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There are several diverse rat models of experimental autoimmune encephalomyelitis (EAE) that can be used to investigate the pathogenesis and regulation of autoimmunity against CNS myelin. The disease course of these models ranges from an acute monophasic disease with limited demyelination to a chronic relapsing or chronic progressive course marked by severe demyelination. These models enable the study of encephalitogenic T cells and demyelinating antibody specific for major neuroantigens such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP), among other important CNS autoantigens. Overall, this unit provides an overview of common methods for induction of active and passive EAE, assessment and analysis of clinical disease, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. This unit also provides a brief discussion of the basic characteristics of these models., (Copyright 2009 by John Wiley & Sons, Inc.)

It has previously been reported that synthetic peptides corresponding to sequences derived from T-cell receptor variable regions identified as dominant in the T-cell-mediated autoimmune disease experimental allergic encephalomyelitis in both the mouse and the rat can down-regulate disease in Lewis rats. In contrast to these results, we have found that immunization of Lewis rats with such peptides in complete Freund's adjuvant prior to induction of experimental allergic encephalomyelitis with myelin basic protein leads to responses ranging from profound disease enhancement to lack of disease. In some cases, enhanced disease was followed by a prolonged neurologic deficit that resembles multiple sclerosis more closely than does acute experimental allergic encephalomyelitis. These findings, on the one hand, support previous results showing T-cell receptor peptide-induced modulation of the disease experimental allergic encephalomyelitis and, on the other, indicate that such immunization is not a reliable method for inducing suppression of encephalitogenic effector cells.

The C-terminal end of the myelin basic protein (BP) molecule (peptide 117.170) was obtained by cleavage with BNPS-skatole, and purified by gel filtration. Peptide 117-170 from guinea pig BP induced experimental allergic encephalomyelitis (EAE) in Lewis rats, whereas the corresponding peptide from bovine BP was inactive. Neither peptide showed more than trace activity when tested in guinea pigs. Guinea pigs and rats treated with peptide 117-170 in incomplete Freund adjuvant were not rendered unresponsive to EAE induced by subsequent challenge with an encephalitogenic emulsion of BP in complete Freund adjuvant. Indeed, peptide-pretreated rats tended to develop clinical EAE significantly earlier than untreated controls.

The first step towards understanding the cellular interaction which results in autoimmune disease is to determine what triggers the recognition between a specific autoimmune antigen determinant and the cellular receptor. In this review, we have focused on the antigen inducing experimental allergic encephalitis (EAE) because the antigen has been characterized and a relatively large body of information on its biological activities has been accumulated. Clearly, a specific allergic encephalitis-producing determinant is present and is represented on a relatively small portion of the molecule. The determinant induces a wide variety of biological reactivities, some of which are classed as cellular mediated. An attempt is made to dissect activities such as blast transformation (BT), migration inhibitory factor (MIF), in vivo delayed type hypersensitivity reaction (DTH) and EAE and to relate them to the structural requirements which the determinants possess. The complexities which arise indicate that subpopulations of cells with different receptor activities may respond selectively and that recognition of the receptor is produced by an EAE determinant consisting of three amino acids in a specific linear sequence. Furthermore, under experimental circumstances the EAE activity can be dissociated from the other activities (BT, MIF, DTH), indicating that while these tests are used generally to follow various human autoimmune disease activities, they may represent the reaction of a broad spectrum of cells.

Mouse myelin basic proteins (MBP) were prepared by ion-exchange chromatography of an acid extract of homogenized, delipidated mouse brain. The use of a linear salt gradient during ion-exchange chromatography gave three peaks, 1, 2, and 3, of MBP. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of these peaks showed that peaks 2 and 3 were a mixture of the large and small forms of mouse MBP, whereas peak 1 was solely the small form. Tryptic peptides of mouse and rat small form MBP were compared by high-performance liquid chromatography. The elution times and absorbances at 210 nm were identical for 16 of 21 peaks derived from small form MBP of the two species. Amino acid analysis showed that 10 corresponding peaks from the small form of mouse and rat MBP had identical amino acid composition. When tested for encephalitogenicity in SJL/J mice, both the mixture of large and small forms of mouse MBP and the small form mouse MBP were highly encephalitogenic.

Hartley guinea pig central nervous system (CNS) myelin has been purified and fractionated into its protein and lipid components. Experimental allergic encephalomyelitis (EAE) was induced in juvenile strain 13 guinea pigs with both lyophilized and fresh 'wet' myelin. However, a larger dose of lyophilized myelin was required to induce chronic EAE. Total myelin lipids, galactocerebrosides, gangliosides, phospholipids or proteolipids were combined with a non-encephalitogenic dose of myelin basic protein (MBP) and injected in juvenile Hartley guinea pigs. No clinical or histological manifestations of disease were observed. Parameters of immune functions indicated that the total myelin lipids augmented cell-mediated immune responses as measured by in vitro lymphocyte transformation and by a significant decrease in the percentage of peripheral early T cells. Only the proteolipids elicited delayed hypersensitivity reactions. Animals that received the phospholipid-MBP combination showed no changes when compared to animals injected with MBP alone. The results suggest that although the myelin lipids did not act synergistically with a non-encephalitogenic dose of MBP to induce EAE, they induced immunological changes and potentiated the immune response to MBP.

Two co-extractable myelin basic proteins (MBP) were isolated and purified from mouse brain and designated: large (L), estimated to be composed of 160-164 amino acid residues, and small (S), estimated to contain 114-115 residues. The two proteins migrate separately in polyacrylamide gel electrophoresis (PAGE) in a pattern similar to rat MBP-S and MBP-L; mouse MBP-L resembles rat MBP-L, human, bovine and guinea pig MBP by PAGE and by amino acid analysis. This report demonstrates for the first time that mouse MPB-L alone, and not mouse MBP-S, is encephalitogenic for guinea pigs and mice.

Encephalitogenic protein fraction (BEC) was isolated from bovine brain tissue by extraction with salt-ethanol mixture at neutral pH, instead of employing dilute mineral acid. The fraction BEC was separated into two fractions. An acid-soluble protein was encephalitogenic and the major component was very alike to the basic protein of myelin (Al). The other was acid-insoluble acidic protein that was not encephalitogenic even at a dose of 100 mug. The acidic protein formed an insoluble complex with Al rotein which was purified by Eylar's method. Encephalitogenic activity of the complex was higher than Al protein in young guinea pigs when injected with complete Freund's adjuvant.However, this enhancement of encephalitogenic activity was not observed in aged guinea pigs. The complex showed higher blastogenic activity than Al protein alone with peripheral blood lymphocytes from guinea pigs immunized with Al protein and complete Freund's adjuvant. These results show that an adjuvant-like acidic protein is present in brain tissue and the complex with Al protein enhances the induction of experimental allergic encephalomyelitis (EAE).

Encephalitogenic, immunogenic properties of the polypeptide fraction of myelin basic protein (FBP) and CNS lesions have been examined in animals with experimental allergic encephalomyelitis (EAE). FBP was isolated from bovine spinal cord using column chromatography. Administration of 1.0 or 0.1 microgram FBP mixed with complete Freund adjuvant caused neurological and histological EAE manifestations in 76% and 26% of guinea-pigs, respectively. Circulating anti-FBP antibodies were not found in sensitized animals, whereas the incidence and intensity of skin reaction of delayed type hypersensitivity to FBP correlated with the development of EAE and the onset of the disease. Perivascular cell infiltration and demyelination noted in the spinal cord and brain of guinea-pigs were similar to those observed after inoculation of the brain white matter or brain tissue homogenate.

A basic protein has been purified from sheep brain. The purified protein sedimented in the analytical centrifuge at 56,000 r.p.m. as an homogenous product. This protein induced an allergic encephalitis when injected into guinea pigs. Some physiochemical properties of the protein were studied: the sedimentation coefficient was 1.52 and the molecular weight was 20,000 +/- 2,000, as estimated by electrophoresis in acrylamide gels containing SDS and urea; the specific extinction coefficient (see article) was 6.01 +/- 0.20. The aminoacid composition of the molecule was determined and its most prominent aspects are a high content of arginine and lysine, the presence of a single tryptophan, the total absence of cysteine and cystine and a blocked N-terminal residue. All these properties are very close to those of human and bovine encephalitogenic proteins.

In order to obtain a clue to understand the induction mechanism of chronic form of EAE we studied the immunized site of EAE animals with immunocytochemical techniques. There were numerous vacuoles associated with inflammatory reactions in the subcutaneous tissue of immunized feet. Immunocytochemical staining with antiserum to myelin basic protein (MBP) disclosed remaining of MBP in and around the vacuoles. The MBP remained more in early stage and less in chronic stage; it remained even on 326th day of postimmunization. A possible role of the remained antigen in chronic EAE was discussed.

A 17-residue peptide (Peptide Y) was isolated from the COOH-terminal end of the basic protein of bovine myelin by peptic digestion. This peptide induced experimental allergic encephalomyelitis in the rhesus monkey. Treatment of Peptide Y with cyanogen bromide released three amino acids from the COOH-terminal end and resulted in a tetradecapeptide (Peptide M) which was also encephalitogenic in the rhesus monkey. The sequence of Peptide M is: Phe-Lys-LEU-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met. Thus a major disease-inducing site active in the rhesus monkey is contained within a 14-residue peptide localized near the COOH-terminal end of the protein. This peptide differs markedly in location and sequence from the 9-residue peptide shown to contain the encephalitogenic determinant for the guinea pig.