Experimental allergic encephalitis. Dissociation of cellular immunity to brain protein and disease production.

The encephalitogenic determinant of brain protein, a nonapeptide having the amino acid sequence Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys, has been characterized and synthesized. In a previous study, analogues of this encephalitogenic peptide were synthesized and some were shown to be encephalitogenic while others were not. Guinea pigs were immunized with encephalitogenic peptides having amino acid sequences different from that in the native protein. These guinea pigs did not show cellular immunity in vivo (skin reactivity) or in vitro (lymphocyte stimulation or macrophage migration inhibition) to the encephalitogenic brain protein (EP) although they did show cellular immunity to the immunizing antigenic peptide. Guinea pigs immunized with an encephalitogenic peptide having the same amino acid sequence as the brain protein, or with a nonencephalitogenic peptide having the same amino acid sequence as the native protein but lacking the terminal lysine, did develop cellular immunity to the EP. Animals immunized with EP showed cellular immunity to this protein, but not to the encephalitogenic peptides. Animals immunized with nonencephalitogenic protein (NEP), prepared by altering the tryptophan residue of EP, did not develop disease but did show cellular immunity in vitro and in vivo to the EP. Animals protected from disease by immunization with NEP similarly showed cellular immunity to EP. Thus, the results suggest a dissociation between cellular immunity to EP and the production of experimental allergic encephalitis (EAE). Animals immunized with the encephalitogenic peptides develop EAE, but do not show cellular immunity to EP, and animals immunized with NEP show cellular immunity to EP but do not develop EAE. A fresh approach to the examination of the pathogenesis of EAE is now possible through the use of these well-characterized antigens.

Experimental allergic encephalomyelitis: synthesis of disease-inducing site of the basic protein.

A highly encephalitogenic peptide whose structure resembles the sequence of amino acids surrounding the single tryptophan residue in the encephalitogenic A1 protein from bovine myelin was synthesized. This peptide is similar in the sequence to peptic peptide E and tryptic T27, derived directly from the A1 protein, and is as active on a molar basis as the A1 protein. The major disease-inducing site of the A1 protein resides in a linear sequence of nine amino acids: H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. This region of the A1 protein is apparently the major encephalitogenic determinant since specific modification of the tryptophan residue in the A1 protein with 2-hydroxy-5-nitrobenzyl bromide destroyed its encephalitogenic activity.

Isolation of a product from the trypsin-digested glycoprotein of sciatic nerve myelin.

When purified rabbit sciatic nerve myelin, whether lyophilized or not, is treated with low amounts of trypsin (25 microgram/ml) for 0.5, 3, or 24 h the resulting protein patterns viewed on sodium dodecyl sulfate (SDS) gel electrophoresis are similar. The most striking feature of the trypsinized myelin is the accumulation of a heavy band at the basic protein position, molecular weight 19 000, which is accounted for as a degradation product of the PO protein, referred to as the TPO protein. The PO protein, the major glycoprotein of sciatic nerve myelin, as well as the 23K and P2 proteins and albumin, an absorbed component, are all partially degraded; most high molecular weight bands are lost. The TPO protein, isolated by gel filtration in 2% SDS on an agarose column, like the PO protein, is highly insoluble in aqueous solvents. It is a glycoprotein (8% carbohydrate), staining with periodic acid-Schiff reagent; containing 3 mannose, 1 galactose, 3 N-acetylglucosamine, 1 sialic acid, and 1 fucose residues and is identical to the nonasaccharide of the parent PO protein. The amino acid composition of the TPO protein, is similar to the PO protein, but has a much higher content of hydrophobic residues and begins with NH2-methionine. This suggests that the PO protein is an amphipathic membrane protein in which its more polar character is confined to the first third of its NH2-terminus. This polar domain is probably positioned above the lipid leaflet where it is accessible to trypsin which cleaves a sensitive lysinyl (or argininyl)-methionine linkage. The more hydrophobic domain (the TPO protein) is buried in the myelin bilayer where it is protected from further tryptic attack. Thus trypsin can serve as a useful probe of myelin structure.

The PO protein. The major glycoprotein of peripheral nerve myelin.

A glycoprotein, referred to as PO protein, was isolated from rabbit sciatic nerve myelin by gel filtration on Agarose 0.5 m in dodecyl sulfate. The purified myelin was first defatted and extracted at pH 2. The water-soluble proteins such as myelin basic protein and P2 protein were extracted leaving a glycoprotein-rich residue, from which the PO protein was isolated. The purified protein showed a single band on gel electrophoresis in dodecyl sulfate when stained with Coomassie Blue of periodic acid-Schiff reagent. The carbohydrate, comprising 6.3% by weight, appears to exist as a nonasaccharide unit having 3 mannose, 3 N-acetylglucosamine, 1 sialic acid, 1 galactose and 1 fucose residue. The polypeptide moiety has a high content of non-polar amino acids. A single amino acid, isoleucine, was found at the NH2-terminal end by dansyl and Edman procedures. The PO protein is the major protein of peripheral nerve myelin.

Allergic neuritis: phospholipid requirement for the disease-inducing conformation of the P2 protein.

The P2 protein, a small, highly ordered basic protein of peripheral nerve myelin, is a potent inducer of allergic neuritis in rats when complexed with phospholipids such as phosphatidylserine. Isolated P2 protein administered without lipid is a poor neuritogen, and if first oxidized with performic acid, aminoethylated in 8 M urea or heat denatured, it loses nearly all activity. When the aminoethylated or oxidized forms are combined with phosphatidylserine, however, they recover essentially full neuritogenic activity. Complexing with lipid also greatly enhances the activity of the heart denatured form. Spleen cells sensitized to the aminoethylated and heated forms of P2 protein show a pronounced mitogenic response to either of these forms as well as to the P2 protein itself, but only when sensitization is initiated with the lipid complex. These data indicate that the lipid complex reverses the distortion acquired by chemical treatment or denaturation and converts the P2 molecule into a conformation approximating that of the native P2 protein in myelin. These studies imply that the neuritogenic domain, while highly sensitive to denaturing conditions, requires interaction with phospholipids in order to attain the most favourable conformation for inducing a cell-mediated response that leads to disease.

Effects of proteins on thermotropic phase transitions of phospholipid membranes.

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three catagories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion. Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (deltaH) accompanied by either an increase or no change in the temperature of transition (Tc). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region. Cytochrome c and A1 protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both Tc and deltaH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer. Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the Tc but did induce a linear decrease in the magnitude of the deltaH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a deltaH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.

Experimental allergic aspermatogenic orchitis. II. Some chemical properties of the AP1 protein of the sperm acrosome.

The AP1 protein, a unique aspermatogenic protein localized in the sperm acrosome, exists as a single polypeptide chain of 136 amino acids, as shown by a single band on gel electrophoresis in sodium dodecyl sulfate and the recovery of the expected 21 to 22 tryptic peptides on peptide mapping. The AP1 protein appears to exist in a compact, highly stable conformation, as shown by its resistance to trypsin hydrolysis. Its aspermatogenic acitivity is not affected by trypsin treatment, by heating at 99 degrees C for 1 h, by 8 M urea, or by acid conditions. After reduction and alkylation, however, the molecule appears to open up, since it becomes hydrolyzable by trypsin and migrates more slowly on gel electrophoresis at pH 2.7 and 8.6. After alkylation, the AP1 protein still migrates as a single band at pH 2.7. The AP1 protein shows microheterogeneity near its isolectric point at pH 8.6; each of five bands shows the same amino acid analysis. Aggregation was not observed following treatment with dimethylsuberimidate. The molecular weight of 15 000, obtained from gel electrophoresis consists of 136 amino acids with a relatively high content of proline, half cystine, glycine, histidine and tryptophan. No galactose, mannose, fucose, glucose, or hexosamines were found; the AP1 protein is thus not a glycoprotein.

Experimental allergic aspermatogenic orchitis. IV. Chemical properties of sperm glycoproteins isolated from guinea pig testes.

Of four glycoproteins isolated from guinea pig testes, two were aspermatogenic (types I and IV) and two (types II and III) were inactive. The glycoproteins were rich in carbohydrate, varying from 41.5% to 49.5% carbohydrate by weight. Each glycoprotein had a unique amino acid composition, but in general low levels of tyrosine, tryptophan, and basic amino acids were found along with relatively high contents of serine, threonine, glutamic acid, and proline. Types I and IV glycoproteins were remarkably stable; their aspermatogenic activity was not affected by urea, trypsin, or heating at 100 degrees C in water or in 1 M HCl for 15 min. Carbohydrate analysis revealed little difference in the monosaccharide compositions of types I and IV glycoproteins, except that only the type I contained sialic acid. In contrast, types II and III glycoproteins lacked sialicacid and fucose and contained much less mannose. Both N-acetylglucosamine and N-acetylgalactosamine were present in all four glycoproteins, and they dominated in the types II and III. Fucose and at least 20-25% of the galactose appeared to occupy terminal positions in type IV glycoprotein as shown by their release after 15 min hydrolysis in 1 M HCl. All of the glycoproteins contained a relatively high percentage of galactose by weight, from 12.6 to 19.3%. The molecular weights of the glycoproteins were estimated by sodium dodecyl sulfate gel electrophoresis to be 47000, 105000 and 18000 respectively for the types I, II, and IV; type III glycoprotein showed two major bands, with molecular weights of 41500 and 22800. All the above molecular weight values are probably overestimated because of high carbohydrate content. The molecular weight of type IV glycoprotein was found to be 13000 by ultracentrifugation; a corrected value of 29000 was calculated for type I glycoprotein.

HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.

BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.

Proliferative response of CD4+ and CD8+ T cell subsets from Hispanics with HIV+ and AIDS: the superantigen hypothesis.

It has been hypothesized that the progressive deletion of CD4+ T cells in the course of infection due to human immunodeficiency virus (HIV) may be mediated in part by interaction with a superantigen inherent in an HIV protein. Consequently, selective loss of CD4+ cells with a T cell receptor V beta-chain capable of interaction with superantigen would produce a CD4+ population less or totally unresponsive to superantigen such as staphylococcal enterotoxins B and A (SEB and SEA respectively), but not to other mitogens such as concanavalin A, anti-CD3 (OKT3), or pokeweed mitogen. We tested this hypothesis by comparing the proliferative response of SEB and SEA with the other mitogens for 25 controls, 20 HIV+, and 15 donors with acquired immune deficiency syndrome (AIDS). We found that peripheral blood mononuclear cells, as well as the CD4+ and CD8+ subsets from both HIV+ and AIDS sources, the degree of suppression of mitogenesis for SEB and SEA was approximately equal to or less than that of the other mitogens. Moreover, suppression of HIV+ CD4+ and CD8+ T cell responses to SEB and SEA was equal (26%). If HIV superantigens exist, our data suggest that they are not responsible for the selective depletion of the CD4+ T cell subset as evaluated by SEB and SEA specificity.

Retrobulbar neuritis. In vitro evidence of sensitization to myelin basic protein in patients without multiple sclerosis.

Thirty-six normal subjects and 34 patients with retrobulbar neuritis were studied with use of the technique of macrophage migration inhibition factor assay and myelin basic protein as antigen. Serial studies were carried out when possible. Normal subjects gave a mean migration index of 100.9+/-9. Eleven patients with retrobulbar neuritis alone gave a mean migration index of 55+/-16 in the first 3 weeks of illness, 89+/-17.3 during the fourth to the twenty-fourth weeks, and 100.9+/-9.0 after the twenty-fourth week. Ten multiple sclerosis patients with retrobulbar neuritis gave values of 61+/-21 in the first 3 weeks of an attack and 92+/-22.8 during the fourth to the twenty-fourth weeks, and 12 other multiple sclerosis patients 24 weeks or longer after an attack gave a value of 101.9+/-12.6. In a mean follow-up period of 1.9 years, only two patients presenting with retrobulbar neuritis alone have had a diagnosis of multiple sclerosis established; three others have weakness and reflex change in one limb only; and four have minor psychiatric problems. One retrobulbar neuritis patient has a family history of multiple sclerosis, but has no neurologic abnormalities. Comparison of these studies in both groups shows no statistical differences and supports the concept that cell-mediated hypersensitization to central nervous system myelin basic protein, however initiated, is a factor in the pathogenesis of retrobulbar neuritis.

Cellular hypersensitivity in attacks of multiple sclerosis. I. A comparative study of migration inhibitory factor production and lymphoblastic transformation in response to myelin basic protein in multiple sclerosis.

Although cell-mediated hypersensitivity to basic myelin protein has been demonstrated in multiple sclerosis with use of cell migration assays, results with the lymphoblastic transformation technique have been inconclusive. However, prospective studies relating results of lymphoblastic transformation to the clinical course of multiple sclerosis have not been reported. In the present study, both lymphoblastic transformation and migration inhibitory factor assays were used, and results related to the temporal course of multiple sclerosis. Results of the present investigation show that cell-mediated hypersensitivity to myelin basic A1 protein is most significant during exacerbations of multiple sclerosis. Responses obtained employing either the lymphoblastic transformation or migration inhibitory factor assay were equally significant. The results support the hypothesis that a factor suppressing deoxyribonucleic acid synthesis is present in multiple sclerosis and is inhibited by the use of steroids.

Induction of allergic neuritis in rhesus monkeys.

Experimental allergic neuritis (EAN) was induced in rhesus monkey (Macaca mulatta) following sensitization with rabbit nerve (PNS) myelin in complete Freund's adjuvant (CFA) or with bovine P2 protein complexed with phosphatidyl serine (P2-lipid) in CFA. The response of monkeys receiving PNS myelin in CFA differed from the previous studies where monkeys developed clinical signs of fatal EAN within 15-20 days following sensitization. The monkeys in this study (6) showed a much longer delay (40-114 days) before the appearance of severe clinical signs, and 4 of the 6 animals survived without further attack (1 year). Monkeys (4) injected with P2-lipid (2:1 ratio; w/w) developed severe clinical signs of EAN which was fetal in 3 cases. Peripheral lymphocytes from monkeys sensitized to the P2-lipid showed a much stronger mitogeneic response to P2 protein than those from the PNS myelin-sensitized monkeys. on quantitation of the circulating anti-P2 antibodies, the P2-sensitized monkeys generally had much titers than those sensitized with PNS myelin.

Comparative mitogenic response of old and young rhesus monkey T cells to lectin from Erythrina cristagalli.

The lectin (EC) from the coral tree, E. cristagalli, while less mitogenic on a molar or weight basis than PHA or ConA, strongly activates both Rhesus monkey and human T cells. The optimal mitogenic concentrations for both Rhesus and human T cells are 0.25, 2.5, and 25 micrograms/ml, respectively, for PHA, ConA, and EC. Aged Rhesus T cells were profoundly suppressed in mitogenic response to EC (approx. 80%) compared to young Rhesus cells. However, in the presence of supplemental interleukin 2 (20 U/ml), the age-related defect was reversed; the average mitogenic response of the old Rhesus T cells was increased sixfold.

Inhibition of T cell mitogenesis by nitrofurans.

A group of nitrofurans (5-nitro-2-furaldehyde, nifuroxime, nitrofurazone, nitrofurantoin, 5-nitro-2-furoic acid and 2-nitrofuran) were evaluated for inhibition of mitogenesis (DNA synthesis) in human peripheral blood T cells. T cells, either triggered by phorbol myristate acetate (PMA) or in the presence of accessory cells, were activated with a specified mitogen [phytohemagglutin (PHA), concanavalin A (ConA), or anti-CD3] and the amount of tritiated thymidine incorporated into DNA was determined. The results obtained indicate that nitrofurans inhibit mitogenesis irrespective of activator. 5-Nitro-2-furaldehyde was much more inhibitory than the other compounds, while 2-nitrofuran was less inhibitory. When the aldehyde group (5-nitro-2-furaldehyde) was replaced by a carboxyl group (5-nitro-2-furoic acid), the inhibitory activity was also reduced greatly. These results show that while the nitro group alone confers inhibitory activity to the furan ring, the group at the 2 position is crucial. In general, the mitogenic response of purified T cells (lacking accessory cells) triggered by PMA (phorbol ester) was inhibited less than that of the T cell-accessory cell system. With the latter, 50% inhibition of T cell mitogenesis was achieved by nifuroxime, nitrofurazone, and nitrofurantoin at 45-51 and 34-39 microM with PHA and ConA respectively. When purified T cells were used, the values were 71-85 and 55-60 microM respectively. For a given drug concentration, mitogenesis was more inhibited when induced by ConA or anti-CD3 than by PHA. The importance of using a single cell system (purified T cells) was emphasized by the interesting finding that only this system showed enhancement of mitogenesis, up to 35-40% at low drug levels. With the exception of the nitrofuraldehyde, the nitrofurans at strongly inhibitory levels were only moderately cytotoxic, exhibiting 62-85% cell survival after exposure to drug for 68 hr. Our results suggest that nitrofurans inhibit T cell mitogenesis by a relatively non-toxic mechanism; these results are comparable to those obtained for mammalian cells under aerobic conditions.

Isolation and partial characterization of the major proteins of rabbit sciatic nerve myelin.

The P0, P1, and P2 proteins were isolated from rabbit sciatic nerve and demonstrated to have molecular weights of 30,000, 18,200, and 12,000, respectively, by polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate. The P1 protein characterized by peptide mapping, optical rotatory dispersion and encephalitogenic activity appears to be quite similar to the CNS myelin basic protein. The P2 protein is distinctly different from the P1 protein as characterized by peptide mapping and optical rotatory dispersion. It appears to have a distinct secondary structure, predominantly of beta-configuration. The P0 protein is distinctly different from either of the basic proteins, especially with respect to its marked insolubility in aqueous solutions. It contains more than 1.0 mole of hexosamine which is not present in either the P1 or P2 protein. Both the P0 and P2 proteins failed to produce any evidence of experimental allergic encephalomyelitis or neuritis when injected into guinea pigs or monkeys. In contrast, the P1 protein produces experimental allergic encephalomyelitis in both species.

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells.

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P2 did not. We demonstrate the presence of PNS myelin proteins P1 (identical to BP) and P2 by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P1 and P2 are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also support the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P2 serum do not.

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. I. Effects of antiserum upon living cultures of nervous tissue.

We studied the effects of antiserum against rat peripheral nervous system (PNS) myelin, rat or chicken central nervous system myelin basic protein (BP), or rabbit P2 protein from PNS myelin on myelinated cultures containing only rat dorsal root ganglion neurons and Schwann cells. While anti-PNS myelin serum consistently produced segmental PNS demyelination, anti-BP serum and anti-P2 serum did not. The culture results suggest that the myelin PNS proteins P1 (identical to basic protein from central nervous system myelin) and P2 are not exposed on the extracellular surfaces of myelin-related Schwann cells in tissue culture.

Multiple sclerosis: sensitization of a myelin basic protein fragment (peptide T) encephalitogenic to primates. A preliminary report.

Myelin basic A1 protein is the sole antigen of the central nervous system capable of inducing experimental allergic encephalitis (EAE), but sensitization with peptide fragments of the molecule may also induce disease. Using the macrophage migration inhibition factor (MIF) assay we have compared sensitization to portions of the molecule active in inducing EAE in monkeys with results obtained concomitantly using the intact protein. Cellular sensitization to human myelin A1 protein, peptide L (residues 1-116), peptide T (residues 117-170), and petide Y (residues 154-170) was studied using the Thor-Rocklin MIF assay system. Lymphocytes of 10 normal subjects, 10 multiple sclerosis patients 0-3 weeks after onset, 10 4 weeks to 3 months after and 10 6 months or longer after onset of an acute exacerbation were assayed. Results of the investigation reveal evidence of cellular sensitization to myelin basic protein encephalitogenic peptide T occurring during attacks of multiple sclerosis. Peptide L, relatively nonencephalitogenic to primates, failed to induce a significant lymphocyte response, whereas peptide Y which is encephalitogenic gave irregular results.

Multiple sclerosis and cell-mediated hypersensitivity to myelin A1 protein.

Cellular hypersensitivity to myelin basic (A1) protein was evaluated using the MIF assay in 246 subjects. Of 100 with multiple sclerosis positive results are seen in relationship to acute exacerbations of illness. Normal control subjects gave a mean value of 100 +/- 9% whereas patients studied within 4 weeks of onset of illness gave a result of 59 +/- 12.5%. A convalescent group studied between 5 and 12 weeks after an attack gave results of 86 +/- 22.2%. A chronic group gave a mean of 91 +/- 8.2%. Positive values were also seen in a number of other patients with central or peripheral nervous system disease especially those with "autoimmune disease". However, results of this study clearly establish a temporal relationship between in vitro evidence of hypersensitivity to A1 protein and clinical expression of disease.