Myelin basic protein serum factor. An endogenous neuroantigen influencing development of experimental allergic encephalomyelitis in Lewis rats.

Age-related concentrations of myelin basic protein serum factor (MBP-SF), an endogenous neuroantigen detected and quantitated by inhibition of binding of rat myelin basic protein (RMBP) antibody with 125I-RMBP reagent antigen and immunochemically indistinguishable from native RMBP in this respect, reach peak levels as high as 21 ng/microliter among 2-3-wk-old normal suckling Lewis rats. Levels then progressively decline to low, usually undetectable levels of less than or equal to 0.6 ng/microliter MBP-equivalents in adult animals by 7 wk of age. MBP-SF levels are inversely related to the age-related increasing capacity of maturing Lewis rats to develop experimental allergic encephalomyelitis (EAE) after sensitization to MBP of syngeneic, but not xenogeneic, origin. MBP-SF appears to be an endogenous neuroimmunoregulatory product of potential importance for immunologic tolerance to autologous RMBP in Lewis rats.

Sequential adsorption analysis of antibodies to neuroectodermal cells by an indirect quantitative method.

An indirect radioimmunoassay was developed for the sequential adsorption analysis of rabbit antibodies raised against rat neuroectodermal cells. The quantitativee relationship between primary adsorbed rabbit antitumor antibodies and secondary adsorbed goat antibodies to rabbit IgG was explored by paired radioiodine analysis. In the final indirect method, the amount of unlabeled rabbit antibody removed by cultured monolayers of cells at each step in a sequence of cells could be determined from an equation relating the unlabeled amount to values of bound 125I-labeled goat antirabbit IgG. To obtain the total quantity of rabbit antibody in a particular antiserum reagent, a sequential adsorption analysis was done with successive steps of homologous cells. To obtain the amount of antibody that was cross-reactive for other cell lines, we included those lines in the first several steps of the sequence. The sequential adsorption profile was considered as a more important indicator of the quality of a particular antiserum reagent than was the total amount of antibody contained in it. Neuroectodermal cell lines used as illustrative examples included subclones of the C6 astrocytoma and of the RN-2 schwannoma.

Monoclonal and polyclonal antibodies to myelin basic protein determinants.

A detailed immunochemical examination of monoclonal and polyclonal antibody responses to myelin basic protein (MBP) and its peptides has revealed the existence of as many as 27 antigenic determinants, many of them conformational. Topological mapping of the potential antigenic determinants onto a model of MBP secondary structure places these determinants within 11 separate regions of the molecule, including those portions that have been found to be encephalitogenic. MBP and its peptides, therefore, fall under the umbrella of the Multideterminant-Regulatory Model of Benjamin et al. (1984). However, in the case of MBP, multideterminant immunogenicity appears to represent mainly an escape from tight regulation through the avenue of conformational change.

Studies of rat antibodies specific for myelin basic protein (MBP). Antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro.

Rats immunized with rat myelin basic protein (MBP) in an encephalitogenic regimen produce antibodies which cooperate with spleen cells from unimmunized rats in the specific lysis of MBP-sensitized chicken erythrocytes (MBP-CRBC) in vitro. Antibodies against MBP capable of participating in antibody-dependent cell-mediated cytotoxicity (ADCC) were detected in serum 9 days post immunization, and prior to the onset of experimental allergic encephalomyelitis (EAE). Measurements based on a sensitive radioimmunoassay technique had previously established that anti-MBP activity emerges by day 9, early enough to participate in events leading up to EAE. The ADCC assay was designed to further characterize functions of immunoglobulins associated with the humoral response to MBP, with emphasis on the cytophilic properties of early antibodies. The assay described was found to be nearly as sensitive as the RIA and could detect specific syngeneic rat anti-MBP at concentrations of about 0.03 pmoles/ml serum. Whether ADCC-type reactions take place in the central nervous system during acute EAE is not known; however, the presence of early cytophilic antibody in serum suggests that humoral immunity may play an ancillary role to that demonstrated for T-cell immunity in EAE.

Polyclonal antibodies to the encephalitogenic neighborhoods of myelin basic protein: singular affinity populations neutralized by specific synthetic peptide probes.

Specific ligand neutralization was used to probe the extent to which singular antibody affinity populations signified specific determinants in the neighborhood myelin basic protein (MBP) encephalitogens. The probes were individual members of a panel of synthetic peptide analogs subsuming encephalitogenic regions. Comparative Scatchard analyses of neutralized and unneutralized antisera helped to identify the particular peptide determinants involved in the original polyclonal antibody responses to the multiple antigenic determinants of encephalitogenic peptides. The range of affinities for an antibody population against a singular MBP peptide determinant was found to be relatively restricted while the range of affinities overall for all populations within a given antipeptide antiserum was found to be relatively wide and invariably discontinuous. Consequently, the individual discontinuous affinity populations could readily be dissected by application of the Rosenthal method of Scatchard curve analysis. It was found that the singular high affinity antibody population (5.6 x 10(7) M-1) of a Lewis rat antiserum to rat encephalitogenic GSLPQKAQRPQDENG (S49) was against a determinant near the N-terminal non-encephalitogenic end of the peptide. Only the low affinity antibody populations were found that had reactivity for determinants within the encephalitogenic region itself. The singular high affinity antibody population (5.97 x 10(7) M-1) of a rabbit antiserum to rabbit encephalitogenic TTHYGSLPQKAQGHRPQDEG (S82) was against a determinant centered about the tyrosyl residue, within the encephalitogenic region for the rabbit, but was completely cross-reactive with a specific circulating endogenous inhibitor. The results obtained with the rat and rabbit EAE sera were consistent with a previously advanced hypothesis that antibodies to determinants within encephalitogenic neighborhoods would effectively block the onset of EAE if high enough in affinity and not neutralized by an endogenous inhibitor.

Heteroclitic antibodies in Fischer 344 rats to a synthetic encephalitogenic myelin basic protein peptide.

Fischer 344 rats, immunized with the synthetic encephalitogenic myelin basic protein peptide YS49 (YGSLPQKAQRPQDENG), produced heteroclitic antibodies that reacted much more extensively and with a much higher affinity with the cross-reacting encephalitogenic guinea pig sequence S49S (GSLPQKSQRSQDENG) than they did with the immunogenic YS49. On the other hand, antisera against S49S reacted in a normal manner with homologous S49S and cross-reacted only poorly with YS49. The phenomenon of heteroclisis in Fischer 344 rats correlated with the greater encephalitogenic potency of the cross-reacting entity. Kibler et al. (J. Exp. Med., 146 (1977) 1323-1331), by comparing the encephalitogenic guinea pig sequence to a less potent analog, had also previously observed what now would be termed a heteroclitic phenomenon at the T cell level in Lewis rats. In their hands, however, as well as in ours Lewis rat antisera against the encephalitogenic peptide region were much too complex to be analyzed with respect to heteroclisis. It was shown in the present experiments that by utilizing the Fischer 344 system one may also readily obtain heteroclisis at the B cell level against encephalitogenic peptides. Neither YS49 nor S49S as immunogen produced detectable antibody in Brown Norway (BN) rats with exception of two immunized with YS49. In those two cases heteroclitic antibodies were obtained that had a very low significant (greater than 3 SD above baseline) antigen binding capacity for S49S and no detectable reactivity for the homologous YS49 ligand.

Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, [125I]S79. A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them.

Among the antibodies contained in a rabbit antiserum to synthetic peptide sequence TTHYGSLPQKAQGHRPQDEG (S82) of bovine myelin basic protein (residues 65-83 plus glycine), was a population reactive with a C-terminal determinant of S82 and cross-reactive with S79 (AQGHRPQDEG) but not S6 (AQGHRPQDENG). This antibody population was purified 153-fold by affinity chromatography from a minicolumn containing S79 coupled to CH-Sepharose 4B(TM) and eluted with 3 M MgCl2. The purified antibodies were then coupled to CNBr-activated Sepharose 4B(TM) and used to purify 125I-labelled, acylated S79 ([125I]S79), 3 M MgCl2 once again having been used to elute the labelled ligand. Sips distribution studies revealed appreciable heterogeneity of binding affinities of unpurified antibodies in their reaction with affinity-purified [125I]S79 or of purified antibodies in their reaction with unpurified [125I]S79 (heterogeneity constant a = 0.34 and 0.36, respectively). In contrast Sips distribution data indicated considerable restriction of binding of the purified antibodies in their reaction with purified labelled ligand (a = 0.92) with an average affinity constant of K0 = 1.56 X 10(8) M-1. The results indicate that the heterogeneous spectrum of binding affinities originally displayed by the unpurified S79-reactive antibodies in their reaction with unpurified labelled S79 was due both to the presence of some antibodies characterized by high affinity binding (K0 greater than 10(9) M-1) and of some labelled ligand with low binding affinity. The affinity chromatographic method as here described should prove advantageous in purifying and eventually characterizing picomolar amounts of serum factors, previously postulated to be fragments of myelin basic protein, that are reactive with reagent antibodies up to an affinity level of 10(8) M-1.

Immunogenicity of synthetic peptide sequences S81 and S82 (residues 68-83 and 65-83) of bovine myelin basic protein. Time-course of antibody responses in rats and rabbits.

The timing and intensity of the antibody responses to the marker determinants of synthetic peptide S81 and S82 sequences of bovine myelin basic protein (MBP) (residues 68-83 and 65-83, respectively) were studied in 20 Lewis rats and 6 rabbits. All rats immunized with either peptide in CFA responded with antibody development. All rabbits immunized with S82 and CFA developed both antibodies and experimental allergic encephalomyelitis. In contrast only one rabbit developed antibodies against S81 and none of the S81-challenged rabbits developed disease. On the basis of extrapolation of linear time-response curves to zero activity, the time of appearance of anti-peptide antibody activity in the Lewis rats was 15.1 +/- 1.7 days after a single immunization, a week longer than the normal latent period before appearance of anti-MBP antibodies. The time of appearance of anti-S82 antibody activity in rabbits exhibiting linear response curves was 18 days, 4 days after a booster immunization with S82 in incomplete Freund's adjuvant. The development of clinical signs of experimental allergic encephalomyelitis occurred within 4 weeks after initial challenge (a few days after boosting) and continued for 8--13 days in all S82-immunized rabbits.

Equilibrium competitive inhibition analysis of synthetic peptide antigens from myelin basic protein as affected by the dual-dilution phenomenon.

It was shown that 125I-labelled and unlabelled forms of synthetic encephalitogenic peptide S82 (residues 65-83 plus glycine) of bovine myelin basic protein (MBP-Bov) were equally competitive in dual-dilution radioimmunoassays with rat- and rabbit-anti-S82 antisera without causing much deviation even at the extremes of the dual-dilution binding curves (solved in terms of total S82). With other antisera the deviations caused by the addition of unlabelled S82 were much greater than would be expected among repetitive assays with labelled antigen alone, and the excessive deviations were usually more prominent in one region of the dual-dilution binding curve than in another. Thus, establishing equivalence between labelled and unlabelled antigen with respect to one antiserum even at several dilutions does not establish proportionate sharing with respect to all antisera at all antigen concentrations. A method of dual-dilution equilibrium competitive inhibition analysis was devised that took this precaution into account. By means of the method, synthetic MBP-Bov peptides representing different parts of the S82 sequence were compared with homologous S82 peptide for their inhibitory effects upon dually diluted [125I]S82-anti-S82 systems. By this process several different S82 determinants were pinpointed, some with high affinity antibodies, others with low affinity antibodies, yet others equally well at high or low affinity.

Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats. Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities.

MBP-SF, previously described as an endogenous myelin basic protein-serum factor in Lewis rats with a suggested function as a neuroautotolerogen, appears not to be a single factor but a heterogeneous collection of serum factors (MBP-SFs), most probably small fragments of MBP, each cross-reactive with a different region of the multideterminant parent molecule. The heterogeneity of the MBP-SFs in any serum sample is defined and limited by the spectrum of binding affinities of the antibody populations represented in a given reagent anti-MBP antiserum. Some samples of normal Lewis rat serum have been found to contain high affinity MBP-SFs which coexist with low affinity anti-MBP antibodies whereas other sera have shown the reversed pattern, viz. low affinity MBP-SFs and high affinity antibodies. Additional sera have been found to contain MBP-SFs of several different affinities. In time-course studies of rats sensitized to neuroantigen-adjuvant a variety of MBP-SFs and anti-MBP antibodies of different affinities may be observed in sequentially collected sera from a given animal. In no animal has any serum sample been found to contain the full spectrum of MBP-SFs. Although some MBP-SFs have been found to increase temporarily during the 2nd week after neuroantigen/CFA sensitization, all MBP-SFs tend to disappear in the 2nd week and to be replaced by anti-MBP antibodies of differing affinities 3-4 weeks following sensitization.

Endogenous myelin basic protein-serum factors (MBP-SFS) and anti-MBP antibodies in a patient with post-herpes simplex virus acute disseminated encephalomyelitis.

The measurement of myelin basic protein serum factors (MBP-SFs) and anti-MBP antibodies in specimens from a patient with post-herpes simplex acute disseminated encephalomyelitis (ADE) is described. Transitory appearance of high affinity anti-MBP antibodies in the absence of detectable MBP-SFs was observed. This pattern was similar to that found previously in acute multiple sclerosis and experimental allergic encephalomyelitis. These findings are consistent with the hypothesis that a possible normal role of MBP-SFs is as neuroautotolerogens, preventing autoreactive lymphocyte clones from damaging myelin.

Comparative levels of endogenous myelin basic protein-serum factors (MBP-SFs) in adult and suckling mice (B6CBAF1 and B6C3HF1, strains) and in neurologically mutant mice of the same genetic background.

Normal adult B6C3HF1 and B6CBAF1 mice contained at least 10 times higher levels (1.17 microM) of endogenous myelin basic protein-serum factors (MBP-SFs) than previously found in adult Lewis rats. In rat MBP-SF levels in the adult (0.03 microM) were much less than in the suckling animals (0.74 microM). In mice, by contrast, the adult (1.17 microM) and suckling (0.75 microM) levels were similar. Suckling mice from 9 different neurologically mutant strains and their clinically normal littermates had MBP-SF levels (0.5 microM) slightly below that of normal suckling mice of the same genetic background (0.75 microM).

Endogenous myelin basic protein-serum factors (MBP-SFs) and anti-MBP antibodies in humans. Occurrence in sera of clinically well subjects and patients with multiple sclerosis.

Sera of normal subjects and patients wtih multiple sclerosis (MS) have been frequently found to contain picomolar quantities of endogenous myelin basic protein-serum factors (MBP-SFs). These serum factors, collectively representing a heterogeneous spectrum, were detected and measured by means of a competitive inhibition radioimmunoassay (RIA) designed to distinguish their respective binding affinities with anti-MBP reagent antiserum. Anti-MBP antibodies in these same normal and patient sera were also detected and their differing binding affinities determined. In general, when sera of normal subjects were found to contain free MBP-SFs, the reagent anti-MBP antibodies in the reagent antiserum used to detect them were of relatively high binding affinity (8 X 10(8) M-1). When normal sera were found to contain free anti-MBP antibodies, the affinities of such antibodies were invariably lower (0.06-0.7 X 10(8) M-1). In contrast, sera of patients with active MS and exhibiting clinical fluctuations in their disease, infrequently contained high or medium high affinity MBP-SFs, whereas higher affinity anti-MBP antibodies were commonly detected. These patterns of MBP-SFs and anti-MBP antibodies in normal and MS human sera resemble those previously observed in studies of normal Lewis rats and rats developing experimental allergic encephalomyelitis (EAE). The findings here reported provide additional support for the view that circulating endogenous MBP-SFs may function as neuroautotolerogens that restrict expansion of MBP-reactive lymphoid cell clones having potentially injurious effector activity for central nervous system (CNS) tissue.