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*
Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands;
Department of Neurological Science, University of Genova, Genova, Italy;
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel;
§
Department of Immunology, Erasmus University of Rotterdam, Rotterdam, The Netherlands; and
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Department of Experimental In Vivo NMR, Image Sciences Institute, Utrecht University, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular analysis of the Mhc class II genes of the common marmoset revealed functional Mhc-DR and -DQ regions, and an apparently inactivated Mhc-DP region (5). On basis of the number of alleles found, it was concluded that the Caja-DQA and -DQB loci are oligomorphic. Moreover, three Caja-DRB loci were found; two loci with limited polymorphism (Caja-DRB1*03 and Caja-DRB*W16) and one monomorphic locus (Caja-DRB*W12). All common marmosets that we have analyzed thus far appeared to share the Caja-DRB*W1201 allele (n > 75). We hypothesized therefore that Caja-DRB*W1201 molecules may function as a major restriction element in the immunopathogenesis of EAE.
The clinical and pathological expression of myelin-induced EAE in marmosets is thought to result from a synergy of cellular and humoral autoimmune reactivity predominantly directed against two Ags, namely myelin basic protein (MBP) (2, 6, 7) and myelin/oligodendrocyte glycoprotein (MOG) (7, 8, 9). Hence, for the purpose of the present study, three marmoset twin couples were randomly selected from our colony; one sibling of each twin was immunized with recombinant human MOG (rhMOG), and the other sibling was immunized with purified human MBP (hMBP). The myelin Ags were emulsified in CFA, but usage of Bordetella pertussis was avoided. The immune systems of twin siblings can be regarded as highly similar given that they are complete bone marrow chimeras due to the sharing of the placental blood stream in utero (10). The cellular and humoral autoimmune responses and the development of clinical and pathological signs of EAE were assessed.
The results show that the rhMOG-immunized monkeys develop severe clinical EAE with specific demyelination of the CNS. The hMBP-immunized monkeys remained asymptomatic and also lacked pathological signs of EAE. The MOG-immunized monkeys proved to share a proliferative T cell response to the same MOG peptide (phMOG14–36), which was found to induce clinical and pathological signs of EAE in four of four monkeys. T cell reactivity to other MOG peptides varied between individual animals.
Activation of phMOG14–36-specific T cell lines appears restricted by the Caja-DRB*W1201 molecule. Anti-MOG IgG molecules appear to bind to peptides contained in two domains of the extracellular domain of MOG, namely between aa 4 and 40 and aa 44 and 76. Because the Caja-DRB*W1201 allele is present in all monkeys, it is concluded that the 100% incidence of demyelinating EAE in an outbred colony of common marmosets can be explained by a uniform immune response to a single encephalitogenic peptide as EAE initiating event.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three marmoset twin couples and four single monkeys were
randomly selected from the outbred colony at the Biomedical Primate
Research Centre. The sex and birth dates (month/year) of the monkeys
are depicted in Table I
. During
experiments, monkeys were housed individually in spacious cages with
padded shelters provided in the cage. The daily diet consisted of food
pellets for nonhuman primates (Hope Farms, Woerden, The Netherlands),
supplemented with rice, raisins, peanuts, marshmallows, biscuits, and
fresh fruit and vegetables. Drinking water was provided ad
libitum.
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Lymphoblastoid B cell lines were generated by transformation of
PBMCs with a cotton-top tamarin EBV (B95-8). Genomic DNA was isolated
from stable growing B cell lines, and exon 2 of the Caja-DRB
genes was amplified by PCR (5). Sequence analysis was
performed on an ABI prism 310 Genetic analyzer (Perkin-Elmer Applied
Biosystems, Foster City, CA) using the ABI Prism dRhodamine terminator
cycle sequencing ready reaction kit (Perkin-Elmer). The
Caja-DRB alleles depicted in Table I
represent the consensus
sequence of at least four separate clones. All 75 common marmosets
tested thus far share the previously described
Caja-DRB*W1201 allele (5).
EAE induction
hMBP was isolated and purified from normal donor brain as described (11). Three animals (9502, 9602, Estrada) were immunized with 1 mg hMBP, emulsified in CFA (Difco Laboratories, Detroit, MI). Escherichia coli-derived rhMOG, representing the N-terminal extracellular domain of human MOG (aa 1–123), was purified as previously described (12). Three animals (9501, 9601, Escudo) were immunized with 100 µg rhMOG as an emulsion in CFA.
Under ketamine anesthesia (6 mg/kg, AST Farma, Oudewater, The Netherlands), all monkeys were injected into the dorsal skin with 600 µl emulsion divided over four locations, two in the inguinal and two in the axillary region. B. pertussis has been used by others to facilitate development of EAE in marmosets (1, 2, 6, 7, 8, 9). In our hands, B. pertussis administration is not essential for myelin or MOG-induced EAE in marmosets. Moreover, injection of B. pertussis around the time of encephalitogenic challenge results in necrotic lesions in the CNS of marmosets (3).
The four single monkeys were immunized with 100 µg of the synthetic MOG peptide phMOG14–36, also emulsified in CFA, and received booster immunizations with 50 µg phMOG14–36 in IFA after 7, 9, and 12 wk.
Clinical diagnosis
A trained observer recorded daily the clinical course of EAE using a previously described semiquantitative scale (3): 0, no clinical signs; 0.5, apathy, loss of appetite, altered walking pattern without ataxia; 1, lethargy and/or anorexia; 2, ataxia; 2.5, mono- or paraparesis and/or brain stem syndrome; 3, hemi- or paraplegia; 4, quadriplegia; 5, spontaneous death attributable to EAE.
For ethical reasons, monkeys were sacrificed when the clinical EAE score of 3 was reached. The highest per day scores in a week were averaged. Moreover, each monkey was weighed at least three times per week to obtain a surrogate disease marker.
Magnetic resonance imaging (MRI)
For in vivo MRI, animals were anesthetized with 30 mg/kg ketamine in combination with 1 mg/kg valium (Diazepam, Kombivet, Etten-Leur, The Netherlands). Acquisitions were performed as described previously in detail (3). Each slide was recorded with a matrix of 512 x 256 data points and a field of view of 4 x 4 cm. The data sets were analyzed on an Apple MacIntosh Performa 630 (Apple Computer, Cupertino, CA) using the public domain National Institutes of Health program.
Neuropathology
Ketamine-anesthetized monkeys were euthanized by an i.v. injection of 400 mg sodium pentobarbital (Euthesate, Apharmo, Duiven, The Netherlands). The brain and spinal cord were excised in toto and fixed for 3 days in 4% buffered formalin, rinsed with PBS containing 0.05% sodium azide, and embedded in paraffin. Small parts of cervical, thoracic, and lumbar spinal cord were postfixed in PBS, 2.5% glutaraldehyde for 2 days, postfixed in 1% osmium tetroxide in PBS, and embedded in Epon. From some animals, the fresh brain was separated into two hemispheres, one being fixed in formalin and the other snap frozen in liquid nitrogen for immunohistochemical analysis. Paraffin sections of formalin-fixed brain and spinal cord were stained with hematoxylin and eosin, Luxol Fast Blue (LFB) combined with periodic acid-Schiff (PAS) for staining of myelin and Bodian for staining of axons. Immunocytochemistry was performed utilizing the immunoperoxidase method of biotin-avidin with the following Abs: mouse anti-human glial fibrillary acidic protein (Biogenex, San Ramon, CA) for astrocytes; rabbit anti-human CD3 (Dako, Glostrup, Denmark) for T cells and mouse anti-human CD20 (Biogenex) for B cells; mouse anti-human macrophage (27E10 and MRP14; BMA Biomedicals, Augst, Switzerland) for macrophages; mouse anti-MAG (CD57, Becton Dickinson, San Jose, CA); anti-human MBP (Biogenex); anti-CNPase (Sigma, St. Louis, MO) for myelin and oligodendrocytes. Semithin sections were stained with toluidine blue. Maturation stage and timing of demyelination were classified according to published criteria (3).
MOG and MBP Ab responses
Sera were collected from animals at the time of necropsy and stored in aliquots at -20°C. The Ab responses of individual monkeys directed to MBP, MOG, and MOG epitopes were analyzed using a slot blot assay. Rhesus monkey MBP (0.5, 1.0, and 5.0 µg), rhMOG (0.25, 0.5, and 1.0 µg) and synthetic overlapping peptides spanning the extracellular domain of MOG (phMOG), were spotted onto a polyvinyl difluoride membrane (Hybond, Amersham, Little Chalfont, U.K.) at a concentration of 0.1, 0.5, and 1.0 µg using a Bio-Dot SF blotting apparatus (Bio-Rad, Richmond, CA) (12). To ensure that all peptides remained bound to the membrane, the blots were immersed with 2.5% glutaraldehyde in PBS for 15 min, washed with PBS for 15 min, and the remaining sites were blocked by incubating the membrane for at least 2 h in PBS containing 3% BSA (PBS/BSA). The blots were then incubated for 1 h with the relevant serum diluted 1:1000 with PBS/BSA 1%, washed four times for 10 min with PBS containing 0.05% Tween 20, incubated for 1 h with rabbit anti-human IgA, IgG, IgM (Dako); diluted 1:14,000 in PBS/1% BSA, washed as described above, and processed for ECL detection according to the manufacturer’s instructions (Amersham).
MOG and MBP T cell responses
At necropsy, PBMC were isolated from venous blood using lymphocyte separation medium (LSM, ICN Biomedical, Aurora, OH). Lymph node cell (LNC) suspensions were prepared from aseptically removed inguinal and axillary lymph nodes. Cultures were set up in HEPES-buffered RPMI 1640 (Life Technologies, Glasgow, U.K.) supplemented with 10% FCS (Flow Laboratories, McLean, VA), 10 mM MEM with nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 2 x 10-4 M 2-ME (all from Life Technologies). PBMC or LNC (2 x 105/well) were seeded into 96-well flat-bottom plates (Greiner, Solingen, Germany) and cultured with rhMOG (10 µg/ml) or hMBP (50 µg/ml). After 48 h, 0.5 µCi/well of [3H]thymidine was added, and incorporation of radiolabel was determined 18 h later using a matrix 9600 beta-counter (Packard 9600, Packard Instrument, Meriden, CT).
Generation of MOG-reactive T cell lines
T cell lines reactive with rhMOG were generated from LNC of MOG-immunized marmosets. For this purpose, single LNC suspensions isolated at the day of necropsy were used as starting material. LNC (106/well) were seeded into 24-well flat-bottom plates (Greiner) and stimulated with 10–15 µg/ml rhMOG. In cycles of 2 or 3 days, one-half of the supernatant was removed, and the cultures were supplemented with fresh medium containing 20 U/ml rIL-2 (Cetus, Amsterdam, The Netherlands). After 14–21 days of culture, the cells were restimulated with rhMOG, using irradiated (50 Gy) autologous EBV-transformed B cell lines as APC.
Peptide specificity of MOG-specific T cell lines
Cells were seeded at 2 x 104 T cells/well into 96-well flat-bottom plates and stimulated with rhMOG or a panel of synthetic overlapping phMOG (1.0 µg/ml) (12, 13). Proliferation was assessed by [3H]thymidine incorporation (0.5 µCi/well) during the final 18 h of a 3-day culture. Incorporated radiolabel was counted as described above. Mean values were calculated from triplicate cultures. T cell lines showing reactivity to a certain phMOG were restimulated with that same peptide at the next round of restimulation until stable growing phMOG-specific T cell lines were obtained.
MHC restriction of phMOG-specific T cell reactivity
The MHC restriction of MOG-induced T cell proliferation was determined by inhibition of responses using mAbs raised to primate MHC isotypes SPVL-3 (anti-DQ), B8.11.2 (anti-DR), B7/21 (anti-DP), PdV5.2 (anti-class II), and W6/32 (anti-class I) at 1:100 ascites dilutions (4). Autologous APC were incubated for 15 min at 37°C with the mAbs and then pulsed with the relevant phMOG for 60 min at 37°C. After extensive washing, the APC were tested for their ability to induce proliferation of specific T cell lines during a 72-h culture. Positive controls consisted of APC pulsed with peptide without mAb and negative controls of APC incubated with mAb without peptide. The restriction elements for presentation of phMOG14–36 were determined by testing T cell proliferation induced by a panel of MHC-typed, EBV-transformed B cell lines from related and unrelated marmosets. Irradiated B cells (50 Gy) were seeded (2 x 104 cells/well) into a 96-well plate, and phMOG14–36 was added. Peptide-induced proliferation of 2 x 104 phMOG14–36-specific T cells was assessed by counting [3H]thymidine incorporation during the final 18 h of a 3-day culture.
Ethics
According to the Dutch law on animal experimentation, the protocol of this study has been reviewed and approved by the Institute’s Animal Care and Use Committee. All experimental procedures with the animals are in accordance with the guidelines of the committee.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All 3 MOG-immunized animals developed clinically manifest
neurological disease (EAE score, 
2), albeit at various times after
immunization. The first clinical signs were observed at 38 days in
Escudo, at 52 days in animal 9601, and at 64 days in animal 9501. The
clinical course of MOG-induced EAE was chronic progressive in all
animals, with each monkey developing complete paralysis of the hind
part of the body (paraplegia; EAE score, 3.0) within 2 wk after disease
onset (Fig. 1A). At this
stage, the monkeys were sacrificed based on ethical considerations.
During the course of the disease, all three MOG-immunized animals lost
more than 15% of their body weight (Fig. 1B).
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). The lesion load of animals 9501
and 9601 differed remarkably, despite similar EAE severity. In the
brain of monkey 9501, several small focal lesions were detected,
whereas in monkey 9601 extremely large lesions were found. No
MRI-detectable lesions could be observed in the brain of monkey
Escudo.
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, a and b), mainly
localized around the wall of the lateral ventricles, in the hemispheric
white matter, in the spinal cord, in the corpus callosum, and in the
optic nerves and tracts. Light microscopic examination revealed a
relevant degree of inflammation within the areas of demyelination,
characterized by perivascular cuffs of mononuclear cells, whereas
granulocytes were absent. Infiltrates were composed of macrophages, T
lymphocytes, and a few B lymphocytes. Inside the cytoplasm of
macrophages, LFB- or PAS-positive myelin degradation products were
detected. Bodian staining did not reveal apparent axonal damage.
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c).
Diffuse infiltration of demyelinated areas by macrophages was present,
but axons were apparently intact (Fig. 3d). Both active and
inactive lesions were present in the same areas, as indicated by the
presence of LFB-positive myelin degradation products and by staining
macrophages with MRP14 (Fig. 3e) or the presence of only
PAS-positive deposits in the macrophages (not shown). Ventral and
dorsal roots belonging to the peripheral nervous system, lying nearby
the severely affected spinal cord, were completely spared. This
indicates that the demyelinating process was restricted to the
myelinated fibers of the CNS (Fig. 3f). Absence of clinical and pathological features of EAE after MBP immunization
MBP-immunized animals developed only mild clinical signs of EAE. Animals 9502 and Estrada showed apathy and loss of appetite (EAE score: 0.5) during a period of 2 wk starting at 10 and 11 wk after immunization, respectively. Monkey 9602 remained asymptomatic during the observation period of 178 days. No weight loss was observed. On in vivo T2-w brain magnetic resonance images recorded 1 mo after immunization, no abnormalities could be detected. However, at 3 mo after immunization, small hyperintense regions were found in the brains of monkeys 9502 and Estrada, but not in the brain of animal 9602. No abnormalities could be detected on postmortem magnetic resonance images, and the neuropathology analysis revealed that no signs of inflammation or demyelination was observed in MBP-immunized animals. The MRI-detectable abnormalities may therefore be an artifact or reflect the edema extravasation associated with perivascular inflammation, which could have been drained by the time the monkeys were sacrificed.
MOG- and MBP-specific Ab responses
In MOG- and MBP-immunized animals, circulating Abs appeared to be
primarily directed against the inciting Ag only (Fig. 4). The epitope specificity of
anti-MOG Abs present in necropsy sera was analyzed using a set of
overlapping 22-mer peptides, spanning the N-terminal extracellular part
of MOG (residues 1–116). The main reactivity of anti-MOG Abs in
all three MOG-immunized monkeys was directed against two separate
regions (Fig. 4). The sera reacted with phMOG4–26, 14–36 and 24–46,
but not to phMOG34–56, indicating that one or more B cell epitopes are
located within aa stretch 4–40. The sera also showed strong reactivity
to phMOG44–66 and 54–76, indicating that one or more epitopes are
contained within aa region 44–76. No Ab reactivity toward rhMOG or
phMOG could be detected in necropsy sera of MBP-immunized animals (not
shown). The preimmune sera showed no reactivity against whole myelin,
rhesus monkey MBP, rhMOG, or phMOG, indicating that Abs were formed
after immunization. Notably, with this technique Ab reactivity against
discontinuous epitopes of MOG are not detected.
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Primary LNC cultures of all three MOG-immunized animals displayed
strong proliferative responses to rhMOG (Fig. 5) but not to hMBP (not shown). In
contrast, LNC from one of the three MBP-immunized animals (Estrada)
displayed a significant proliferative response to hMBP (Fig. 5). In
conclusion, the different clinical expression and radiological
manifestation of EAE as well as the clear immunological and
histopathological differences found in MOG- vs. MBP-immunized siblings
prompted us to study the MOG-induced EAE in greater detail.
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After two rounds of restimulation with rhMOG, the epitope
specificities of LNC-derived cultures were analyzed using a set of 10
overlapping 22-mer phMOGs (spanning residues 1–116). Positive
responses, stimulation index 4, are depicted by shaded boxes (Fig. 6A). Subsequently,
peptide-specific T cell lines were generated. After another two rounds
of culture with the specific peptides, the lines were checked for
peptide specificity. As shown in Fig. 6B, only one T cell
line could be established from monkey 9501, reactive with phMOG14–36
[9501 (14–36)] and with phMOG24–46. From animal 9601, 4 different T
cell lines could be generated: 9601 (4–26); 9601 (14–36); 9601
(24–46); and 9601 (74–96). Line 9601 (14–36) proved reactive to
phMOG24–46. Line 9601 (24–46) recognizes a different epitope in that
it is not responding to phMOG14–36. Finally, T cell line 9601 (74–96)
is reactive with a distinct epitope (Fig. 6B). From Escudo,
three lines were established (eso (14–36), eso (24–46) and eso
(34–56)) responding to at least two distinct epitopes (Fig. 6B). All presently described in vitro-generated T cell lines
were CD4+CD8- with high
surface expression of Caja-DR molecules reflecting their activated
state.
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The fine specificities of T cell lines were delineated using
smaller peptides. The fine specificity of the phMOG4–26-reactive T
cell line was defined at residue p4–11 (Fig. 7A), of phMOG74–96-reactive T
cells at p81–96 (Fig. 7B) and of phMOG14–36-reactive T
cells to p24–36 (Fig. 7C). In conclusion, all three
MOG-immunized marmosets share T and B cell reactivity to phMOG14–36
(Figs. 5 and 6B). To determine the MHC restriction elements
that control the T cell reactivity to p24–36, autologous APC were
exposed to mAbs directed against primate Mhc class II
isotypes before they were pulsed with phMOG14–36. Peptide-induced
proliferation could be inhibited by both mAbs B8.11.2 and PdV5.2 but
not by mAbs SPV-L3, B7/21, and W6/32 (Fig. 8). Hence, it is concluded that p24–36
is presented to the T cell lines in the context of Caja-DR
molecules.
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), was
tested for presentation of phMOG14–36 to Ag-specific T cell lines.
phMOG14–36-specific T cell proliferation could be induced by APC from
all marmosets (Fig. 9). Because
Caja-DRB*W1201 is shared by all marmosets tested in our
population (5), it is the most likely restriction element
of p24–36 presentation. This assumption is supported by the fact that
transformed B cell lines from the cotton-top tamarin, a new world
monkey closely related to the common marmoset but lacking the
equivalent of the Caja-DRB*W1201 allele (14),
were incapable of p24–36 presentation to the T cell lines.
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To investigate whether the peptide phMOG14–36 is involved in the
initiation of EAE, four common marmosets were immunized with the MOG
peptide emulsified in CFA, avoiding administration of B.
pertussis. Fig. 10 shows the
clinical course of the EAE (Fig. 10A) and the primary LNC
responses to rhMOG and phMOG14–36 (Fig. 10B). The results
show that immunization with phMOG14–36 induces clinical signs of EAE
in all four monkeys as well as a cellular immune reaction to the
peptide and rhMOG. The time of onset and the course of clinical
signs appeared to differ between individual monkeys, as was also found
in MOG-immunized monkeys. As could be expected, the earliest responder
(QY) displayed the highest proliferative response to MOG protein and
peptide. Histopathology analysis of the brain confirmed the diagnosis
of EAE, as perivascular cuffs of mononuclear cells could be found (not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Where the relevance of the autoimmune reaction to MBP and proteolipid protein for the immunopathogenesis of MS is disputed, evidence is accumulating that T cell and Ab reactivity to MOG play an important, most likely synergistic role. MS patients appear to display a significantly higher level of T cell reactivity to MOG than control individuals (12, 13, 18, 21). Moreover, anti-MOG Abs are localized in CNS areas where myelin disintegration and lesion formation are taking place (8, 17). Finally, in its pathological expression, MOG-induced EAE closely resembles MS (8, 9, 21, 22). However, the Ab dependence of EAE has been challenged by the observation that B cell knockout mice also develop clinical signs (23, 24).
Our present results show that in common marmosets MBP is antigenic; MBP-specific T and B cell responses were detected but were only weakly encephalitogenic. The lack of clinical signs and pathological evidence of EAE in our MBP-immunized marmosets seems to contrast with published data (1, 2). It should be emphasized, however, that we avoid usage of B. pertussis, which was found an essential component of the EAE induction protocol by these authors (1). The reason is that, besides a direct effect on the blood-brain barrier permeability, B. pertussis administration to marmosets immunized with human myelin in CFA appeared to cause lesions by necrosis rather than specific demyelination of CNS white matter (3). This effect might be related to potentiation and/or polarization of the MBP-specific T cell response by B. pertussis (25, 26).
The present analysis of the proliferative responses of LNC-derived T
cells to rhMOG and the panel of MOG peptides shows that all
MOG-immunized monkeys share a T cell reactivity to a single MOG
epitope, p24–36. The epitope is contained in the encephalitogenic
phMOG14–36 peptide and presented in the context of Caja-DRB*W1201
molecules. The T cell epitope seems not to concur with any of the thus
far identified immunodominant T cell epitopes in humans or rodents
(18, 27). An individually variable response was found
against other MOG epitopes, p4–11, p31–40, and p81–96. Preliminary
data from similar cross-presentation studies, as in Fig. 9, indicate
that these MOG peptides are likely presented by other Caja-DR molecules
(unpublished results). Binding of anti-MOG Abs proved to be
confined to two regions within the rhMOG molecule, namely p4–40 and
p44–76. Our results of T cell and Ab epitope mapping are in line with
data from Genain et al. (5, 6, 28).
On the basis of the data discussed thus far, we conclude that phMOG14–36 may contain critical T and B cell epitopes for the initiation of EAE in common marmosets. The observation that four of four common marmosets immunized with this peptide emulsified in CFA develop clinical EAE strengthens this assumption. Computer modeling of the three-dimensional conformation of MOG predicts that the 14–36 peptide is exposed on the surface of a homodimer and thus freely accessible to Ab binding (29). The fact that all common marmosets share the Caja-DRB*W1201 molecule, which functions as a major restriction element of the T cell reaction to phMOG14–36, underlies the 100% incidence of severe demyelinating EAE in common marmosets. The EAE-initiating event in myelin-immunized common marmoset monkeys may thus be a remarkably uniform event, namely, the Caja-DRB*W1201-restricted activation of phMOG14–36-specific CD4+ T cells. The subsequent spreading of the T and B cell reactivity to other MOG-epitopes appears to vary between individual monkeys, reflecting the outbred nature of this species. This unique feature, together with the possibility of adoptively transferring T cell lines between fraternal siblings (1) makes the common marmoset a unique model for the detailed analysis of pathophysiological pathways in EAE and MS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bert ‘t Hart, Biomedical Primate Research Centre, Department of Immunobiology, Lange Kleiweg 139, 2288GJ Rijswijk, The Netherlands.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cells; LFB, Luxol Fast Blue; MBP, myelin basic protein; hMBP, human myelin basic protein; MRI, magnetic resonance imaging; MS, multiple sclerosis; rhMOG, recombinant human myelin/oligodendrocyte glycoprotein; PAS, periodic acid-Schiff; phMOG, human MOG peptide; T2-w, T2-weighted.
Received for publication September 13, 1999. Accepted for publication April 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N. K. U. Koehler, C. P. Genain, B. Giesser, and S. L. Hauser The Human T Cell Response to Myelin Oligodendrocyte Glycoprotein: A Multiple Sclerosis Family-Based Study J. Immunol., June 1, 2002; 168(11): 5920 - 5927. [Abstract] [Full Text] [PDF]
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L. Boon, H. P. M. Brok, J. Bauer, A. Ortiz-Buijsse, M. M. Schellekens, S. Ramdien-Murli, E. Blezer, M. van Meurs, J. Ceuppens, M. de Boer, et al. Prevention of Experimental Autoimmune Encephalomyelitis in the Common Marmoset (Callithrix jacchus) Using a Chimeric Antagonist Monoclonal Antibody Against Human CD40 Is Associated with Altered B Cell Responses J. Immunol., September 1, 2001; 167(5): 2942 - 2949. [Abstract] [Full Text] [PDF]
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2; not shown). In contrast, LNC from
only one of three MBP-immunized animals showed a proliferative response
to hMBP. Results are means of triplicate assays and are expressed as
stimulation indexes (stimulation with Ag/stimulation without Ag).
