takenaka and Y. Nagai, Experimental allergic encephalomyelitis - - Suppression with SD-170, a new synthetic drug, Jap. J. Exp. Med.. 51 (1981) 293-298. ;~eith, A.B. and J.R. McDermott, Optimum conditions for inducing chronic relapsing experimenL'~J allergic encephalomyelitis in guinea pigs, J. Neurol. Sci., 46 (1980) 353-364. Keith, A.B., R. Arnon, D. Teitelbaum, E.A. Caspary. and H.M. Wisniewski, The effect of Cop 1, a synthetic polypeptide, on chronic relapsing experin~ental allergic encephalomyelitis in guinea pigs, .T. Neezol. Sci., 42 (1979) 267-274. Liutk, R.P. and B. Zwelman, In vitro an~ in vivo immune responses to homologous myelin basic prc~ein in experimental allergic encephalomyelitis, Cell. lmmunol., 11 (7.974) 212-220. Madrid, R.E., H.M. Wisniewski, K. lqb~d, R.K. Pullarkat and H. Lassmann, Relapsing experhr ~:nUd allergic encephalomyelitis induced with isolated myelin and wi~h myelin basic protein plus myelin lipids, J. Neurol. Sci., 50 (1981) 399-411. Nggal, Y,, K. Akiyanm, K. Suzuki, S. Kotani, S. Watanabe, T. Shimono, T. Shiba, S. Kusumoto, F. lkuta and S. Takeda, Minimum structural requirements for encephalitogen and adjuvant in the induction of experimental allergic encephalomyelitis, Cell. lmmunol., 35 (1978) 158-167. Ortiz-Ortiz, L. and W.O. Weigle, Cellular events in the induction of experimental allergic enc~ halo~ye~ tis in mrs, J. Exp. Med., 144 (1976) 604-616. Paterson, P.Y., Experimental allergic encephalomyelitis in rats by means of lymph node ce~ls, J. Exp. Med., 111 (1960) 119-136. ~atine, C.S. an~l U. Traugott, The pathogenesis and therapy of multiple sclerosis is based upon the reqnireuumt of a combination of myelin antigens for autoimmune 4emyelination, J. blearohnmuno~ 2 (1982) 83-91. Ralne, C.S., D.H. Snyder, M.P. Valsamis and S.H. Stone, Chronic experimental allergic enceph tlomyelitis in in'3red guinea pigs, an ultrastructur,al study, Lab. Invest., 31 0974) 369-380. aalne, C.S., U. 'rraugott and S.H. Stone, Suppression .of chronic aller~c encephalomyelitis - R,~evance to m~tip~ ~ e r o ~ S c ~ 201 (1978) 445-448. Salvin, S.B., M,~. Gregg and R.F. Smith, Hypersensitivity in new born guinea pigs, J. Exp. Med., 115 (1962) 707-7~.

304 Shaw, C.-M., E.C. Alvord, Jr., J. Kalm ~ d M.W. Kits, ,~rr4~afion of ~ t a l ~ ane~halomyelitis with delayed-t~pe skin se~tsitivityto slp~if~ h o ~ w ~mmpimiitostm,Ann. N.I". Acaui. Sci., 122 (196S) 318-331. Snyder, D.H., M.P. Vahamls, S.H. Stone ~nd C.S. ~ Progressive demyelination and reparative. phenomena in chronic cxp~rimml~! allerBic¢ n ~ ~ t i s , J. N~mol~lth. Exp. bTcn~roL,34 (1975) 209-221. Stone, S.H. and E.M. Lerner, !i, Chronk di~mnthtated alkrgic a~ephalomyelitis in $t~nea pits, ;~nn. N.Y. Ac.ad. Sci., t22 (1965) 227-241o Stone, S.H., E.M. Lerner and £H. Goode, Jr., Acute and ¢hron;¢ auto~.mmune~ y e l i t i s ~ AS,c, strain, and sex d~.l~endeney,Pro¢. See. Exp. Biol. Med., 132 (1.069) 341-344. Tabira, T., Y. Itoyama and Y. Kuroiwa, Chronic r e l a p ~ experimental 1tiler'Bita n c ~ e i ! t i s in strain 13 suinea pip - - Presetwe of myelin basic pr,ztein in the local tissue IonS after the immunization, Jap. J. Exp. Med., 52 (1982) 131-.137. Waksman, B.H., Evidence favorinS ~ r ~ m ~ ~ ~ ~ U~y~S ~ ~ ~dlergicencephalomyelitis - - An expet'imeatel model of multiple w.lerosis. In: M~W. ~ .and E.C. Alvord, Jr. (Eds.), Allergic Encephalomyelitis, Charles C. Thomas, .f~gfleld, IL, 1959, pp. 419-443. Wi~niewski, H.M. and A.B. Keith, Chronic relapsing e x ~ t a l ~ enet,phelom3zlitis ~ An experimental model of multiple sclerosis, Ann. NeuroL, 1 (1977) 144-148. Wisniewski, H.M., R.E. Madrid, H. La~matm, D.S. Deshmukh and K. lqbal, Search for antiltetgs) and immunological mechanisms responsib.~¢for extensive demyelituttion and relapse~ in experim'.,ntal allergic encephalomyeliti~ (EAE). In: A. Boese (Ed.), Search for the Cause of Multiple Sclero~$ and Other Chronic Diseases of the Central Nervous System, Verht$ Chemic, Weinheim, 1980, pp. $~-95.