Allergic encephalomyelitis: passive transfer prevented by encephalitogen.

Allergic encephalomyelitis was produced in rats by passive transfer of lymph node cells from donors immunized intradernmally with nleural tissute or an encephalitogenic basic protein pluts adjulvants. The same basic protein, injected intravenously into the recipients before or after transfer of lymph node cells, prevented the disease. Even established lesions were reversed. Inhibition by basic protein was specific for encephalomyelitis; it had no effect onz passive transfer of allergic adrenalitis.

Transfer of experimental allergic encephalomyelitis with guinea pig peritoneal exudate cells.

Incubation with specific antigen, myelin basic protein, greatly enhances the ability of guinea pig peritoneal exudate cells to transfer experimental allergic encephalomyelitis. Reproducibly successful transfers are obtained with 10(7) cells. With this relatively small number of cells, in vitro studies to determine the immunologic mechanisms involved in the disease process are now possible.

Myelin basic protein: location of multiple independent antigenic regions.

Immnunization of guinea pigs with homologous myelin basic protein induces antibodies that differ in their ability to bind specific peptide fragments of the protein. Antiserums with differing specificities made it possible to demonstrate at least three mutually exclusive antigenic sites in the protein molecule. One of these sites is located between residues 44 and 89, another between 90 and 116, and the third between 117 and 170.

Encephalitogenic activity of bovine basic proteins.

Two basic proteins isolated from bovine white matter in connection with a study of the protein-bound phosphoinositides of central nervous system tisstue have been tested for encephalitogenic activity. The biological activity of these proteins, which is equivalent to that of basic encephalitogenic proteins isolated in other laboratories, suggested that they are identical.

Myelination inhibition factor: dissociation from induction of experimental allergic encephalomyelitis.

Sensitization of guinea pigs with purified myelin basic protein induces experimental allergic encephalomyelitis (EAE) but does not induce a serum factor which inhibits myelin formation in vitro. This factor, induced by some unidentified constituent of whole central nervous system tissue, should not be characterized as a component of "EAE serum."

Species restriction of a monoclonal antibody reacting with residues 130 to 137 in encephalitogenic myelin basic protein.

A monoclonal antibody (immunoglobulin G1) has been produced that reacts against myelin basic protein present in or extracted from the brains of many mammals-with certain important exceptions. Because of known species differences in amino acid sequences of basic protein and of certain peptide fragments, the binding site for this particular antibody appeared likely to include residues 130 to 137. Confirmation of this hypothesis was obtained by amino acid composition of the major immunoreactive peptides produced by thermolysin digestion of human basic protein and isolated by high-performance liquid chromatography.

Peptide specificities of myelin basic protein-reactive human T-cell clones.

Forty myelin basic protein (BP)-reactive T-cell clones were isolated from a patient with multiple sclerosis and used to identify human T-cell recognition sites on the BP molecule. At least three sites have been identified: one in the N-terminal half of the molecule (residues 1-97), one in the C-terminal (residues 98-170), and one which spans residues 97-98. The clones exhibited a marked preference for the C-terminal half of the molecule. No cross-reactivity with measles virus was detected. These clones will be useful for both the further delineation of the human T-cell recognition sites on BP and the generation of anticlonotypic monoclonal antibodies.

Enhanced transfer of experimental allergic encephalomyelitis with Lewis rat lymph node cells.

Lewis rat lymph node cells (LNC) are greatly enhanced in their ability to transfer experimental allergic encephalomyelitis (EAE) after culture with myelin basic protein (BP). As few as 10(6) LNC transfer disease after culture with antigen. In contrast to spleen cells, enhanced transfer with LNC is not seen after culture with concanavalin A. Neither cell homogenates nor culture supernatants were capable of transferring EAE. Removal of B-cells had no adverse effect on disease transfer. LNC capable of enhanced transfer were found in rats after recovery from, as well as at the height of, clinical disease.

Immunocytochemistry of myelin basic proteins in adult rat oligodendroglia.

Immunocytochemical demonstration of myelin basic protein (MBP) in oligodendroglia of the developing rat and human central nervous system has been reported. However, reaction with MBP antiserum was detectable only prior to and during the early phase of myelination. The lack of reaction with still actively myelinating and mature oligodendroglia has been puzzling. We report here that by the use of mild fixation and by a modification of the staining technique previously used for detection of MBP on vibratome sections, staining of oligodendroglia in sections of adult rat brain has been achieved.

Cell surface endothelial proteins altered in experimental allergic encephalomyelitis.

Endothelial cells (EC) are increasingly being considered as important participants in the early evolution of inflammatory and immune responses in experimental allergic encephalomyelitis (EAE). We have found that a mouse monoclonal antibody, which reacts with the luminal plasma membrane of central nervous system endothelium, detects an alteration in the blood-brain barrier (BBB) in lesions in Lewis rats with EAE. Anti-endothelial barrier antigen (EBA) reacted with microvessels in normal rat brain and spinal cord. This reaction was abolished in 'EAE' microvessels surrounded by inflammatory cells. In rats that had recovered from one attack most EC reacted with the antibody, indicating that EBA was reexpressed during recovery. However, blood vessels in areas with residual inflammatory lesions were negative. The biochemical changes that lead to this absence of antibody binding and the cells or mediators responsible for producing this change are not yet known. However, anti-EBA should provide a useful tool for exploring molecular mechanisms underlying BBB breakdown.

Evidence for multiple human T cell recognition sites on myelin basic protein.

Myelin basic protein (BP)-specific T cell clones were used to study human T cell recognition sites on the BP molecule. Proliferation assays performed with a panel of xenogeneic BPs of known amino acid sequence and with large peptide fragments of human and guinea pig BPs demonstrated ten different patterns of reactivity. The data provide evidence for at least four different human T cell epitopes within the C-terminal half of the BP molecule, three within the N-terminal half, and three located within the central portion of the molecule. The results indicate that attempts to inhibit anti-BP responses in vivo in an antigen-specific manner will require the suppression of multiple T cell populations.

Experimental allergic encephalomyelitis in rabbits. A major encephalitogenic determinant within residues 1-44 of myelin basic protein.

Experimental allergic encephalomyelitis could be induced in rabbits by injection in Freund's complete adjuvant of either peptide 1-44 or peptide 45-87 of rabbit myelin basic protein. In order to localize the encephalitogenic determinant present in peptide 1-44, several smaller derivative peptides were prepared and examined. Peptic peptide 15-44 and thrombic peptide 1-31 were as active as peptide 1-44, whereas peptic peptides 1-14 and 18-38 and BrCN peptide 22-44 were virtually inactive. Weak activity was shown by BrCN peptide 1-21. These results provide evidence that a major encephalitogenic determinant present in peptide 1-44 lies within sequence 15-31. The encephalitogenic activity of peptide 15-44 was essentially destroyed by oxidation of methionine-21 to methionine sulfoxide; methylation of Met-21, on the other hand, appeared to be relatively ineffective in eliminating the encephalitogenicity of peptide 1-44.

A neurochemical and immunocytochemical study of P2 protein in human and bovine nervous systems.

The anatomical distribution of P2 protein was studied in human autopsy tissue. Spinal cord (SC) and peripheral nerve (PN) were stained by the peroxidase-antiperoxidase method with antisera to bovine P2, glial fibrillary acidic protein, and myelin basic protein (BP). P2 antiserum did not stain all of the myelin in the PN. The staining was randomly distributed and discontinuous along a given myelinated axon. P2 antiserum also stained SC myelin in a pattern similar to the PN. Only a fraction of the sheaths stained, in contrast to BP antiserum that stained all myelin sheaths in both the SC and PN. P2-positive myelin was distributed throughout the SC white matter, including an occasional myelinated fiber in the SC grey matter. P2 and BP antisera did not stain regions of demyelination in a case of idiopathic polyneuritis, while adjacent myelinated PN stained normally. Absorption of the P2 antiserum with P2, bovine PN or bovine SC (carefully dissected to eliminate PN contamination) nullified the specific staining in both the PN and SC; however, absorption with BP or hemispheric myelin did not eliminate P2 staining. The P2 antiserum formed a single immunodiffusion line with pure P2 and acid extracts of bovine SC and PN myelin, but not with an acid extract of bovine hemispheric myelin. Electrophoresis of defatted bovine SC produced a distinct band corresponding to P2. Therefore, three lines of evidence, immunocytochemical, immunodiffusion and electrophoretic, suggest that P2 is present in PN and SC but not in hemispheric myelin.

LPS augments adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat.

Adoptive transfer of experimental allergic encephalomyelitis (EAE) is enhanced after in vitro culture of myelin basic protein (BP)-sensitized lymphoid cells with BP. Addition of lipopolysaccharide (LPS) to the culture further augments transfer of EAE to a level 5 times greater than that achieved with cells activated only with BP. Neither the proliferative response of a BP-specific cell line nor the production of IL-2 by BP-sensitized lymphoid cells in response to BP was augmented by the addition of LPS to the culture. Augmentation of EAE was also observed if recipients received simultaneous injections of BP-sensitized lymph node cells (BP/LNC) cultured with BP (BP-activated) and normal spleen cells cultured independently with LPS (LPS/Spl-C). To analyze the effect of contact between these two cell populations in vivo, we mixed the two cell populations in vitro at reduced cell concentrations. When BP-activated BP-LNC were mixed with LPS-Spl-C in vitro, a marked synergistic proliferative response was observed. Irradiation of BP-activated BP/LNC abrogated this synergistic response, whereas irradiation of LPS/Spl-C did not, suggesting that the proliferating population was in the BP/LNC and that the LPS/Spl-C enhanced their proliferation. These results indicate that LPS exerts its effect through BP-nonspecific cells and that these cells enhance transfer of EAE by augmenting the proliferation of the BP-specific cells in vivo after transfer.

A comparison of demyelinating and myelination-inhibiting factor induction by whole peripheral nerve tissue and P2 protein.

Sera from rabbits sensitized with whole bovine spinal roots demyelinated and inhibited myelination in both peripheral and central nervous system tissue cultures. Antisera directed against the peripheral nerve myelin basic protein, P2, demonstrated no antimyelin activity in vitro. These results suggest that demyelinating and myelination-inhibiting factors are directed against some peripheral nerve component(s) other than the P2 protein.

Failure of serum from recovered rats to prevent enhanced adoptive transfer of experimental allergic encephalomyelitis.

The rat is unique among species used for research on experimental allergic encephalomyelitis (EAE) because of the spontaneous recovery which occurs routinely after severe, almost fatal disease. The mechanism of recovery has never been adequately explained although it has been suggested that suppressor cells might play a role in this phenomenon. In another immune system (contact sensitivity) anti-idiotypic antibodies obtained during the recovery phase have been shown to have a protective effect. Adoptive transfer of EAE, which can be markedly enhanced by incubation of sensitized cells with antigen in vitro, offers a convenient tool for investigating mechanisms of recovery. With this system, we have attempted to suppress transfer of disease with serum obtained from recovered rats. In spite of various manipulations of the experimental protocol, including the use of serum plus complement before and after incubation of cells with antigen, we have been unable to demonstrate suppression of disease. We and others recently reported that cells from recovered rats are also capable of enhanced transfer. This permitted the use of autologous serum from individual cell donors. Even in this strictly autologous system, however, no inhibitory effect of serum could be detected.

The multiple causes of multiple sclerosis: the importance of age of infections in childhood.

The geographic distribution of multiple sclerosis (MS) may relate to the age of initial exposure and degree of sensitization to common viruses or bacteria which have proteins with epitopes (antigenic determinants) which are homologous with potentially encephalitogenic peptides in central myelin proteins, such as basic protein and proteolipid protein. Comparable homologies may exist for the as-yet-undefined nonencephalitogenic myelin antigen(s) which evoke demyelinating factors (probably complement-fixing antibodies). Many of these homologous epitopes occur in microorganisms that also possess adjuvant activity for evoking not only the sensitized T-cells but also the antibodies that cross-react with the target antigens in central myelin. If sufficient sensitization to myelin basic protein or proteolipid protein occurs, especially in infections of young adults, the individual develops acute disseminated encephalomyelitis, exactly comparable to ordinary acute experimental allergic encephalomyelitis (EAE). If very young children are infected, however, practically complete resistance develops, and neither acute disseminated encephalomyelitis nor MS follows. In between these two extremes, especially in slightly older children in whom insufficient sensitization occurs to induce acute disseminated encephalomyelitis, the individual may become resistant to acute disseminated encephalomyelitis, but susceptible to chronic relapsing or progressive disseminated encephalomyelitis, otherwise generally recognized as MS. This is exactly comparable to a recently described variant of chronic EAE in which demyelinating antibodies and large subpial plaques of demyelination occur. The similarity of this form of chronic EAE or chronic disseminated encephalomyelitis to one form of MS is emphasized.

Autoimmunity in multiple slcerosis: do we have an experimental model?

Experimental autoimmunity of the CNS has been well characterized--the antigen has been identified, effector cell specificity has been defined, and the relationship between cellular sensitization and antibody production has been partially clarified. In the guinea pig, experimental allergic encephalomyelitis (EAE) is induced by one injection of myelin basic protein in complete Freund's adjuvant (BP/CFA). If BP/CFA is preceded by repeated injections of basic protein in incomplete Freund's adjuvant (BP/IFA), EAE is not induced; the guinea pigs survive and ultimately produce antibody. Induction and prevention of EAE as well as antibody induction by this schedule are dependent on the presence of the intact encephalitogenic (T-cell) site in the polypeptide used for sensitization and preimmunization. In contrast, B cell sites (those peptide sequences which bind antibody) are independent of the T-cell site. At least 5 specific antigenic regions (B-cell sites) have been demonstrated in the BP molecule. High mycobacteria levels bypass the specificity requirement of helper T-cells but cannot bypass the specificity requirement of effector T-cells. In spite of the sophisticated immunologic techniques available, our knowledge of humoral and cellular sensitivity in multiple sclerosis (MS) patients is very limited. The experimental demonstration of an analogy between EAE and MS is weak: a) Demonstration of BP-sensitized cells or BP-specific antibodies in peripheral blood of MS patients has not been successful. b) Anti-myelin serum factors reported to be associated with both disease states (experimental autoimmunity and MS) are clearly not identical. Nevertheless, successful treatment of EAE in animals by BP/IFA injections has encouraged consideration of clinical trials to test the therapeutic value of BP injections in MS patients. If successful, the question will be answered: if unsuccessful, the dilemma still remains.