GFAP mRNA fluctuates in synchrony with chronic relapsing EAE symptoms in SJL/J mice

Activation of astrocytes and hypertrophy of their processes is a result of a number of pathological conditions in the central nervous system. Astrocytic gliosis is especially prominent in multiple sclerosis (MS), where astrocytic fibers form a dense matrix around demyelinated axons. Experimental allergic encephalomyelitis (EAE), a laboratory model for MS, is also accompanied by astrocytic hyperactivity. We have previously shown the formation of plaque-like structures which stain heavily for glial fibrillary acidic protein (GFAP) in the brains and spinal cords of SJL/J mice after several episodes of chronic relapsing EAE (Smith and Eng: J Neurosci Res 18:203, 1987). To further investigate the mechanisms of this phenomenon, we have measured the levels of mRNA for GFAP throughout the course of three episodes and recoveries of EAE in the SJL/J mouse. Mice were immunized with spinal cord homogenate and subsequently developed EAE. After recovery they were again immunized at appropriate intervals, resulting in successive episodes of EAE, with partial or complete recovery between the paralytic stages. At appropriate times in the course of the different stages of EAE, spinal cords were dissected and RNA was prepared from each spinal cord. RNA Was analyzed by Northern blots to determine the levels of mRNA for GFAP and, as a control, for the 70 kDa neurofilament (NF-L). With the onset of the first EAE episode GFAP mRNA in spinal cords from animals with mild symptoms increased to sixfold the control level (P < 0.02) and to 20-fold in those with paralysis (P < 0.01). With recovery, the GFAP mRNA level decreased to twice the control. With each subsequent episodes, a chronic but stable neurological deficit was established, with GFAP mRNA at about eightfold the control levels (P < 0.01). Over the course of several episodes, the GFAP rose to about 2.8 times the control, while vimentin increased by a factor of 3.6. Thus multiple episodes of EAE resulted in upregulation of GFAP mRNA and accumulation of GFAP, which are associated with astrocyte activation and hypertrophy. Similar events may occur in the human demyelinative disease MS, where multiple episodes of inflammatory cell invasion occur, resulting in a neurological deficit. © 1995 Wiley-Liss, Inc.     

Delayed,relapsing experimental allergic encephalomyelitis in mice: Role of adjuvants and pertussis vaccine

The effect was studied of varying the dosage of mycobacteria and pertussis vaccine in the immunization emulsion on the development of delayed, relapsing experimental allergic encephalomyelitis (DR-EAE) in SJL/J mice. DR-EAE could be induced with dosages of mycobacteria from 0.2 mg to 1.0 mg per mouse. Animals given pertussis vaccine from different batches and different sources all developed DR-EAE with similar percentages of animals sick, animals relapsing, and delay to onset. Mice immunized without pertussis vaccine also developed DR-EAE but had fewer relapses and a longer delay to onset. The role of antigens and adjuvants in relapsing EAE and analogies with multiple sclerosis are discussed.     

Adoptive transfer of murine relapsing experimental allergic encephalomyelitis

Relapsing experimental allergic encephalomyelitis (EAE), an autoimmune disorder resembling multiple sclerosis, has been produced by inoculating SJL/J mice with spinal cord or myelin basic protein in appropriate adjuvants. To determine whether initially sensitized lymphocytes or the persistence of antigen depots in the animal were responsible for the relapsing episodes of inflammatory demyelination, adoptive transfer studies were undertaken utilizing lymphocytes from relapsing EAE-immunized donors transferred directly or after in vitro culture. In direct-transfer studies donor lymphocytes produced clinical and pathological signs of relapsing EAE in 3 of 7 recipients of lymph node lymphocytes and 1 of 5 recipients of splenic lymphocytes. In vitro culture of lymphocytes in myelin basic protein or T cell growth factor prior to transfer increased both the incidence of disease and the number of animals having relapses, and allowed transfer with fewer lymphocytes. Because all animals had delayed onset of disease, this study demonstrates that the ability to develop relapsing inflammatory demyelination is transferable with lymphocytes and does no require the presence of antigen.     

Novel monoclonal antibodies against proteolipid protein peptide 139-151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions

Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with epitopes of the proteolipid protein (PLP), a major myelin constituent, forms a useful model for the study of multiple sclerosis (MS). In addition, MS patients display PLP-specific T- and B-cell responses, suggesting that PLP reactivity is relevant to pathogenesis.Here, the generation and characterization of a panel of mouse monoclonal antibodies (Mab) against PLP139-151, the prominent encephalitogenic sequence in SJL/J mice is described. Five Mab were generated by conventional immunization of an SJL/J mouse and hybridoma generation. These Mab reacted well with the PLP139-151 peptide in ELISA and belonged to the IgG2a and IgG2b subclasses, consistent with CD4+ T helper 1-cell-supported antibody formation. The Mab also efficiently detected PLP peptide-BSA conjugates in Western blot, confirming their multi-assay applicability.The Mab were subsequently used to determine the occurrence of demyelination in brains of MS patients and marmoset monkeys with EAE. Immunohistochemistry on both paraffin and frozen sections demonstrated a homogeneous expression of PLP139-151 in normal myelin, and a complete absence in lesions containing demyelinated areas, confirming that the Mab can be used as a general myelin marker. In active demyelinating MS lesions, the Mab visualized the peptide in the cytoplasm of macrophages containing phagocytosed myelin.In conclusion, this panel of Mab against the encephalitogenic PLP139-151 epitope forms a useful tool for further study of autoantigen expression, demyelination/remyelination and the staging of lesional activity in MS patients, as well as in EAE models in distinct animal species.     

[3H]Thymidine labeling of astrocytes in experimental allergic encephalomyelitis

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [³H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11–15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [³H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.     

[H]Thymidine labeling of astrocytes in experimental allergic encephalomyelitis

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [³H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11–15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [³H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.     

PLP不同肽段诱导EAE模型的对比研究

目的建立不仅与多发性硬化(multiple sclerosis，MS)临床表现、病理特征接近而且病程相似的较为理想的复发一缓解型实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)模型。方法应用髓鞘蛋白脂质蛋白(proteolipid potein，PLP)多肽的两种免疫优势表位肽段PLP139-151和PLP178-191免疫雌性SJL／J小鼠，制作复发缓解型EAE(relapse remitting experimental autoimmune encephalomyelitis，RR—EAE)模型，观察其体重及神经功能评分的变化，应用HE、Luxol fas tblue髓鞘染色等方法观察模型的组织形态学改变。结果两种PLP肽段免疫的小鼠发病均具有缓解一复发的特点，出现明显的神经系统体征；小鼠发病时脑和脊髓组织显示明显的血管鞘形成、卫星现象和炎性细胞浸润以及脱髓鞘改变。结论PLP两种肽段均可诱发RR-EAE模型，这与临床MS的缓解一复发病程更相似，更能表现MS的临床特点，是研究MS的较为理想的动物模型。     

Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis

Spinal cord sections from Lewis rats with acute experimental allergic encephalomyelitis (EAE) showed greatly increased staining of astrocytes when stained immunocytochemically for glial fibrillary acidicc protein (GFAP). Fibrous processes in white matter were heavily stained early in the course of the disease when paralysis was first evident (10–12 days after injection of guinea pig spinal cord myelin), then protoplasmic astrocytes were stained in the gray matter and became more heavily stained at 20 dats post-injection. The stained astrocytes were evenly distributed throughout the tissue, and did not correspond to the sites of the lesions. Spinal cord slices of control and EAE rats were incubated with [³H]amino acids, then cytoskeletal proteins were prepared in an enriched fraction, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands counted for radioactivity. In the EAE rat all cytoskeletal proteins, including the neurofilaments, vimentin, microtubules, GFAP and actin, showed increased uptake of radioactive amino acids. Immunoprecipitation of GFAP with specific antiserum showed increased radioactivity in the complex beginning at day 10 when cellular infiltration was beginning in the EAE animals. As the disease became acute, the radioactivity in the immunoprecipitated GFAP increased, in some cases to very high levels, then by day 18 when recovery was underway, the radioactivity had fallen to normal levels. Possible agents causing metabolic activation of protein synthesis in EAE animals include stimulating substances elaborated by infiltrating lymphoid scells, and the generalized edema accompanying the demyelinative condition. The activation of GFAP protein staining and metabolism in EAE might serve as a model for the activated growth of astrocyte processes which cause the severe gliosis seen in multiple sclerosis.     

Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-α), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-α suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN-γ secretion in both donors and recipients. IFN-α acts orally by modifying the encephalitogenicity of donor spleen T cells.     

PSGL-1 is dispensible for the development of active experimental autoimmune encephalomyelitis in SJL/J mice

The adhesion molecule P-selectin glycoprotein ligand (PSGL)-1 has been suggested to be involved in the immunopathogenesis of multiple sclerosis (MS). However, in C57BL/6 mice PSGL-1 was found to be dispensible for the development of MOG(aa35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS. To study, if involvement of PSGL-1 to EAE pathogenesis can be observed in another common mouse model, we backcrossed PSGL-1(-/-) mice for at least 12 generations into the SJL/J background and compared PLP(aa139-151) induced EAE in PSGL-1(-/-) SJL/J mice versus wild-type SJL/J mice. Here, we demonstrate that PSGL-1(-/-) SJL/J mice exhibited EAE pathogenesis indistinguishable from wild-type SJL/J mice. Our present study underscores and emphasizes previous observations that PSGL-1 is dispensible for EAE pathogenesis.     

Myelin basic protein and myelin-associated glycoprotein in chronic, relapsing experimental allergic encephalomyelitis

One-micron plastic sections of spinal cords from SJL/J mice with chronic relapsing experimental allergic encephalomyelitis (EAE) were reacted immunocytochemically with antiserum to myelin basic protein and myelin-associated glycoprotein. The distribution of myelin basic protein and myelin-associated glycoprotein in myelin sheaths was compared in acute and chronic areas of demyelination. No difference in the size of the lesion was seen with the two antisera. Myelin-associated glycoprotein was seen periaxonally in both normal myelin sheaths and sheaths which showed extensive splitting and ballooning as seen with toluidine blue stain and myelin basic protein antiserum. At least at the level of the light microscope, myelin basic protein antiserum gave intense staining of myelin while antiserum to myelin-associated glycoprotein showed little or no affinity to stain the myelin sheath itself, in contrast to other recent electron microscope observations. A few myelin basic protein or myelin-associated glycoprotein-containing oligodendrocytes were seen in lesion areas and remyelination by oligodendrocytes was rare. These observations are in agreement with findings from other models of EAE and multiple sclerosis where a primary loss of myelin has been implicated.     

Chronic relapsing experimental allergic encephalomyelitis in SJL mice following the adoptive transfer of an epitope-specific T cell line

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in SJL mice following the adoptive transfer of a T cell line derived from mice immunized with a synthetic peptide corresponding to residues 89-100 of the guinea pig myelin basic protein (MBP) molecule. This cell line proliferated to both the peptide and MBP and induced CREAE characterized by a series of relapses with eventual stabilization. Central nervous system (CNS) inflammation and demyelination were prominent neuropathologic features of both the acute and relapsing phase of the disease. The chronic phase was characterized by CNS lesions containing chronically demyelinated fibers with remyelination and some fiber drop-out, but little inflammation. The induction of CREAE in SJL mice by a cell line specific for residues 89-100 demonstrates that T cell recognition of this epitope is required for successful disease induction in this strain of mouse.     

Sequence 104–117 of myelin proteolipid protein is a cryptic encephalitogenic T cell determinant for SJL/J mice

Immunization of animals with myelin proteolipid protein (PLP) causes experimental autoimmune encephalomyelitis (EAE), a disease model that shares many features with human multiple sclerosis (MS). The SJL/J (H-2⁵) mouse is widely used in EAE studies because of its high disease susceptibility. Previous studies have shown that sequences 139–151 HCLGKWLGHPDKF and 178–191 NTWTTCQSIAFPSK represent distinct co-immunodominant encephalitogenic determinants of PLP for SJL/J mice. In the present study, we identify a third distinct PLP encephalitogenic peptide for SJL/J mice. Following immunization with PLP 104–117 KTTICGKGLSATVT, 10/14 SJL/J mice developed clinical and histological EAE with a mean time of onset of 38 days (18–65 days). T cell lines generated from SJL/J mice immunized with p104–117 were predominantly (> 90%) CD3⁺, CD4⁺, αβTCR⁺, CD8^dim, γδTCR^dim T cells and responded in an Ag-specific, I-As-restricted manner to p104–117. Upon adoptive transfer of 16−40 × 10⁶ T line cells, EAE was produced in naive SJL/J recipients 20–34 days after transfer. The delayed onset of both active and passive disease may be related to the non-immunodominant, cryptic nature of p104–117 in SJL/J mice. Lymph node cells from SJL/J mice immunized with either whole PLP or with pooled encephalitogenic PLP peptides responded to challenge with the immunodominant PLP determinants p139–151 and p178–191 but did not respond to p104–117. The existence of three distinct PLP encephalitogenic T cell determinants for SJL/J mice suggests that susceptibility to EAE and perhaps MS may be related to promiscuous T cell recognition of multiple myelin protein determinants.     

T cell lines selected with synthetic peptides are highly encephalitogenic in SJL/J mice

T cell lines were selected from basic protein (BP)-immunized SJL/J mice using synthetic peptides encompassing the major SJL/J encephalitogenic determinant. Synthetic peptide-derived T cell lines proliferated in response to BP, the 89-169 peptidase fragment of BP and the synthetic peptides, pM87-99, pM90-99 and pM91-99. These lines transferred a demyelinating and chronic relapsing form of experimental autoimmune encephalomyelitis (EAE) into naive mice, and EAE induced by synthetic peptide-derived lines was more severe than that induced by whole BP-derived lines. This study demonstrates that T cell lines selected with synthetic peptides are encephalitogenic in SJL/J mice and offers an improved means for selecting SJL/J encephalitogenic T cell lines.     

Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors

The proinflammatory Th1 cytokine, tumor necrosis factor-α (TNFα), the cell death signaling molecule FasL, and several extracellular matrix degrading metalloproteinases have been implicated in the pathogenesis of multiple sclerosis (MS). The latter enzymes, as well as TNFα-converting enzyme and FasL-converting enzyme, can be blocked by matrix metalloproteinase inhibitors (MMPIs). In this study, we show that a potent MMPI was clinically effective in an animal model for MS, experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse. Efficacy was remarkable, as indicated by blocking and reversal of acute disease and reduced number of relapses and diminished mean cumulative disease score in chronic relapsing animals. Also, demyelination and glial scarring were significantly decreased in MMPI-treated mice with chronic relapsing EAE, as was central nervous system gene expression for TNFα and fasL. It is interesting that expression of the beneficial cytokine interleukin-4 (IL-4) was increased, and IL-4 was expressed on glial cells. The relevance of these compounds for MS was underscored by their ability to specifically inhibit TNFα shedding and cytotoxicity of myelin-autoreactive human cytotoxic CD4⁺ T-cell clones. This is the first report to show a positive effect by MMPIs on chronic relapsing EAE, its central nervous system cytokine profile, and on TNFα shedding by human myelin-autoreactive T cells.     

Sex differences in experimental autoimmune encephalomyelitis in multiple murine strains

Multiple sclerosis (MS) is more prevalent in women than men. We evaluated seven different mouse strains commonly used in the study of autoimmune diseases, for sex differences in the disease course of experimental autoimmune encephalomyelitis (EAE). Greater severity of EAE was observed in the female SJL immunized with two different peptides of myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) as well as in the female ASW relative to males. Female NZW mice showed a greater incidence of EAE than males. However, male B10.PL and PL/J mice showed more severe disease than females. No sex differences were noted in the C57BL/6 or NOD strains.     

Treatment of relapsing experimental autoimmune encephalomyelitis with T cell receptor peptides

Restricted T cell receptor (TCR) VB gene usage by T cells for recognition of antigens involved in the production of experimental autoimmune encephalomyelitis (EAE) offers the possibility of selective immunotherapy. We determined the preferential VB gene usage of lymph node-derived clones from SJL/J mice to recognize the encephalitogenic epitope PLP 139-151 and from PL/J mice to recognize the newly described encephalitogenic epitope PLP 43-64. In addition, the VB gene usage for recognition of PLP 139-151 by T cell lines derived from SJL/J spinal cords was analyzed. Lymph node-derived SJL/J lines and clones specific for PLP 139-151 expressed VB2, VB4, and VB17a preferentially, and PL/J lines and clones specific for PLP 43-64 expressed VB2 and VB8.2 preferentially. A VB4+ SJL/J clone and a VB8.2+ PL/J clone were encephalitogenic. Encephalitogenic SJL/J lines derived from spinal cord expressed VB2, VB10, VB16, and VB17a preferentially, with a predominance of VB2. Candidate TCR peptides were synthesized and tested from the VB gene families VB4, VB8.2, and VB17a, based on our data and previous data on BP-induced EAE in mice. Treatment of relapsing EAE (R-EAE) in SJL/J mice with VB4 and VB17a peptides reduced clinical and histological disease severity, and treatment of R-EAE in (PLxSJL)F1 mice with VB4 and VB8.2 peptides also reduced clinical and histological disease. The use of TCR peptide therapy may have applications for the treatment of human autoimmune diseases such as multiple sclerosis. © 1993 Wiley-Liss, Inc.     

Astrocytic reactivity and intermediate filament metabolism in experimental autoimmune encephalomyelitis: The effect of suppression with prazosin

In either actively or passively transferred experimental autoimmune encephalomyelitis (EAE), increased immunocytochemical staining of glial fibrillary acidic protein (GFAP) in astrocytes was detected early in the disease process in both the gray and white matter of the spinal cord. Staining was not restricted to areas of perivascular mononuclear infiltration, and was observed at all levels of the cord. This enhanced staining pattern was delayed in rats in which clinical signs of EAE had been suppressed by treatment with the alpha 1-adrenoceptor antagonist prazosin. This glial reaction in EAE was not accompanied by increased GFAP synthesis, as measured by in vitro labeling of spinal cord slices, nor an increase in GFAP content, as measured by densitometry of intermediate filament fractions separated by polyacrylamide gel electrophoresis. Total protein synthesis was increased, with vimentin being labeled especially heavily; in prazosin-treated EAE animals, the increase in total protein synthesis was reduced and delayed.     

Treatment of relapsing autoimmune encephalomyelitis with T cell receptor Vβ-specific antibodies when proteolipid protein is the autoantigen

Monoclonal antibodies (mAbs) directed against the Vβ chain of the T cell receptor (TCR) of pathogenic T cells have been used to treat acute murine experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (BP). We evaluated anti-Vβ mAb for the treatment of relapsing EAE (R-EAE) induced in SJL/J mice by the myelin proteolipid protein (PLP) peptide 139–151. Spinal cord mononuclear cells isolated from mice immunized for R-EAE with PLP 139–151 were shown to express a predominance of Vβ2 and Vβ17 during acute and relapsing disease. T cell lines specific for PLP 139–151 were magnetically sorted to express 80–90% Vβ2. These Vβ2-enriched lines induced typical relapsing demyelinating EAE in naive recipient mice. SJL/J mice with R-EAE induced by a PLP 139–151-specific T cell line expressing 88% Vβ2 were treated with anti-Vβ2 mAb. Anti-Vβ2 mAb markedly reduced clinical and histological disease severity when given at the time of cell transfer or when given at clinical disease onset. In contrast, anti-Vβ mAbs showed only a mild clinical effect on R-EAE induced by immunization with PLP 139–151 or R-EAE induced by immunization with PLP 139–151 or R-EAE transferred by a PLP 139–151-specific T cell line expressing multiple Vβs. A cocktail of mAbs directed against Vβ2, Vβ4, and Vβ17 significantly reduced the numbers of spinal cord T cells expressing these Vβs during acute EAE but had little effect on disease course, suggesting that pathogenic T cells expressing other Vβs were producing disease. These findings may have implications for the treatment of multiple sclerosis with Vβ-selective therapy. © 1996 Wiley-Liss, Inc.     

The adoptive transfer of chronic relapsing experimental allergic encephalomyelitis with lymph node cells sensitized to myelin proteolipid protein

In this report we describe the transfer of chronic relapsing experimental allergic encephalomyelitis (EAE) with in vitro-stimulated lymph node cells (LNC) from SJL/J mice immunized with human myelin proteolipid protein (PLP). No additional immune enhancing procedures were applied in the transfer recipients. Clinical and histological EAE was transferred with 10-30 X 10(6) LNC to 27/28 mice. The LNC proliferated in vitro to PLP, but not to myelin basic protein (MBP), and induced delayed-type hypersensitivity. Enrichment for lymphoblasts by Ficoll centrifugation was essential for the disease development. The clinical course usually showed an early episode of acute paralytic illness, followed by chronic relapsing disease, and resembled the transfer of EAE using MBP-specific cells, both clinically and histologically.