close

Вход

Забыли?

вход по аккаунту

?

Chronic experimental allergic encephalomyelitis produced by bovine proteolipid apoprotein Immunological studies in rabbits.

код для вставкиСкачать
Chronic Experimental Allergc
Encephalomyelitis Produced by Bovine
Proteolipid Apoprotein: Immunological
Studies in Rabbits
Franca Cambi, MD,"? Marjorie B. Lees, PhD,*$ Rosemarie M. Williams, DVM,$
and Wendy B. Macklin, PhD*$
A chronic experimental allergic encephalomyelitis (EAE) has been produced in rabbits sensitized with bovine white
matter proteolipid apoprotein. Eleven of 12 animals developed clinical disease one to six months after immunization
with a single dose of the apoprotein. The clinical course was characterized by posterior ataxia, flaccid paralysis
progressing to spastic paralysis, and incontinence. Spontaneous relapses and remissions were observed in 3 rabbits.
Histologically, acute and chronic encephalomyelitis accompanied by primary demyelination were observed. Serum
antibody production, assayed by both an enzyme-linked immunosorbent assay and an electroblot procedure, did not
correlate with either the clinical course or the histopathological findings. Delayed hypersensitivity to proteolipid
apoprotein was observed in all rabbits prior to the onset of clinical signs. The data suggest that lymphocytes specifically
sensitized to the proteolipid may be involved in the pathogenesis of the demyelination in chronic EAE.
Cambi F, Lees MB, Williams RM, Macklin WB: Chronic experimental allergic encephalomyelitis produced
by bovine proteolipid apoprotein: immunological studies in rabbits. Ann Neurol 13:303-308, 1983
Chronic experimental allergic encephalomyelitis (EAE)
is a useful model for understanding the pathophysiology of demyelination and the immune mechanisms involved [ l l , 29, 331. Currently, it provides tht. best
available model for both the clinical and the histopathological aspects of multiple sclerosis. The chronic
experimental disease is obtained in either a progressive
or a relapsing form after immunization of young guinea
pigs with whole neural tissue, and is characterized by
both inflammation and demyelination. Myelin basic
protein (MBP), the major encephalitogenic protein of
the central nervous system (CNS), does not account
for all the features of neuroautoimmune diseases; MBP
alone induces an inflammatory encephalitis with little
or no demyelination, whereas demyelination is the major characteristic of the chronic forms of EAE as well as
of multiple sclerosis {S, 251. It can be inferred, therefore, that other neuroantigens, acting alone or in cooperation with MBP, may be important in eliciting or
maintaining demyelination. The CNS contains a second major myelin protein, proteolipid protein C6, 161,
which occurs in amounts as great as or greater than
MBP 1141. Unlike MBP, which is an extrinsic mem-
brane protein localized at the cytoplasmic surface, proteolipid protein is an intrinsic protein that mainly is
embedded within the lipid bilayer, where it is undoubtedly involved in maintaining the integrity of the myelin sheath. However, a portion of the proteolipid protein is exposed at the external surface of the myelin
lamellae, where it is in contact with the extracellular
space (21.
In a previous paper [32 J we reported the production
of a delayed-onset, chronic progressive EAE in rabbits
injected with bovine proteolipid apoprotein (APL).
The histopathological picture was consistent with primary demyelination. In the present paper we show that
bovine APL is capable of inducing either a progressive
or a relapsing chronic demyelinating disorder in rabbits. The immunological features of this disease have
been examined, and we report data for both humoral
and cellular responses to the two myelin proteins, APL
and MBP.
From the 'Biochemistry Department, Eunice Kennedy Shriver Center for Mental Retardation, Maltham, MA 02254, the Departnients
of iNeurology and tBiologicd Chemistry, Harvard Medical School,
Boston, MA 02115, and the $Department of Pathology, Tufts University School of Veterinary Medicine, Boston, MA 0 2 1 11.
Received May 1I , 1982, and in revised form July 19. Accepted for
publication July 21, 1982.
Materials and Methods
APL was prepared by dialysis of a total lipid extract of bovine
white matter against neutral and acidified chloroform-
Address reprint requests
Dr Cambi,
Trapel" Rd,
MA 02254,
E. K. Shriver
2oo
303
methanol [15]. The apoprotein was converted to a watersoluble form by the method of Sherman and Folch-Pi [27).
Possible contamination of the preparation with trace amounts
of MBP was assessed by radioimmunoassay [ 4 ] and by an
electroblot procedure 13 I}. The former was found to be unsuitable for quantitative determination of MBP because hydrophobic compounds such as proteolipid protein interfere
with the assay [20].Electroblot analysis was carried out by Dr
John H. Carson using a high-titer MBP antiserum under conditions permitting the detection of 2 ng of MBP. By this
analysis no MBP was detected in a 25 )*g sample of APL;
thus, if any MBP is present, it is at a level less than 0.0087f.
Sixteen 2-month-old female New Zealand/White rabbits
(ARI Breeding Labs, Avon, MA) were maintained in individual cages in an environmentally controlled room. They were
fed a commercial pelleted diet with free access to food and
water. Twelve rabbits were immunized with a mixture consisting of 1 mg of APL in 0.5 ml of water emulsified with an
equal volume of complete Freund’s adjuvant (Difco, Detroit,
MI). The emulsion was administered subcutaneously in several sites along the back. Four control rabbits were injected
with a comparable emulsion, with water substituted for the
APL.
The animals were observed daily, weighed once a week,
and bled once a month from the ear vein for the immunological studies. Neurological examination was carried o u t daily
once clinical signs appeared. At different stages of disease,
individual animals were perfused by intracardiac catheterization with a mixture of 4% paraformaldehyde and 3 2 glutaraldehyde in 0.1 M phosphate buffer for histological studies.
Routine sections were taken from brain, brainstem, and
spinal cord, and additional samples were taken at the level of
any gross lesion. Dorsal root ganglia were included with the
cervical sections. Slides were stained routinely with Luxol fast
blue and counterstained with hematoxylin and eosin.
Antibody titers against APL and MBP were quantitated in
serum samples using an appropriate enzyme-linked immunosorbent assay (ELISA) for each of the proteins. Antibodies
against APL were determined by an ELISA as described by
Macklin and co-workers [l91. Anti-MBP antibodies were
determined by a modification of the method of Sires and
colleagues [28}. To reduce nonspecific binding, 1 9 bovine
serum albumin, fraction V (Sigma Chemical Co., St. Louis,
MO), was added to the antirabbit horseradish peroxidase
conjugated immunoglobulin G (IgG) (Cappel Laboratories,
Cochranville, PA), which was used at a dilution of 1 : 1,000.
In both assays, H r O r and o-phenylenediamine (Eastman
Kodak, Rochester, NY) in 0.01 M citrate-phosphate buffer,
p H 6, were used as the substrate [19]. The same serum
samples were also screened by an electroblot procedure [ 181.
Sodium dodecyl sulfate (SDS) polyacrylamide gels of myelin
5 w , Millipore
were transferred to nitrocellulose paper (0.4
Corp, Bedford, MA) in a Hoefer Transphor apparatus, as
described elsewhere [IS]. To increase transfer of protein,
two layers of SDS-soaked filter paper were added to the
cathodal side of the sandwich.
To detect cell-mediated immunity to APL and MBP, delayed hypersensitivity was assessed by skin testing. The basic
procedure was to inject 0.5 ml of solution containing 300 pg
.
of antigen in water intradermally in a shaved area in each
flank. The injection site was inspected 24,48, and 72 hours
304 Annals of Neurology
Vol 13 N o 3
March 1983
later. The cross-sectiondl diameter of erythema and the degree of induration were recorded.
Results
C l i n i d Objewaations
The onset of neurological signs occurred between one
and two months after immunization in 9 of the 12
experimental rabbits. These animals are a homogeneous group in that they all showed substantially the
same clinical pattern. The onset of disease was characterized by loss of proprioception and posterior ataxia,
indicating lesions in the posterior columns of the spinal
cord. Flaccid paresis o r frank paralysis, o r both, in the
hind legs, documented by decreased reflexes, was followed by spastic paralysis, hyperreflexia, and clonus.
T h e neurological symptoms indicate that lower levels
of the spinal cord were affected initially, with later involvement of higher levels. Various degrees of bladder
dysfunction were noted in the late stages of the disease.
Severe atrophy, localized in the hind legs and paravertebral muscles, was a common finding at the terminal
stages, but no dramatic loss of weight was recorded. Six
of the animals were killed and subjected to histological
examination at different times after the onset of clinical
symptoms (one, two, and three months). T h e 3 additional animals in this group showed clear-cut relapses
and remissions over the six-month period of observation (Table). Two of the 12 rabbits had a delayed onset
of disease with neurological signs appearing six months
after immunization. T h e clinical signs in these animals
were substantially the same as in the animals previously
described: loss of proprioception and slow progression
of paralysis over the three-month period of observation. O n e of the experimental rabbits showed no clinical signs of disease until autopsy, when mild inflammatory cell infiltration along penetrating vessels and mild
to moderate demyelination in lumbar and thoracic cord
regions were found. All control animals were normal.
Histological 0bwmztion.s
Histologically, all animals showed consistent lesions
characterized by lymphoplasmacytic meningomyelitis
accompanied by demyelination and gliosis. T h e lesions
were localized in the spinal cord, predominantly in the
thoracolumbar segments, and were characterized by
different stages of progression. No parenchymal inflammatory lesions o r demyelination was found in the
brain, although some rabbits had a mild meninb’
’itis over
the cerebral hemispheres. T w o of the rabbits with a
chronic relapsing disease course were killed following a
relapse, arid these showed, in addition to the features
we have described, a recent severe inflammatory lesion
in the spinal cord consisting of a necrotizing transverse
myelitis at the thoracolumbar level extending for eight
to ten spinal segments (Fig 1). Optic neuritis with demyelination and gliosis was found in 4 animals, 2 of
’
Clinical Course and Delayed Hypersensitivity i n Rabbits Injected with Bovine Pvoteolipid Apoprotein
Rabbit
No.
Onset of
Neurological
Signs
(mo postinjection)
1
2
1
1
3
4
5
1
6
7
8
9
10
11
12b
1
1
1Y2
1
2
1
6
6
None
Onset of Positive Skin Test toZa
Type of
Chronic
Disease
Proteolipid
Apoprotein
(days postinjection)
Myelin Basic
Protein
(days postinjection)
Progressive
Relapsing
Kelapsing
Relapsing
Progressive
Progressive
Progressive
Progressive
Progressive
Progressive
Progressive
None
20
20 (6 mm + + )
20 (18 mm + +)
20 (15 mm + )
20
20 (10 mm + )
20 (20 mm + + )
20 (20 mm + + )
20
60(10mm ++)
20 (13 mm + )
20 (10 mm + + )
Negative
Negative
Negative
Negative
Negative
75 (15 mrn
Negative
Negative
Negative
Negative
Negative
Negative
+
+
+ +)
~
+
"Values in parentheses are diameter of erythema and degree of induration ( + = mild, + = strong)
bAutopsy revealed mild inflammatory cell infiltration along penetrating vessels and mild to moderate demyelination in lumbar and thoracic cord
regions
which had chronic relapsing disease (Fig 2). Peripheral
nerves were unaffected in all rabbits.
Immzlnological Observations
A delayed hypersensitivity to APL, as assessed by skin
tests, was observed in all animals prior to the onset of
neurological signs (see the Table). In 11 of the 12 rabbits, erythema and induration appeared as early as 20
days postinjection. In contrast, a positive skin test for
F i g I . Transverse section at the level of the thovacolumbarspinal
cord showing marked liquefactive necrosis in central areas of both
white and gray matter (arrows). (Luxol fast bbe, H 6 E ; x 20).
MBP was present in only 1 of the animals, and this
appeared two and a half months after initial sensitization. All other rabbits were negative for MBP.
Among the 9 rabbits with early onset of disease, 5
(nos. 1, 3, 5 , 8, and 9) began to produce APL antibodies at the time of appearance of neurological signs,
1 (no. 2) had a delayed onset of appearance of APL
antibodies, and in the other 3 (nos. 4 , 6 , and 7) no APL
antibodies could be detected in any of the samples (see
Fig 2). In the 2 rabbits with delayed onset of clinical
signs (nos. 10 and ll), APL antibody production was
also evident at the time of appearance of the neurological deficits. The clinically normal rabbit (no. 12) had
APL antibodies in the initial sample taken one month
Cambi et al: Proteolipid Apoprotein and Chronic EAE
305
I : 5000
I : 4000
Antibody
Titer
'
I 3000
I :2000
I: 1000
I : 200
I month
2 months
5 months
6 months
Time Post Immunization
after immunization, but no antibodies were present in
any of the subsequent samples. Antibodies against
MBP were detected only in rabbit 1, and these appeared one month postimmunization at the same time
and at the same titer as the APL antibodies. Subsequent bleedings of this animal were all negative for
MBP. Rabbit 2 showed antibodies against MBP five
months after the appearance of clinical symptoms. All
other animals were negative for MBP antibodies. The
appearance of both APL and MBP antibodies was also
followed in all animals using an electroblot procedure.
An example of the results in 1 animal is shown in
Figure 3. The data obtained using this technique are
essentially in agreement with those obtained by ELISA.
No relationship was observed between the APL
antibody titer and the clinical course or severity of the
disease (Fig 4).For example, rabbits 3 and 4 showed
the same course of clinical signs, but no. 4 produced no
antibodies, whereas no. 3 showed a constant antibody
titer of 1: 1,000 beginning one month postimmunization. Because both of these animals had a relapsing
course, it is clear that the APL antibodies do not correlate with exacerbations and remissions of the disease.
Discussion
Acute EAE has been induced in a number of animal
species by immunization with either whole nervous tissue, myelin, MBP, or encephalitogenic MBP fragments
[lo, 231. Only in the case of whole tissue is demyelination a significant feature. Chronic EAE is characterized
by a progressive or relapsing course accompanied histologically by both inflammation and demyelination,
making it a good model for multiple sclerosis. In contrast, immunization with MBP alone fails to produce a
chronic course and induces acute disease with little or
no demyelination. The inflammation and demyelination produced by whole tissue must therefore involve
different antigens. In adult guinea pigs, the histological
pattern produced by MBP can be modified by the presence of helper antigens such as galactocerebroside or
total myelin lipids combined in the same emulsion
306 Annals of Neurology
Vol 13 No 3
March 1983
Fig 2. Antiprotealipid antibody titers in rabbit sera at dqkrent
times after immunization with apoprotein as described in
Methods. Numbers refer to individual rabbits described in the
Table.
Fig 3. Electroblot anulysis of serum from rabbit 10. fa) SDSpolyactylamide gel (10 to 22%) of myelin stained with Coomassie blue. (6) Nitrocellulosestrips incubated with 1:200 diiution
of antiserum obtained (1) one month. ( 2 ) two months. (3)five
months. and ( 4 )six months postimmunization. Antibodies to
proteolipid became evident at six months, concomitant with the
appearance of neurological signs. (PLP = proteolipid protein;
MBP = nzyeiin basic protein.)
1:5000Y
Titer
1:2000
c
Signs
\
Acute
EpiSode
Rmission
Months Post Immunization
F i g 4. Clinical course and antibody titers to major myelin proterns in rabbit 3. (Open bars, pvoteolipid apoprotein; solid bars,
myelin basic protein.)
1241. The combination of MBP with another antigen
results in demyelination, whereas the individual antigens do not. Additional evidence that the demyelination observed in chronic EAE in guinea pigs is related
to components other than MBP is the appearance during relapses of antibodies directed against an unidentified myelin antigen, designated M2 112, 131.
The present study shows that rabbits immunized
with a major myelin protein, namely proteolipid protein, develop a chronic disease with the same c-linical
and histological features as that produced by whole
tissue. The occurrence of demyelination as a consequence of sensitization to a single myelin component
implies that this protein may play a major role in the
pathophysiology of the demyelination. The one c-onsistent immunological finding in these experiments was
that delayed hypersensitivity was observed prior to the
appearance of clinical signs. This observation points to
a pathogenetic role for a cell-mediated response to proteolipid in myelin breakdown. Whereas proteolipid
apoprotein appears to be a strong inducer of cellmediated immunity, serum antibody production varied
among animals and does not correlate with either the
course of the disease or the histopathological pattern. It
remains to be determined whether the antibodies produced in some animals show demyelinating activity in
vitro. Antibodies to proteolipid protein isolated b y elution from SDS polyacrylamide gels do not cause demyelination of aggregating cell cultures 111.
Regardless of the humoral pattern, immunological
events occurring within the CNS may play a somewhat
different role. The brain occupies a segregated position
with respect to immunological factors, and serum antibodies may not reflect processes within the bran. Intracerebral synthesis of IgG has been demonstrated in
several diseases, including chronic EAE and multiple
sclerosis 121, 301. One possibility is that intracerebral
synthesis of antibodies to proteolipid can occur and
that these antibodies interact with cell-mediated processes to amplify the myelin damage. Another aspect
of the observed phenomenon may be related to the
hydrophobic properties of the apoprotein. Both glycolipids and lipoproteins have been shown to modulate the responses of lymphocytes [5, 17, 221, and the
hydrophobic character of the proteolipid may cause it
to act in a comparable way. The topographical distribution of the proteolipid in the membrane may also contribute to the observed effects. Because the proteolipid
is an intrinsic membrane protein, it presumably participates in lipid-protein interactions important for the
maintenance of bilayer integrity. However, a portion of
the protein is at the surface of the membrane, where it
can interact with the extracellular environment [2]. In
particular, immunocompetent cells specifically sensitized to the proteolipid might interact with these exposed regions. Subsequent attack of the protein by
inflammatory factors, such as proteolytic enzymes or
complement, would destabilize the myelin membrane
c3, 71.
The possibility of small amounts of contamination of
APL by MBP is extremely difficult to rule out. However, electroblotting ensures that the APL used in
these experiments does not contain detectable MBP; if
there is any MBP contamination, the amount injected
into each animal is less than 0.8 pg. To the best of our
knowledge, 15 pg is the smallest amount of MBP that
has resulted in disease 191. Immunologically, if MBP
were to play a role, one would expect to see a cellmediated immunity to this antigen. Such sensitization
was observed in only 1 animal, and this was two and a
half months after the initial sensitization. The response
of this animal may have been a consequence of multiple skin tests. Alternatively, it is possible that myelin
breakdown had occurred, with a consequent immunological response to released products. A similar
situation has been observed in mice, in which humoral
antibodies to MBP could be detected beginning 20
days after cutting the sciatic nerve 1261.
Supported by Grants N S 16945 and HD 04147 from the National
Institutes of Health. Animals used in this study were maintained in
accordance with the guidelines of the Committee on Animals of the
E. K. Shriver Center and those prepared by the Committee on Care
and Use of Laboratory Animals of the Institute of Laboratory Animal
Resources, National Research Council (DHEW Publication no.
[NIH] 78-23, revised 1978).
The authors thank David Kelley and Mary Beard for care of the
animals and Cindy Goldman and Vibah Goyal for histopathological
tissue processing.
References
1. Agrawal HC, Hartrnan BK, Shearer WT, et al: Purification and
immunohisrochemical localization of rat brain myelin proteolipid
protein. J Neurochem 28:495-508, 1977
2. Braun P: Molecular architecture of myelin. In Morel1 P (ed):
Myelin. New York, Plenum,1977, pp 91-116
3. Cammer W, Bloom BR, Norton WT, et al: Degradation of basic
Cambi et al: Proteolipid Apoprotein and Chronic EAE
307
protein in myelin by neutral proteases secreted by stimulated
macrophages: a possible mechanism of inflammatory demyelination. Proc Natl Acad Sci USA 75.1554-1558, 1978
4.Cohen SR, McKhann GM, Guarnieri MA: A radioimmunoassay
for myelin basic protein and its use for quantitative measurements. J Neurochem 25:371-176, 1975
5. Curtiss LK, Edgingron TS: Immunoregulatory plasma low density lipoprotein: the biologic activity and receptor-binding
specificity is independent of neutral lipids. J Immunol
126:1008-1012, 1981
6. Folch-Pi J. Stoffyn P: Proteolipid from membrane systems. Ann
N Y Acad Sci 195:86-107, 1972
7. Hirsch HE: Proteinases and demyelination. J Histochem Cytochem 29:425-430, 1981
8. Hoffman PM, Gaston DD, Spitler LE: Comparison of experimental allergic encephalomyelitis induced with spinal cord, basic
protein and synthetic encephalitogenic peptide. Clin Immunol
Immunopathol 1:364-371, 1973
9 . Kibler RF, Shapira R, McKneally S, et al: Encephalitogenic protein: structure. Science 164:577-580, 1969
10. Kies MW: Experimental allergic encephalomyelitis, In Gaul1 GE
(ed): Biology of Brain Dysfunction. New York, Plenum, 1973,
pp 185-224
1 1 . Lassmann H, Wisniewski HM: Chronic relapsing experimental
allergic encephalomyelitis. Clinicopathological comparison with
multiple sclerosis. Arch Neurol 36.490-497,1979
12. Lebar R, Vincent C: Studies on autoimmune encephalomyelitis
in the guinea pig. 111. A comparative study of two autoantigens
of central nervous system myelin. J Neurochem 32:145 1-1460,
1979
13. Lebar R, Vincent C: Studies on autoimmune encephalomyelitis
in the guinea pig. IV. Relative independence of acute and
chronic diseases. Immunol Lett 2:239-244, 1981
14. Lees MB, Paxman SA: Myelin proteins from different regions of
the CNS. J Neurochem 23:825-831, 1974
15. Lees MB, Sakura JD: Proteolipids. In Marks N , Rodnight R
(eds): Research Methods in Neurochemistry. New York,
Plenum, 1979, vol 4, pp 354-3’0
16. Lees MB, Sakura JD, Sapirsrein VS, et al: Structure and function
of proteolipids in myelin and non-myelin membranes. Biochim
Biophys Acta 559:209-230, 1979
17. Levy JA, Ibrahim AB, Shirai T, et al: Dietary fat affects immune
response, production of antiviral factors, and immune complex
disease in NZBiNZW mice. Proc Natl Acad Sci USA 79:19741978, 1982
308 Annals of Neurology
Vol 13
No 3
March 1983
18. Macklin WB, Braun PE, Lees MB: Electroblot analysis of the
myelin proteolipid protein. .J Neurosci Res 7 : l - 1 0 , 1982
19. Macklin WB, Lees MB: Solid-phase immunoassays for quantitation of antibody to bovine proteolipid apoprotein. J Neurochem
38:348-355, 1982
20. Macklin WB, Lees MB, Cohen SR, et al: Hydrophobic compounds interfere in radioimmunoassay for basic protein in myelin. Clin Chem 27:742-744, 1981
21. Mehta PD. Lassmann H , Wisniewski HM: Immunological studies of chronic relapsing EAE in guinea pigs: similarities to multiple sclerosis. J Immunol 12’:334-338, 1981
22. Miller H C , Esselman WJ: Modulation of the immune response
by antigen-reactive lymphocytes after cultivation with gangliosides. J Immunol 115:839-843, 1975
23. Paterson PY: Neuroimmunologic diseases of animals and humans. Rev Infect Dis 1:468-482, 1980
24. Raine CS, Traugott U, Farooq M, et al: Augmentation of immune-mediated demyelination by lipid haprens. Lab Invest
45:174-182, 1981
25. Raine C S , Traugott U, Iqbal K, et al: Encephalitogenic properties of purified preparations of oligodendrocytes as tested in
guinea pigs. Brain Res 142:85-96, 1978
26. Schwartz M, Sela BA, Eshhar N : Antibodies to gangliosides and
myelin autoantigens are produced in mice following sciatic nerve
injury. J Neurochem 38: 1132-1 135, 1982
27. Sherman G, Folch-Pi J: Rotary dispersion and circular dichroism
of brain “proteolipid” protein. J Neurochem 17:597-605, 1970
28. Sires LR, Hruby S, Alvord EC Jr, et al: Species restrictions of a
monoclonal antibody reacting with residues 130 to 137 in encephalirogenic myelin basic protein. Science 2 14:8’-89, 198 1
29. Stone SH, Lerner EM 11: Chronic disseminated allergic encephalomyelitis in guinea pigs. Ann N Y Acad Sci 122:227-241, 1965
30. Tourtellotte WW, Ma BI: Multiple sclerosis: the blood-brainbarrier and the measurement of de novo central nervous system
IgG synthesis. Neurology 28(suppl):76-83, 1978
31. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA
76:4350-4354, 1979
32. Williams RM, Lees MB, Cambi F, et al: Chronic EAE induced
in rabbits with bovine white matter proteolipid protein. J
Neuropathol Exp Neurol 41:508-521, 1982
33. Wisniewski HM, Keith AB: Chronic relapsing experimental allergic encephalomyelitis: an experimental model of multiple
sclerosis. Ann Neurol 1:144-148, 1977
Документ
Категория
Без категории
Просмотров
3
Размер файла
613 Кб
Теги
experimentov, bovine, allergic, rabbits, producer, proteolipid, encephalomyelitis, studies, immunologic, chronic, apoproteins
1/--страниц
Пожаловаться на содержимое документа