Autoimmunity Against Myelin Oligodendrocyte Glycoprotein Is Dispensable for the Initiation Although Essential for the Progression of Chronic Encephalomyelitis in Common Marmosets

April 25, 2008

By Jagessar, S Anwar Smith, Paul A; Blezer, Erwin; Delarasse, Cecile; Pham-Dinh, Danielle; Laman, Jon D; Bauer, Jan; Amor, Sandra; Hart, Bert ‘t

Abstract To elucidate the pathogenetic significance of myelin/ oligodendrocyte glycoprotein (MOG)-specific autoreactivity in a genetically and immunologically heterogeneous nonhuman primate model of multiple sclerosis, we analyzed experimental autoimmune encephalomyelitis (EAE) in the outbred common marmoset (Callithrix jacchus). One sibling each of 5 bone marrow chimeric marmoset twins was immunized with myelin derived from wild-type (WT) C57BL/6 mice (WT myelin); the other sibling was immunized with myelin from MOG- deficient C57BL/6 mice (MOG^sup -/-^ myelin). One twin pair developed acute EAE simultaneously; the 4 remaining twin siblings immunized with WT myelin developed chronic progressive EAE, whereas siblings of these 4 monkeys remained free of clinical disease signs. Many EAE-related abnormalities were identified in the CNS of both groups by magnetic resonance imaging and histologic analysis, but mean percentages of spinal cord demyelination were lower in monkeys immunized with MOG^sup -/-^ myelin (8.2%) than in WT myelin- immunized animals (40.5%). There was a strong correlation between the development of overt clinical EAE and seropositivity for anti- MOG antibodies, but blood and lymph node T-cell proliferative responses showed no relationship to disease. These results indicate that the initiation of CNS inflammation and demyelination can take place in the absence of detectable autoimmunity against MOG, but the clinical progression and histopathologic severity depends on the presence of antibodies against MOG in this multiple sclerosis model.

Key Words: Autoimmunity, Common marmoset, Demyelination, Experimental autoimmune encephalomyelitis, Myelin/oligodendrocyte glycoprotein

INTRODUCTION

Experimental autoimmune encephalomyelitis (EAE) is a widely studied model of the human neuroinflammatory disease multiple sclerosis (MS). Experimental autoimmune encephalomyelitis can be induced in a wide range of genetically susceptible laboratory animal species, including rodents and primates, by immunization with myelin or myelin components in strong adjuvants (1, 2). Previously, we showed that the induction of chronic EAE in Biozzi ABH mice strictly depends on the presence of myelin/oligodendrocyte glycoprotein (MOG) in the immunization inoculum. Although Biozzi ABH mice immunized with myelin from MOG-deficient (MOG^sup -/-^) C57BL/6 mice (3) exhibited a short limited episode of neurologic deficit, they did not develop the typical relapsing/remitting disease course observed in Biozzi mice immunized with MOG containing myelin from wild-type (WT) mice (4). Furthermore, the addition of only a minute amount of recombinant mouse MOG (rmMOG) to the MOG^sup -/-^ myelin preparation used for immunization resulted in the induction of a relapsing/ remitting EAE course. Whether the critical role of MOG for induction of chronic EAE is specific to the inbred Biozzi ABH mouse EAE model or if the phenomenon also holds true in a model in a more complex genetically outbred species more closely related to humans is not known. Experimental autoimmune encephalomyelitis in the common marmoset (Callithrix jacchus) provides a valid MS model of MS in which this question can be addressed (5).

Myelin oligodendrocyte glycoprotein is a highly conserved, quantitatively minor constituent of CNS myelin and is exposed on the outermost lamellae of the myelin sheath and on the surface of mature oligodendrocytes (6-8). Because of a lack of MOG expression within the thymus, autoreactive T lymphocytes likely escape negative selection and are present in the normal immune repertoire (3, 9, 10). After immunization of marmosets with recombinant human MOG^sub 1-125^ (rhMOG) in adjuvant, MOG-specific autoreactive T cells contribute to the induction of EAE (10, 11). Compared with more abundant CNS myelin proteins such as myelin basic protein (MBP) and proteolipid protein (PLP), MOG is the most potent inducer of inflammatory demyelinating disease in this species. Immunization with rhMOG or synthetic MOG peptides also induces T-cell-mediated EAE in susceptible rodent strains (4, 12-14) and in nonhuman primates (11, 15, 16). This disease model reproduces many of the clinical and histologic features of MS. Furthermore, the ability of anti-MOG antibodies to increase myelin uptake by macrophages, enhance demyelination, and augment clinical disease in rodents (17) and in nonhuman primates (16) to a greater extent than humoral responses against other myelin antigens is unique.

The aim of this study was to determine the extent to which the presence of MOG in the myelin inoculum is required for the induction of EAE in the common marmoset. This was tested in marmoset twins that, due to their natural bone marrow chimerism, are immunologically highly similar (18, 19). One sibling of each twin was immunized with myelin from WT C57BL/6 mice, the other was immunized with myelin from MOG^sup -/-^ C57BL/6 mice produced in 1 of our laboratories (3). We found that T-cell reactivity against MOG (i.e. proliferation) was present in both siblings of each twin pair, but that anti-MOG antibodies were substantially reduced or absent in the siblings immunized with MOG^sup -/-^ myelin. In 1 twin pair, acute clinical EAE developed simultaneously in both siblings. Of the remaining 4 twin pairs, the siblings immunized with WT myelin developed overt chronic EAE, whereas this was observed in none of the siblings immunized with MOG^sup -/-^ myelin. The clinical data were confirmed using high-definition magnetic resonance brain imaging (MRI) and histopathologic analysis, showing that, although substantial abnormalities were observed in the monkeys immunized with MOG^sup -/-^ myelin, CNS demyelination was less severe than in the WT myelin-immunized animals. These results indicate a strong influence of anti-MOG antibodies on the development of chronic autoimmune demyelinating disease.

MATERIALS AND METHODS

Animals

Ten healthy adult male common marmosets were purchased from the outbred colony maintained at the Biomedical Primate Research Centre (Rijswijk, The Netherlands). To reduce variation between the 2 experimental groups, 5 nonidentical twin pairs were used. Despite genetic differences inherent to the outbred nature of this species, twins are immunologically highly similar due to naturally occurring bone marrow chimerism. All animals were given a complete physical, hematologic, and biochemical health screen prior to experimental selection. During the experiments, twin siblings were pair-housed in spacious cages and were under intensive veterinary supervision. The daily diet consisted of commercial food pellets for New World monkeys (Special Diet Services, Witham, Essex, UK) supplemented with rice, raisins, peanuts, marshmallows, fresh fruit, and live insects. Drinking water was provided ad libitum. According to the Netherlands’ law on animal experimentation, the procedures of this study have been reviewed and approved by the institute’s experimental ethics committee. The housing, care, and biotechnical handlings were in conformity with guidelines of this committee.

Antigens

Myelin was purified from the spinal cords of WT and MOG^sup -/-^ mice as previously described (4). Wild-type mice were bred at the Biomedical Primate Research Centre, and MOG^sup -/-^ mice were obtained from Dr. D. Pham-Dinh (Universite Pierre et Marie Curie, Paris, France). Protein concentrations were determined using the Bradford technique (20). Synthetic 23-mer MOG peptides corresponding to the extracellular domain of human MOG were purchased from ABC Biotechnology (London, UK). Recombinant mouse MOG^sub 1-116^ and rhMOG were produced, and myelin was isolated as previously described (4, 21, 22). Human MBP (hMBP) was a kind gift from Dr. J. M. van Noort (TNO-PG, Leiden, The Netherlands).

Induction of EAE

Experimental autoimmune encephalomyelitis was induced under ketamine anesthesia (40 mg/kg; AST Pharma, Oudewater, The Netherlands) by a single subcutaneous inoculation of 300 [mu]l myelin in water (10 mg/ml) emulsified with 300 [mu]l complete Freund adjuvant (Difco Laboratories, Detroit, MI) into 4 sites of the dorsal skin (11). Animals were clinically scored twice daily by trained observers using a previously described semiquantitative scale (23). Plasma samples were collected at various postsensitization days and stored at -20[degrees]C for determination of antibody reactivity with myelin preparations by Western blotting or myelin proteins by enzyme-linked immunosorbent assay (ELISA). Animals were killed when they reached the humane end point (EAE score >/=3) or otherwise at the predetermined study end point. Humane end point criteria are discussed in detail elsewhere (24).

Postmortem Examination

At the time of necropsy, the monkeys were first deeply sedated by an injection of ketamine (50 mg/ml saline, i.m.) at a dose of 100 [mu]l/kg body weight and subsequently euthanized by infusion of pentobarbital sodium (Euthesate; Apharmo, Duiven, The Netherlands). The brain, spinal cord, spleen, and lymph nodes from inguinal and axillary regions were aseptically removed. Spleen and lymph nodes were processed for preparation of mononuclear cell (MNC) suspensions. Representative samples of all organs were snap-frozen in liquid nitrogen or fixed with 4% buffered formalin. Frozen tissues were stored at -80[degrees]C. After at least 7 days’ fixation in formalin, the tissues were transferred into buffered saline containing sodium azide (Sigma-Aldrich, Gillingham, UK) for stabilization prior to MRI (25). To assess the lesion load in the brain, MRI was performed on formalin-fixed brains as described previously (23, 25). The frozen and fixed tissues were processed for examination with histologic and immunohistochemical techniques as described (23, 26, 27).

Histopathology and Immunohistochemistry

After formalin fixation, samples of the brain, spinal cord, and peripheral nerves were embedded in paraffin and processed as described previously (28). In brief, in each animal, the cerebrum and cerebellum were divided into 7 or 8 coronal sections; the spinal cord was sectioned transversely into 10 to 15 pieces. This material was embedded in 3 to 4 paraffin blocks. The extent of inflammation, demyelination, and axonal abnormalities were evaluated on 3- to 5- [mu]m-thick sections stained with hematoxylin and eosin to visualize infiltrating cells, Luxol fast blue combined with periodic acid Schiff for myelin and myelin degradation products, and with Bielschowsky silver impregnation for axons.

For immunohistochemical staining, 3- to 5-[mu]m-thick paraffin sections were deparaffinized in xylene and transferred to 90% ethanol. Endogenous peroxidase was blocked by 30-minute incubation in methanol with 0.02% H^sub 2^O^sub 2^. Sections were then transferred to distilled water via a 90%, 70%, and 50% ethanol series. Before staining with antibodies, antigen retrieval was performed as follows: paraffin sections were pretreated in a household food steamer device (MultiGourmet FS 20; Braun, Kronberg/ Taunus, Germany) by a 60-minute incubation in a plastic Coplin jar filled with EDTA (0.05 mol/L) in TRIS buffer (0.01 mol/L; pH 8.5). To detect Immunoglobulin (Ig)G and IgM, antigen retrieval was performed by incubation with 0.03% protease from Streptomyces griseus (Sigma, St. Louis, MO) for 15 minutes at 37[degrees]C. After antigen retrieval, the sections were incubated with 10% fetal calf serum (FCS) in 0.1 mol/L phosphate-buffered saline (FCS/PBS). Primary antibodies for CD3 (DakoCytomation, Hamburg, Germany) and PLP (Serotec, Oxford, UK) were applied in FCS/PBS at 4[degrees]C overnight. Immunoglobulin G and IgM were detected by staining with biotinylated anti-human-IgG and anti-human-IgM antibodies (DakoCytomation). After washing with PBS, secondary antibodies in PBS/FCS were applied for 1 hour at room temperature. Biotinylated secondary antibodies were used at a concentration of 1:200 (donkey- anti-rabbit, sheep-anti-mouse; Amersham Pharmacia Biotech, Uppsala, Sweden). As a third step, avidin peroxidase (1:100; Sigma) was used. Labeling was visualized with 3,3′ diaminobenzidine-tetra- hydrochloride (Sigma).

Quantification of Inflammation and Demyelination

In hematoxylin-eosin-stained sections, the degree of inflammation was expressed as an index that was calculated as the average number of inflamed blood vessels per spinal cord cross section (inflammatory index; n = 10 to 15 spinal cord cross sections per monkey). The degree of demyelination was quantified in Luxol fast blue/periodic acid Schiff-stained sections in each monkey on 10 to 15 spinal cord cross sections by overlay of a 100-point morphometric ocular grid and counting the amount of normal and demyelinated white matter.

Magnetic Resonance Imaging

High-definition T^sub 2^-weighted images were made of formalin- fixed brains as previously described (25). Previous studies showed that the usually sharply demarcated hyperintense abnormalities present in the brain white matter of EAE-affected monkeys, but reference samples of non-EAE monkeys do not represent demyelinated lesions (23, 25).

All experiments were performed on a 4.7-T horizontal bore nuclear MR spectrometer (Varian, Palo Alto, CA) equipped with a high- performance gradient insert (12-cm inner diameter; maximum gradient strength, 500 mT/m). A home-built solenoid coil (4 windings; [empty set], 35 mm) was used for radio-frequency transmission and signal reception. Brain specimens were immersed in a nonmagnetic oil (Fomblin; perfluorinated polyether; Solvay Solexis, Weesp, The Netherlands) to prevent unwanted susceptibility artifacts. Forty- seven contiguous T^sub 2^-weighted transversal slices of 0.75 mm were collected with the following characteristics: repetition/echo time, 3,000/15 milliseconds; field of view, 2.5 x 2.5 cm^sup 2^; matrix, 128 x 128; zero-filled, 256 x 256; in-plane resolution, 195.3 x 195.3 [mu]m, 2 transitions.

Individual image sets were registered using the Medical Image NetCDF package (McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada). For quantitative analysis of the area of high signal intensity in the white matter (i.e. lesions), the free available Medical Image Processing, Analysis and Visualization (version 2.7.101; 2006; National Institutes of Health, Bethesda, MD) package was used. Regions of interest with abnormal decreased signal intensities were automatically outlined in all slices containing white matter structures using the level-set method of Medical Image Processing, Analysis and Visualization. The volumes of the region of interest were calculated.

Magnetic resonance imaging data are expressed as mean +- SEM where appropriate. Statistical analyses were performed using the statistical software package Sigmastat (version 3.11; 2004). Data were evaluated by 2-way repeated-measures analysis of variance, followed by the Student-Newman-Keuls post hoc test. p < 0.05 was considered statistically significant.

T-Cell Proliferative Responses

According to the Institute’s guidelines, the maximum blood volume that can be collected from a primate without damaging its health is 0.7% of the body weight per month for a single collection or a total of 1% when collections are spread over multiple time points. For an adult marmoset of 350 g, this represents a maximum of 3.5 ml, thereby limiting the number of tests that can be performed in this model. Mononuclear cells from heparinized peripheral blood mononuclear cell (PBMC) or lymphoid organs (lymph node cell) were isolated using lymphocyte separation medium (ICN Biomedical, Inc., Costa Mesa, CA) as previously described (11). Mononuclear cells (1 x 10^sup 6^ cells/ml) were cultured in RPMI media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml of penicillin, 100 [mu]g/ml of streptomycin, 5 mmol/L of 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid, and 5 x 10^sup -5^ mol/L of beta- mercaptoethanol (Invitrogen, Gibco BRL, Glasgow, UK), with 10 [mu]g/ well MOG peptides, rmMOG, rhMOG, or WT myelin for 72 hours. Proliferation was measured by the incorporation of ^sup 3^H- thymidine (Amersham Biosciences, Buckinghamshire, UK) after addition at 1 [mu]Ci/ well during the last 18 hours of culture and expressed as mean counts per minute +- SD of triplicate cultures. Stimulation indices were calculated from measured counts per minute in cultures stimulated with antigen divided by counts per minute in cultures without antigen.

FIGURE 1. The experimental autoimmune encephalomyelitis course in marmosets immunized with myelin from wild-type (WT) or myelin/ oligodendrocyte glycoprotein (MOG)-deficient (MOG^sup -/-^) mice. One twin each of 5 marmoset twin pairs was immunized with myelin from WT mice in complete Freund adjuvant (CFA) and the other twin with myelin from MOG^sup -/-^ mice in CFA. The graphs depict clinical scores of each monkey and the percentage of weight loss from Day 0 weight as a surrogate disease marker. The horizontal axis is days postsensitization (PSD). The disease-free survival times between the 2 groups differ significantly (logrank test; p = 0.0273).

Generation of MOG-Reactive T-Cell Lines

Lymph node MNCs were stimulated ex vivo with WT myelin, MOG^sup – /-^ myelin, or rhMOG to establish T-cell lines. Briefly, lymph node cells (10^sup 6^ cells/well) were seeded onto 24-well plates (Greiner Bio-one, Frickenhausen, Germany) and stimulated with 10 [mu]g/ml of antigen. Every 2 to 3 days, half of the culture supernatant was removed and replaced with fresh medium containing 20 U/ml of recombinant human interleukin (IL) 2 (Proleukin, Chiron Corporation, Emeryville, CA). After 14 to 21 days of culture, the T- cell lines were transferred into 96-well flat-bottom plates (Greiner Bio-one) and tested for proliferation against a panel of 23-mer overlapping peptides derived from the human MOG extracellular domain using irradiated immortalized B-cell lines as antigen-presenting cells for restimulation (11). Antigen- and peptide-reactive T-cell lines were characterized for cytokine responses by ELISA (U-Cytech, Utrecht, The Netherlands).

Western Blotting

Total WT or MOG^sup -/-^ myelin proteins were obtained by solubilization of mouse CNS samples in 80% tetrahydrofuran-20% water- 0.1% trifluoroacetyl acid, and subsequent delipidation was performed by repeated ether precipitation and subjected to sodium dodecylsulfate-polyacrylamidegel electrophoresis. The purified myelin protein fractions (3 [mu]g) or rmMOG (4 [mu]g) were solubilized in 15 [mu]l NuPAGE LDS Sample buffer containing NuPAGE Sample Reducing Agent (Invitrogen, Carlsbad, CA) was applied to a 4% to 12% gradient Bis-Tris gel (Invitrogen, CA). A semidry blotting system (Ancos, Hoejby, Denmark) was used to test reactivity of immune sera.

Samples were immunoblotted with preimmune or necropsy sera from marmosets immunized with WT or MOG^sup -/-^ myelin. Protein bands were confirmed by monoclonal antibodies; anti-MOG Z12 (generated in our laboratory [29]), rabbit anti-myelin-associated glycoprotein (kindly provided by N. Gregson, London, UK), mouse anti-2,3-cyclic nucleotide 3-phosphodiesterase (Chemicon, Temecula, CA), mouse anti- PLP (Chemicon), mouse anti-MBP (Dako, Glostrup, Denmark), and rabbit anti-oligodendrocyte-specific protein (Abcam, Cambridge, UK). The primary antibodies were detected with the appropriate secondary antibodies, including rabbit anti-mouse Ig/horseradish peroxidase, rabbit anti-human Ig/horseradish peroxidase, or goat anti-rabbit Ig/ horseradish peroxidase (all from Dako) and developed using enhanced chemiluminescence (Amersham, UK). The computerized system Chemidoc XRS and PDQuest (Bio-Rad, Hercules, CA) was used to calculate the molecular weight and density of the bands. TABLE. Survival, MRI, and Histologic Summary

FIGURE 2. Slice distribution of lesion load in postmortem brain magnetic resonance (MR) images. High-definition T^sub 2^-weighted MR images were made of formalin-fixed total brains. A semiquantitative assessment of the amount of hyperintense abnormalities per slice of 0.5-mm thickness is given as minor (light gray cells; [black square]), moderate (gray cells; [black square]), and high (black cells; [black square]). The arrows indicate the slices depicted for each monkey in Figure 3. Total lesion volumes for individual monkey are given in the Table. DOK, day of killing.

Enzyme-Linked Immunosorbent Assay

Microlon plates (Greiner Bio-one) were coated overnight at 4[degrees]C with 10 [mu]g/ml of rmMOG, rhMOG, or hMBP in PBS. The plates were washed twice in PBS-Tween and blocked for 1 hour at 37[degrees]C with 2% bovine serum albumin/PBS. After blocking, 100 [mu]l of diluted plasma (1:200) in 1% bovine serum albumin/PBS was added and incubated for 2 hours at 37[degrees]C. Individual animal preimmunization plasma was used as a negative control. After washing in PBS-Tween, the plates were incubated for 1 hour at 37[degrees]C with alkaline phosphatase-conjugated rabbit anti-human IgG (Abcam) or goat-anti-monkey IgM mu-chain (Rockland, Gilbertsville, PA). The reaction product was visualized using p-nitrophenyl phosphate-Tris buffer (Sigma-Aldrich, Gillingham, UK), and the absorbance read at 405 nm. Absorbances more than the mean plus 3 SD of the activity measured in preimmune sera were interpreted as positive.

FIGURE 3. Magnetic resonance imaging-detectable abnormalities in formalin-fixed brains. The brains of monkeys immunized with myelin from wild type (left panels) and their twins immunized with myelin from myelin oligodendrocyte glycoprotein-deficient mice (right panels) were mildly fixed in buffered formalin, after which T^sub 2^- weighted images were made to visualize experimental autoimmune encephalomyelitis-related abnormalities. Three slices representative of the whole brain are shown for each monkey. The slices correspond to those indicated with arrows in Figure 2. The inserts are enlargements of the boxed areas.

RESULTS

Clinical Scores and Body Weights

The EAE scores and patterns of weight loss of the 10 monkeys are depicted in Figure 1. All 5 marmosets immunized with myelin from WT mice developed overt neurologic signs of variable severity and different extents of weight loss. In 1 monkey (M03015), the disease remitted after a short episode of incomplete hindlimb paralysis and did not exacerbate during the remainder of the 180-day observation period. We observed a similar variable disease course in our previous studies in which EAE was induced with human CNS myelin (23). By contrast, only 1 of the 5 marmosets immunized with myelin from MOG^sup -/-^ mice (M02115) developed an overt neurologic deficit. In the other 4 monkeys, there were minimal signs of EAE and moderate or no weight loss. The acute EAE course and dramatic weight loss in monkey M02115 coincided with the course of its twin sibling (M02114) that had been immunized with WT myelin. This suggests that the acute EAE in this twin was not induced by MOG and was likely caused by an immune response to other myelin components.

FIGURE 4. Inflammation, demyelination, and antibody staining in spinal cord. Spinal cord histology and immunohistochemistry of a representative twin pair of monkeys immunized for experimental autoimmune encephalomyelitis with wild-type (WT) myelin (M03007; A, C, E, G) and myelin oligodendrocyte glycoprotein-deficient (MOG^sup – /-^) myelin (M03006; B, D, F, H; both x 15), hematoxylin and eosin staining (A and B). Arrowheads indicate blood vessels with perivascular inflammation. The rectangles in (A and B) enclose the area of CD3 staining shown in (C and D), respectively. (C and D) (120 x ) show infiltration of CD3-positive T cells in the spinal cord in both monkeys. (E and F) (15 x ) show Luxol fast blue/ periodic acid Schiff stain of spinal cord sections adjacent to those in (A and B), respectively. Marked subpial demyelination affecting more than 50% of the white matter is evident in monkey M03007 (WT myelin immunized). In M03006, (MOG^sup -/-^ myelin immunized), demyelination is also present but is less marked. (G and H) (210 x ) show staining for immunoglobulin (Ig)M, detecting deposition of IgM antibodies in spinal cord lesions of the animals immunized with WT myelin (G) and the MOG^sup -/-^ myelin (H). The arrowhead in (H) indicates an IgM-positive plasma cell in the meninges.

MRI and Histology

High-definition T^sub 2^-weighted postmortem images were made of the brains of each monkey to visualize white matter abnormalities. Moreover, the extent of inflammation and demyelination were quantified (Table).

Total lesion volumes per monkey are shown in the Table. To demonstrate the distribution of lesions in different brain regions in the 2 groups of monkeys, the lesion load per slice is indicated semiquantitatively in Figure 2, and representative slices corresponding to the arrows in Figure 2 are shown in Figure 3. The data show that MRI-detectable abnormalities were scattered throughout the brain white matter in both groups of monkeys. Quantitation of the lesion areas of twins show that with the exception of the acute EAE twins M02114/M02115, the total lesion volume of monkeys immunized with WT myelin brain lesions was larger than those induced with MOG^sup -/-^ myelin. The MRI findings were supported by histologic analysis of the spinal cord (Fig. 4; Table). They demonstrate that immunization with WT and MOG^sup -/-^ myelin induced similar amounts of inflammation (mean inflammatory index, 3.2 [range, 1.1-5.1] and 2.4 [range, 0.4-4.0], respectively). In contrast, immunization with MOG-containing myelin induced more demyelination (group mean, 40.5% [range, 14-56]) than immunization with MOG^sup -/-^ myelin (group mean, 8.2% [range, 0-20.3]). In 2 of the latter monkeys (M02115, M03006), more than 10% of the spinal cord white matter was demyelinated. Inflammation and demyelination in the spinal cord of the twin monkeys M03007 and M03006 are shown in Figure 4. Demyelination was also detectable in the optic nerves, although the extent was greatest in the monkey immunized with WT myelin (Fig. 5). The day of killing varied considerably among the individual monkeys, and long survivors have had more time to accumulate CNS lesions (Fig. 2; Table).

FIGURE 5. Demyelination in optic nerve. Demyelination in the optic nerve of the twin pair M03007 and M03006 that had been immunized with wild-type (WT) myelin (A, C) or myelin oligodendrocyte glycoprotein-deficient (MOG^sup -/-^) myelin (B, D), respectively. (A and B) (18 x ) show proteolipid protein (PLP) staining in the optic nerve close to the eye. (A) The optic nerve is almost completely demyelinated. The rectangle encloses the border of the demyelinated area that is shown enlarged in (C) (240 x ). (B) There is some demyelination in peripheral parts of the optic nerve, indicated by the rectangle and shown enlarged in (D) (240 x ). Arrowheads in (C) and (D) indicate macrophages with PLP degradation products in their cytoplasm.

Antibodies

The presence of anti-myelin antibodies in immune sera of each monkey was tested by immunoblotting. Antibody levels against hMBP, rhMOG, and rmMOG were tested with ELISA.

FIGURE 6. Serum antibody levels in marmosets immunized with wild- type (WT) myelin and myelin oligodendrocyte glycoprotein-deficient (MOG^sup -/-^) myelin. Immune sera collected at necropsy were tested for antibodies against WT myelin and MOG^sup -/-^ myelin by Western blotting (A). Immunoglobulin (Ig)M and IgG antibodies against the myelin proteins recombinant human MOG^sub 1-125^ (rhMOG), recombinant mouse MOG (rmMOG), and human myelin basic protein (hMBP) were measured with enzyme-linked immunosorbent assay (B, C, respectively). Antibodies against whole myelin (A). Freshly isolated myelin from WT and MOG^sup -/-^ C57/BL6 mice (3 [mu]g loaded per lane) was fractionated by electrophoresis through a 4% to 12% Bis- Tris gel. In addition, rhMOG and rmMOG (both 4 [mu]g per lane) and molecular weight marker preparation were loaded on the gel. A gel from representative twin pair is shown in the upper portion. After fractionation, the gels were blotted with 200-fold diluted necropsy serum from all twin siblings. The densitograms show the reactivity of necropsy serum from monkeys immunized with MOG^sup -/-^ (gray bars) or WT myelin (black bars). Immunoglobulin M (B) and IgG (C) antibodies against myelin proteins. Sera were collected at the indicated time points. Enzyme-linked immunosorbent assay plates coated with rhMOG, rmMOG, or hMBP were incubated with plasma at 200- fold dilution for IgM and IgG. The results are given in arbitrary units (i.e. as fold increase compared with preimmune sera).

Blotting Results

Western blots from a representative twin pair (M02055 and M02056) are shown in Figure 6A. The densitograms (lower portion of figure) show that serum from the WT myelin-immunized monkey contained higher reactivity on the blots than the serum from the MOG^sup -/-^ myelin- immunized monkey. As anticipated, antibodies recognizing the native MOG band in the WT myelin (lane B) and the rmMOG band in lane A were only found in serum from the WT myelin-immunized animal. ELISA Results

Data from IgM and IgG antibody analyses are presented in pairs for all monkeys in Figures 6B (IgM) and C (IgG). Induction of IgG antibodies against hMBP was observed in all monkeys, although differences among twin pairs and between twins were observed. For example, anti-MBP IgG levels were barely detectable in M03007 but were much higher in M03006. Moreover, antibodies appeared significantly earlier in M03015 than in M03016.

Induction of antibodies against MOG was only found in the monkeys that developed clinical EAE. In all 5 monkeys immunized with WT myelin, these were of the IgG isotype, whereas in the 1 monkey (M02115) that developed overt EAE after immunization with MOG^sup -/ -^ myelin, only anti-MOG IgM antibody reactivity was detected.

Antibody Deposition in the CNS Tissues

To assess whether the different autoantibody profiles in the serum samples were reflected in the CNS, lesions were analyzed for the presence of IgM and IgG deposits and assessed in a semiquantitative manner. The Table shows that CNS lesions of WT myelin-immunized monkeys had considerably greater antibody staining than those in MOG^sup -/-^ myelin-immunized monkeys. Representative IgM antibody deposits in spinal cord lesions are shown in Figures 4G and H. We do not know whether or not the deposited antibodies are directed against MOG.

Cellular Responses

Serial venous blood samples were collected periodically for the isolation of PBMC to probe reactivity with WT and MOG^sup -/-^ myelin, rmMOG, rhMOG, hMBP, and the overlapping 23-mer MOG peptides. Furthermore, at necropsy, MNC suspensions were prepared from the spleen and the various lymph nodes. As observed in previous studies (28), the distribution of autoreactive T cells within draining lymphoid tissues may vary during the progression of EAE; therefore, separate compartments were tested.

Peripheral Blood Mononuclear Cell

In none of the monkeys was there significant (stimulation index >2) proliferation against the MOG peptides (not shown), but proliferation against WT myelin was present (Fig. 7A). Proliferation levels were somewhat higher in siblings immunized with MOG^sup -/-^ myelin than in those immunized with WT myelin.

Lymph Node and Spleen MNCs

As was the case in PBMC, marked proliferative responses against the MOG peptides were not observed in spleen or lymph node MNCs (data not shown), but proliferative responses to MOG proteins, hMBP, and myelin were detected. The data in Figure 7B show that there is not a consistent and clear difference in proliferation in response to myelin or MOG between twin siblings. Remarkably, we observed in each twin similar response levels against rmMOG and rhMOG in siblings immunized with WT or MOG^sup -/-^ myelin. We suspect that these responses are triggered by myelin-loaded antigen-presenting cells that drain from the CNS to the spleen (30).

Cytokines

Various cytokines, that is, IL-2, IL-12, IL-13, tumor necrosis factor, and interferon-gamma, were determined in culture supernatants of spleen and axillary lymph node cells stimulated with WT myelin or rmMOG. A high degree of variation of cytokine levels was observed, but no consistent differences were found between twin siblings immunized with WT MOG or MOG^sup -/-^ myelin (data not shown).

DISCUSSION

The experiments reported here were conducted in the EAE model in the common marmoset, a small neotropical primate species. Experimental autoimmune encephalomyelitis in the marmoset was first proposed as a nonhuman primate model for MS approximately a decade ago (31). In addition to its outbred nature, the marmoset has several immunologic similarities with humans. Of particular relevance to modeling of MS are similarities at the level of genes encoding the variable elements of T-cell receptors (32) and Igs (33), major histocompatibility complex class II molecules (34, 35), T-cell and antigen-presenting cells, costimulatory molecules, and cytokines (36). Moreover, cross-reactivity of monoclonal antibodies against human CD markers with marmoset leukocytes has been demonstrated (37). The clinical and neuropathologic similarities between the marmoset EAE model and MS have also been previously reviewed (5, 38). There is an ongoing debate m the literature on the question whether patients presenting with clinically isolated symptoms, which are also seropositive for anti-MOG or anti-MBP antibodies, have an increased chance to develop clinically definite MS in later life (39-41). The present results show that immunization of marmosets with MOG^sup -/-^ mouse myelin induces overt clinical EAE only in 1 of 5 monkeys. However, with MRI and with histology, abnormalities typical for EAE were detected in the brain and the spinal cord of the MOG^sup -/-^ immunized monkeys, in which only 1 monkey in this group resulted in overt clinical EAE. One abnormality observed in the 1 monkey that developed EAE was the presence of anti- MOG IgM antibodies, although it was immunized with MOG^sup -/-^ myelin. We conclude that antibodies against MOG are dispensable for the induction of early pathogenic mechanisms, but that they strongly impact the full development of overt clinical EAE. Essentially, similar results have been reported for the chronic relapsing EAE model in Biozzi ABH mice, that is, that mice immunized with MOG^sup – /-^ myelin display a short episode of neurologic deficit and CNS inflammation but lack the subsequent relapses typically found in mice immunized with WT myelin (4).

FIGURE 7. Proliferation of mononuclear cells (MNCs) in peripheral blood and lymphoid organs. (A) Blood MNC responses to wild-type (WT) myelin and recombinant human myelin oligodendrocyte glycoprotein^sub 1-125^ (rhMOG). Venous blood samples of 1 ml were collected at the indicated PSDs to isolate MNCs. Mononuclear cells were cultured in triplicate with WT myelin or rhMOG, and after 18 hours, incorporation of ^sup 3^H-thymidine was determined as a measure of proliferation. Results are expressed as S1 versus cultures without antigen. (B) Lymph node and spleen MNC responses against myelin and myelin proteins. At necropsy, the spleen and axillary and inguinal lymph nodes were aseptically removed, and MNC suspensions were prepared. Mononuclear cells were cultured with WT myelin, MOG^sup -/ -^ myelin, rhMOG, recombinant mouse MOG, or human myelin basic protein, and proliferation was determined by the incorporation of ^sup 3^H-thymidine during the final 18 hours of 3-day cultures. Results are expressed as S1 versus cultures without antigen. Only S1 values above the dotted lines are considered relevant. MOG^sup -/- ^, MOG-deficient myelin; PSD, postsensitization day; S1, stimulation index. The maximum blood volume that can be obtained from an adult marmoset each month without harming the monkey’s health is 1% of the body weight (i.e. 3.5-4 ml). Because of this, and the fact that blood samples cannot be pooled in this outbred model because each animal must be regarded as unique, extensive longitudinal immunologic testing is not feasible. We therefore tested PBMC only for reactivity with WT myelin and at some time points also against rhMOG. Comparison between PBMC of twin siblings showed that in 4 twin pairs (i.e. other than M03015/ M03016), the proliferative responses against WT myelin were higher in siblings immunized with MOG^sup -/-^ myelin than in the twin immunized with WT myelin. By contrast, we observed that in most twin pairs (i.e. other than M02114/ M02115), MNCs from lymphoid organs of WT myelin-immunized monkeys showed a higher proliferative response against the myelin preparations and with the myelin proteins rhMOG, rmMOG, and hMBP. These data indicate that opposite results can be obtained depending on the compartment analyzed.

Mononuclear cell reactivity with MOG peptides was not observed in any monkeys, although such responses are normally present in monkeys immunized with 100 [mu]g rhMOG (11), that is, the amount estimated to be present in 3 mg of myelin inoculum used for EAE induction (4). We previously proposed that the full-length MOG in its natural glycosylated conformation as present in the myelin inoculum may dampen T-cell epitope spreading (42). This yin and yang paradigm postulates that (self-)glycoproteins inhibit the capacity of dendritic cells to activate autoreactive T-cells by binding to certain C-type lectin receptors (CLRs) via their glycan epitopes. Support for this hypothesis comes from the observation that mice can be rendered tolerant to EAE by targeting of the immunizing antigen to the CLR DEC-205 (43). Preliminary experiments show that the binding of mouse myelin to DC-SIGN, a CLR expressed on primate DC, is mediated via MOG (unpublished observation). Moreover, ligands of the CLR DC-SIGN such as ManLam from mycobacteria antagonize DC maturation and IL-12p40 production (44). Thus, myelin binding to DC- SIGN may reduce the capacity of DC to induce the autoaggressive T cells that drive the progression of EAE. The observation that B- cell activation occurs (relatively) independent of Toll-like receptor activation (45) may explain that autoantibody production was not inhibited.

Our data show that development of overt clinical EAE is associated with seropositivity for anti-MOG antibodies. The observation that demyelination is less severe in monkeys immunized with MOG^sup -/-^ myelin is consistent with the previous observation in the marmoset EAE model that passively transferred anti-MOG antibodies amplify demyelination (16). Our data clearly show, however, that in monkeys immunized with MOG^sup -/-^ myelin, significant demyelination can take place. Therefore, we conclude that demyelination is not absolutely dependent on anti-MOG antibodies. The remarkable finding that anti-MOG IgM antibodies were present in monkey M02115, although this monkey had been immunized with MOG^sup -/-^ myelin, also has a precedent in the literature. McFarland et al (46) reported that anti-MOG antibodies are occasionally induced in marmosets immunized with the MBP/PLP chimeric protein MP4, and that EAE developed only in the monkeys in which anti-MOG antibodies had been formed.

In conclusion, the current data obtained in marmosets together with previously reported data in the Biozzi ABH mouse illustrate that autoimmunity against MOG, although it is a quantitatively minor component of CNS myelin, plays a prominent role in the development of chronic EAE. These data support the concept that seropositivity for anti-MOG antibodies is a significant risk factor for chronic encephalomyelitis. We tend to agree with Gaertner et al (47), however, that serologic assays tailored to the detection of antibodies against glycosylation variants of MOG may be more informative for MS than those detecting only the nonglycosylated (recombinant) protein.

ACKNOWLEDGMENTS

The authors thank Fred Batenburg for superb biotechnical support, Drs. Jaco Bakker and Gerco Braskamp for veterinary care, and Dr. Rogier Hintzen (Erasmus Medical Center Rotterdam) for critical reading of the article.

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S. Anwar Jagessar, BSc, Paul A. Smith, PhD, Erwin Blezer, PhD, Cecile Delarasse, MSc, Danielle Pham-Dinh, PhD, Jon D. Laman, PhD, Jan Bauer, PhD, Sandra Amor, PhD, and Bert ‘t Hart, PhD

From the Department of Immunobiology (SAJ, PAS, SA, BtH), Biomedical Primate Research Centre, Rijswijk; Department of Immunology (JDL, BtH), Erasmus Medical Centre Rotterdam; Imaging Science Institute (EB), University Medical Centre Utrecht, Utrecht, The Netherlands; Institut National de la Sante et de la Recherche Medicale (INSERM) (CD, DPD), Universite Pierre et Marie Curie, Hopital de la Salpetriere, Paris, France; Division of Neuroimmunology (JB), Center for Brain Research, Medical University of Vienna, Vienna, Austria; and MS Centre ErasMS (SAJ, JDL, SA, BtH), Rotterdam, The Netherlands.

Send correspondence and reprint requests to: Bert A. ‘t Hart, PhD, Department of Immunobiology, Biomedical Primate Research Centre, PO Box 3306, 2280GH Rijswijk, The Netherlands; E-mail: hart@bprc.nl

This work has been financially supported by the Dutch MS Research Foundation via the program grant ErasMS and Project Grant No. 00- 417MS.

S. Anwar Jagessar and Paul A. Smith have contributed equally to the work.

Drs. Sandra Amor and Bert ‘t Hart share senior authorship.

Topics: Health Medical Pharma, Neurology, Nervous system, Glycoproteins, antibodies, B cell, Antibody, Oligodendrocyte, Monoclonal antibodies, Experimental autoimmune encephalomyelitis, Myelin, multiple sclerosis, Myelin oligodendrocyte glycoprotein, Immune system, demyelination

Autoimmunity Against Myelin Oligodendrocyte Glycoprotein Is Dispensable for the Initiation Although Essential for the Progression of Chronic Encephalomyelitis in Common Marmosets

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Autoimmunity Against Myelin Oligodendrocyte Glycoprotein Is Dispensable for the Initiation Although Essential for the Progression of Chronic Encephalomyelitis in Common Marmosets

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