Remitting-relapsing EAE in nonhuman primates: a valid model of multiple sclerosis.
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Animals↗
Biomedical subjects
S Hruby
Publications and source records attributed to S Hruby.
Magnetic resonance imaging of experimental allergic encephalomyelitis in primates.
The potential use of quantitative MRI to characterize early, as well as late, immune-mediated lesions of multiple sclerosis (MS) has been investigated. Experimental allergic encephalomyelitis (EAE) was induced in 4 male Macaca fascicularis monkeys, and the development of the disease followed using quantitative MRI at 0.15 Tesla. Serial scans were recorded daily, beginning at day 9 after inoculation. Lesions were detected before the onset of clinical signs, due to an elevation in the spin-lattice (T1) and spin-spin (T2) relaxation times. The T1 and T2 values from the lesions were shown to increase over time, indicative of progressive change at a molecular level. The appearance of any new lesions and any changes in existing ones were noted; this allowed dating of the lesions postmortem. Pathological correlation showed the long T1 and T2 values to be associated with the presence of inflammation, demyelination and haemorrhagic necrosis. Microscopically similar lesions had the same MRI characteristics. In addition, these studies showed the oldest lesions to be the most haemorrhagic; this is contrary to the belief that haemorrhage is a secondary event in EAE. The results show that quantitative MRI is sensitive to variable pathology, and has the potential for use in characterizing the pathological progression in MS.
Animals↗
Myelin basic protein and its free and bound antibodies in cerebrospinal fluid. All three must be determined on each specimen.
Myelin basic protein (BP) in the cerebrospinal fluid (CSF) is an important marker of brain damage, especially of white matter, but low or "normal" values can be misinterpreted if the CSF is not also examined for free and bound antibodies to BP. BP has many epitopes (antigenic determinants) and is very susceptible to fragmentation by proteolytic enzymes that are frequently very active in CSF, especially in patients with neurological diseases. This combination of factors permits BP to be degraded as it is released from myelin and to allow antibodies to those epitopes destroyed by enzymatic action to persist free in the CSF. Other free anti-BP antibodies may simply represent antibody excess. The relatively frequent formation of soluble antigen-antibody complexes with other epitopes on BP permits the existence of anti-BP antibodies bound to BP also in CSF. In addition to these factors that contribute to low values of free BP in CSF, if the analyses are not performed promptly on CSF collected in plastic tubes (polystyrene but preferably polypropylene), the "natural" adhesiveness of BP (immediately to glass, slowly to plastic) can remove it from the CSF, resulting in artifactually low values which can also be misinterpreted as "normal".
Animals↗
Chronic remitting-relapsing experimental allergic encephalomyelitis induced in monkeys with homologous myelin basic protein.
A chronic remitting-relapsing form of experimental allergic encephalomyelitis (EAE) has been produced in monkeys sensitized to homologous myelin basic protein in Freund's complete adjuvants by the technique of suboptimal treatment after the onset of disease. Not only does the clinical course resemble that of human multiple sclerosis more closely than does the clinical course of acute EAE, but so also does the histological reaction, with more-nearly pure demyelination, rather than the hyperacute hemorrhagic-necrotic lesions that occur so commonly in untreated monkeys with ordinary acute EAE.
Animals↗
New monoclonal antibodies reactive with defined sequential epitopes in human myelin basic protein.
Three new IgG monoclonal antibodies are described which recognise sequential epitopes of the human myelin basic protein (MBP) molecule in amino acid sequences 36-50, 64-75 and 80-89. Two of the secreting hybridomas were prepared by immunisation of mice with synthetic peptides. This procedure appears to generate antibodies of similar affinities to those made using intact myelin basic protein as the immunogen. It has the advantage that antibodies to preselected regions of the molecule can be made at will and the problem of subsequent epitope localisation is simplified. It is possible with synthetic peptides to generate antibodies of specificities which it would be impossible to achieve by immunisation with intact myelin basic protein. The monoclonal antibodies described here should be useful tools in studies of myelin catabolism in vivo and in vitro. Of particular interest is our Clone 22, making an antibody which reacts equally well with intact human MBP and synthetic peptide sequence 80-89 in liquid phase assays. Antibodies of this rare specificity have been claimed to be able to react with the peptides of myelin basic protein found in the spinal fluid of patients with multiple sclerosis.
Antibodies, Monoclonal↗
Anti-CD4 monoclonal antibody therapy of experimental allergic encephalomyelitis in longtailed macaques.
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Animals↗
Fluctuations of T- and B-cell subsets in basic protein-induced experimental allergic encephalomyelitis (EAE) in long-tailed macaques.
Experimental allergic encephalomyelitis (EAE) was induced in long-tailed macaques (Macaca fascicularis) by inoculation of autologous myelin basic protein (BP) in complete Freund's adjuvant. Natural killer (NK) cell activity and lymphocyte subsets detected by one- and two-color immunofluorescence were monitored longitudinally in these animals. A decrease in NK cell activity was detected at the onset of clinically defined disease. During the preclinical phase of EAE (5-7 days before the onset of clinical signs) the absolute number of T helper (CD4+) and T suppressor (CD8+) cells in the peripheral blood decreased significantly. Analysis of peripheral blood B cells revealed a selective depletion of IgD+ B cells and a corresponding increase in the number of IgD- B cells prior to and during the onset of clinical signs. Total B-cell numbers were not significantly different between EAE and normal groups. The increased proportion of IgD- B cells in BP-sensitized animals corresponded with the appearance of high titers of circulating anti-BP antibodies. Thus two-color analysis of B-cell subsets may be a sensitive indicator of B-cell activation and of abnormal immune status in EAE. Changes in lymphocyte subsets in macaques with EAE are compared with those in humans with multiple sclerosis.
Animals↗
In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis.
The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freund's adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.
Animals↗
Monoclonal antibodies reactive with myelin basic protein.
New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22-34, 75-82, 83-96, 118-131 and 125-131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75-82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys75 (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala76 by Ser (as in all other species of BP) and either deletion of Gln77 (as in human, monkey and rabbit BP) or His78 (as in the guinea pig and rat BP) or substitution of Pro82 by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10-20 times the reactivity of chicken BP but still only 20-50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.
Amino Acid Sequence↗
Region-specific immunoassays for human myelin basic protein.
Three monoclonal antibodies reactive with human myelin basic protein have been used to develop immunoradiometric assays for this protein. Clone 1, a mouse IgG2a, is reactive with an epitope in the region 129-138. Clone 2, a mouse IgG1, is reactive with the region 119-131. Clone 12, a rat IgG, is reactive with the region 86-96. Competition experiments show that the reactions of Clone 1 and Clone 2 are mutually exclusive, probably because of steric effects. In contrast, when either Clone 1 or Clone 2 react they cause minimal interference with the subsequent binding of Clone 12. Less than 1 ng/ml of myelin basic protein can be detected in each of the two immunoradiometric assays developed. Clone 12 can also be used on its own in a competitive immunoassay to detect around 2 ng/ml. Using an extraction technique before the assay, serum or plasma can also be investigated. Assays for defined regions of myelin basic protein should prove valuable in identifying the products of myelin catabolism in patients with demyelinating disease.
Amino Acid Sequence↗
Evidence for specific polypeptide chain folding in myelin basic protein from reactions between fragments of the protein and monoclonal antibodies.
The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Early detection of experimental allergic encephalomyelitis by magnetic resonance imaging.
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Animals↗
Sites in myelin basic protein that react with monoclonal antibodies.
The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.
Amino Acid Sequence↗
Epitopes in myelin basic protein reactive with monoclonal antibodies.
The epitopes (antigenic determinants) evoked by guinea pig or monkey myelin basic protein (BP) for 7 monoclonal antibodies (MAb) have been localized to 4 different sequences of the BPs extracted from various species. The first pair of epitopes is included within residues 1-14 and reacts with 2 rat IgG MAbs having slightly different specificities. The second pair of epitopes overlap within the sequence 86-100, requiring the Phe(91)-Phe(92) residues. One includes only residues 90-100 and reacts with a rat IgG MAb, the other is longer and reacts with a mouse IgM MAb. The third epitope lies within residues 114-124 (specifically including the single Trp) and reacts with a rat IgG MAb. A cross-reacting epitope in the amino half of the molecule is present in certain species (human, bovine and guinea pig). The fourth pair of epitopes includes residues 131-140 and is severely species-restricted. A mouse IgM MAb reacts practically only with guinea pig BP. Another IgG MAb reacts with human, chimpanzee, monkey, bovine and rat-18.5 kDa BPs, to a much lesser extent with rabbit and rat-14 kDa BPs and not with guinea pig, pig or chicken BPs. A cross-reacting epitope in the amino half of the molecule may be present in the more reactive species.
Animals↗
Myelin basic protein and its antibodies in the cerebrospinal fluid in experimental allergic encephalomyelitis, multiple sclerosis and other diseases.
Our studies have revealed no essential differences between EAE in monkeys and MS and other CNS diseases in humans. High concentrations of BP occur early, especially if no anti-BP antibodies are also present. Lower concentrations of BP follow and may be associated with the presence of anti-BP antibodies. In EAE these antibodies come from the relatively strong peripheral sensitization to BP and enter the CSF through a damaged blood-brain barrier; in MS they come from the relatively weak immunologic stimulation probably evoked by previous attacks of the disease. Proteolytic enzymes also enter the CSF and produce peptide fragments of BP whose differing antigenic compositions permit antibodies to some fragments to coexist with other fragments unrelated antigenically but detected as "BP" in vitro. Since oligoclonal immunoglobulins (IgG) occur more often in MS than in other diseases, one can expect to find BP, anti-BP antibodies and oligoclonal IgG more often in MS, but even the combination is not specific for MS. Consideration of the temporal and immunochemical relationships as well as of the differential diagnosis provides a basis for the understanding of the significance of BP, its antibodies and other immunoglobulins in the CSF.
Animals↗
The onset of experimental allergic encephalomyelitis as defined by clinical, electro-physiological and immunochemical changes.
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Animals↗
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.
Amino Acid Sequence↗
Has myelin basic protein received a fair trial in the treatment of multiple sclerosis?
Autosensitization to some central nervous system antigen still remains one of the best hypotheses for the continuing pathogenesis of multiple sclerosis (MS). Enough is now known about the cause, pathogenesis, and treatment of experimental allergic encephalomyelitis (EAE) to test this hypothesis. Reports of therapeutic failure of the encephalitogen myelin basic protein (BP) in the treatment of MS have their counterparts in similar therapeutic failures in EAE. Only highly inbred strain 13 guinea pigs respond consistently to BP therapy, and this only when BP is administered in relatively high doses. Noninbred guinea pigs respond much less well to simple BP therapy, and monkeys hardly at all. In both strains of monkeys so far studied, a nonspecific adjunctive factor--an antibiotic in Macaca mulatta and a steroid in Macaca fascicularis--is also required. Accordingly, human trials of the therapeutic efficacy of BP in MS should include its administration in large concentrations together with an adjunctive agent.
Adjuvants, Immunologic↗