Immunologic activity of myelin basic protein in strain 2 and strain 13 guinea pigs.

The resistance of Strain 2 guinea pigs to experimental allergic encephalomyelitis (EAE) induced by inoculation with whole CNS tissue in complete Freund's adjuvant (CFA) has been confirmed. The resistance is even more pronounced when myelin basic protein (BP) is used in attempts to induce EAE. Strain 2 guinea pigs are also resistant to an immunization schedule (multiple injections with BP in IFA followed by a single injection of BP in CFA) known to induce significant levels of antibody in susceptible strains. The poor response of Strain 2 guinea pigs to BP is not the result of lack of specific B cells--antibody equivalent to that produced by Strain 13 animals is obtained when the inoculum contains 0.5 mg BP and 2.5 mycobacteria.     

Prevention of experimental allergic encephalomyelitis by bacterial lipopolysaccharides: inhibition of cell-mediated immunity

The immunization of Lewis rats with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant inhibits the development of experimental allergic encephalomyelitis (EAE) in these animals. These protected animals fail to manifest significant in vivo delayed-type hypersensitivity skin tests and in vitro lymphocyte proliferative responses to BP. Our results indicated that LPS induces a nonspecific reduction in immune reactivity of BP in Lewis rats.     

Immunomorphologic features of experimental allergic encephalomyelitis induced by the administration of tryptophan peptide

Three patterns of EAE with different morbidity and mortality rates were induced in the guinea pigs inoculated with various doses of tryptophane peptide (TP) and complete Freund's adjuvant. TP-sensitized animals manifested the delayed type hypersensitivity (DTH) reactions to TP and circulating anti-TP and -BPF antibodies were not found, polypeptide fraction of myelin basic protein (BPF while a A correlation was revealed between the DTH-reactions and EAE development. Intracutaneous TP and BPF injections at the early period before the EAE onset resulted in reduction of morbidity rate from 90 to 50 per cent. For the first time with the help of modified Marchi method demyelination has been shown to be highly marked in CNS tissue process in CNS can be caused by cell-mediated immune of animals given TP. It is supposed that the demyelinating reaction to encephalitogenic fragment of BP molecule. The data indicate a possibility of EAE inhibition by means of TP and BPF injections in saline solution.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

THE ENCEPHALITOGENIC ACTIVITY AND MYELIN BASIC PROTEIN CONTENT OF ISOLATED OLIGODENDROGLIA

Abstract— Oligodendroglia isolated from frozen human brain induced EAE in the guinea pig when injected with Freund's complete adjuvant. An acid extract of the isolated cells was highly encephalitogenic. The disease was clinically and histologically similar to EAE induced with CNS white matter or myelin basic protein(MBP). The isolated cells were essentially myelin-free but contained a large amount of myelin basic protein as determined by polyacrylamide gel electrophoresis. It was shown that the cells can absorb ¹²⁵I-MBP during isolation and the possibility that contamination from extra cellular MBP is responsible for the encephalitogenic activity is considered.     

Transfer of allergic encephalomyelitis with spleen cells from donors sensitized with myelin basic protein in incomplete Freund's adjuvant

Myelin basic protein (BP) emulsified in incomplete Freund's adjuvant (BP/IFA) is relatively nonencephalitogenic in Lewis rats. Furthermore, repeated injections of BP/IFA prevent subsequent induction of experimental allergic encephalomyelitis (EAE) by BP emulsified in complete Freund's adjuvant (BP/CFA). In spite of this, spleen cells from rats injected repeatedly with BP/IFA transfer EAE after they are cultured with BP almost as effectively as BP/CFA spleen cells. However, unlike the latter, BP/IFA spleen cells do not proliferate in response to BP in culture. Furthermore, BP/IFA spleen cells are unable to transfer EAE after culture with concanavalin A (Con A), in contrast to BP/CFA spleen cells. Both populations of spleen cells undergo a strong proliferative response to Con A in culture. For BP/IFA cells, at least, a proliferative response to BP in vitro is not a prerequisite for enhanced transfer of EAE in Lewis rats.     

Suppression of experimental allergic encephalomyelitis in Lewis rats treated with myelin basic protein-liposome complexes: clinical, histopathological, and cell-mediated immunity correlates

Guinea pig myelin basic protein (MBP) was inserted into phosphatidylserine liposomes and Lewis rats were injected by the intracardiac (ic) route with 75 microgram doses of MBP-liposomes according to various schedules. After challenge with 75 microgram guinea pig MBP in complete Freund's adjuvant, the rats were followed for clinical signs, were tested for delayed hypersensitivity (DTH) and lymphocyte transformation (LT) to MBP. The animals were sacrificed 30 days after challenge and the central nervous system tissue was examined for histological modifications. Rats treated with two injections of MBP-liposomes, 7 days before and 7 days after challenge, showed the highest degree of protection from clinical manifestations. Histological lesions were not significantly reduced. DTH reactions to MBP were all positive, regardless of treatment. LT assays were positive overall in only 50% of the animals tested. The response to rat MBP was significantly lower than to guinea pig MBP, especially in the groups treated with MBP-liposomes. Adoptive transfer of spleen cells from MBP-liposome-treated donors reduced the clinical scores of actively induced EAE in syngeneic recipients by 40-50%. These results suggest that at least one mechanism responsible for antigen-specific protection in EAE by MBP-liposomes operates through active suppression transferable by spleen cells.     

Correlation Between 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase Activity and Demyelination In Vitro Using a Syngeneic System

Cultures of myelinated SJL/J fetal mouse spinal cord were incubated with serum and lymphoid cells from syngeneic animals with experimental allergic encephalomyelitis (EAE) induced by syngeneic spinal cord homogenate (SSCH) in complete Freund's adjuvant or others injected with complete Freund's adjuvant alone. After 24 or 48 h of exposure, demyelination was determined by light microscopic examination and quantification of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. Cultures exposed to spleen or lymph node cells from SSCH-sensitized animals showed the greatest alterations in myelin and decreases in 2',3'-cyclic nucleotide 3'-phosphohydrolase activity whereas serum from these animals had less effect. Cells and serum from complete Freund's adjuvant-injected control animals also induced structural changes in myelin that were significantly less than changes induced by cells and serum from animals with EAE. These experiments show that lymphoid cells and serum obtained from SJL/J mice with acute EAE affected myelin biochemistry and morphology in syngeneic CNS cultures.     

Brain and Spinal Cord Levels of Histamine in Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord homogenate emulsified with Mycobacterium tuberculosis-enriched complete Freund's adjuvant (CFA). Control rats were inoculated with CFA alone. Control and EAE rats were killed on days 7, 9, 11, and 13 postinoculation, and regional brain and spinal cord levels of histamine were determined. No regional differences in histamine content between control and EAE rats were seen on day 7 or 9 postinoculation. However, depending on the region, EAE rats exhibited significantly higher levels of histamine in their CNS on day 11 or 13 postinoculation or on both. Thus, regionally and temporally specific increases in brain and spinal cord levels of histamine develop concomitant with or just after the appearance (on day 10 postinoculation) of clinical signs of acute EAE, a finding suggesting that histamine may be involved in the development or expression of acute EAE in Lewis rats.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Studies on autoimmune encephalomyelitis in the guinea pig. II. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor.

Complement-dependent demyelinating activity of whole brain homogenate (WBH)-induced experimental allergic encephalomyelitis (EAE) sera was tested on long term tissue cultures of in vitro myelinated fetal guinea pig cerebellum. Complement-fixing (CF) auto-antibodies were shown to be the responsible agents, as demonstrated in experiments where all reagents belonged to the same species: guinea pigs of outbred (Hartley) and even of inbred (S2 or S13) strains. These antibodies were of the IgG2 class as shown by Sephadex G-200 and DEAE cellulose fractionation experiments. The corresponding auto-antigen was present in the homogenate and myelin of the central nervous system (CNS) tissue. It was different from the encephalitogenic basic protein of CNS myelin (BP), as shown in experiments where the demyelinating auto-antibodies were induced, detected, and absorbed by WBH or by CNS myelin but not by BP. They were neither induced by nor cross-reacting with cerebroside and peripheral nervous system (PNS) tissue.     

Autoimmune effector cells. VI. Transfer of experimental allergic encephalomyelitis with spleen cells activated in mixed lymphocyte cultures

Although spleen cells (SpC) from Lewis rats that have been immunized with guinea pig myelin basic protein in complete Freund's adjuvant do not transfer experimental autoimmune encephalomyelitis (EAE) to syngeneic recipients, we report that effector cells of EAE can be activated in SpC suspensions during mixed lymphocyte reactions (MLR) to allogeneic SpC. Effector cell activation correlates with interleukin 2 (IL 2) production. The results are consistent with the hypothesis that autoreactive cells may be generated as a result of an immune response to exogenous antigens.     

It is still not for the old iron: adjuvant effects of carbonyl iron in experimental autoimmune encephalomyelitis induction

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis. Dark Agouti rats immunized with spinal cord homogenate (SCH) and carbonyl iron (CI), as an adjuvant, develop severe hyperacute form of EAE. They succumb to EAE earlier and have higher clinical scores and lethality rate in comparison to counterparts immunized with SCH + complete Freund's adjuvant. There is no difference in the number of cells or in histological presentation of the CNS infiltrates of rats immunized with the two adjuvants. However, there are more granulocytes, NK and NKT cells, and less CD4(+) T cells in the spinal cord infiltrates of SCH + CI-immunized animals. Nitric oxide (NO)-generating enzyme inducible NO synthase have higher expression in spinal cord of SCH + CI-immunized rats, and this corresponds to more intensive nitrotyrosine formation in the CNS tissue of these rats. Abundant infiltration of granulocytes and NK cells into the CNS and excessive generation of peroxynitrite within the CNS of SCH + CI-immunized rats might account for the severe neurological deficits induced by immunization with CI. These factors should be closely examined in the fulminant forms of multiple sclerosis and acute disseminated encephalomyelitis, as they could represent a promising targets for therapy.     

Peptide length requirement for experimental allergic encephalomyelitis in guinea pigs

Three peptides overlapping the tryptophan region of bovine CNS myelin basic protein were synthesized by the solid phase procedure of Merrifield. These were the nonapeptide H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, the octapeptide H-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, and the heptapeptide H-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. They were tested for encephalitogenic activity in guinea pigs with either Freund's complete adjuvant containing M. tuberculosis or muramyl dipeptide in incomplete Freund's adjuvant at doses of 10 microgram per animal. The results show that deletion of one or two residues from the amino-terminal end of the nonapeptide destroyed the ability of the shorter peptides to induce clinical but not histological signs of EAE.     

Chronic permeability of the central nervous system to mononuclear cells in experimental allergic encephalomyelitis in the Lewis rat.

In order to assess whether experimental allergic encephalomyelitis (EAE), a putative animal model for multiple sclerosis (MS), is an ongoing chronic disorder, we have studied the permeability of spinal cords of Lewis rats with EAE to 3H-uridine- or 3H-thymidine-labeled lymphoid cells obtained from thymuses of naive donors or from draining lymph nodes of donors injected with guinea pig spinal cord + complete Fruend's adjuvant (CFA), guinea pig myelin basic protein + CFA, or with CFA alone. During the acute clinical phase of EAE there is a high-level infiltration of 3H-thymidine- or 3H-uridine-labeled cells into the spinal cords. After clinical recovery from EAE up to 58 days post-inoculation, there is a low-level infiltration of 3H-thymidine-labeled cells into the spinal cords. A similar infiltration into the spinal cords by 3H-uridine-labeled cells was not detected. Donor cells from animals immunized with CFA alone showed similar levels of infiltration into the spinal cords of animals with EAE as donor cells from animals immunized with the encephalitogenic emulsion. Spinal cords from recipients immunized with CFA alone showed no increased permeability to labeled cells. Heat-killed labeled cells did not migrate into the spinal cords of animals with EAE. We conclude that a) EAE is a chronic disease and in this regard is a valid model for MS; and B) in the chronic phase of EAE, recently divided cells (3H-thymidine-labeled cells) show higher levels of migration into the target tissue than 3H-uridine-labeled cells.     

Chronic relapsing experimental allergic encephalomyelitis: Identification and dynamics of T and B cells within the central nervous system

Using a monoclonal antibody against guinea pig T cells and anti-guinea pig immunoglobulins, T- and B-cell dynamics were studied by immunofluorescence in situ in the central nervous system (CNS) of animals with untreated and treated chronic relapsing experimental allergic encephalomyelitis (EAE). Treated animals were given a series of injections of either myelin basic protein (MBP) in incomplete Freund's adjuvant (IFA) or MBP and galactocerebroside in IFA. Within the CNS, T and B cells showed distinct distribution patterns in untreated chronic relapsing EAE, similar to that recently described in acute EAE. T cells were predominantly localized within the CNS parenchyma and B cells were mainly found in perivascular areas. B-cell infiltrates were more extensive than in acute EAE and, although most were centered around blood vessels, some were also detectable in the parenchyma. IgG, C₃, and albumin deposits were common. These observations suggest an age-dependent difference in the immune response. In treated chronic EAE, the disease process was apparently arrested and T- and B-cell infiltrates in the white matter were negligible. Therefore, it appears that the present treatment protocol prevents lymphocytes from entering the CNS parenchyma.     

Specificity of antisera from Lewis rats immunized with encephalitogenic fragment 43-88 of guinea pig myelin basic protein.

Inbred Lewis rats were immunized with encephalitogenic fragment 43-88 of guinea pig myelin basic protein emulsified in complete Freund's adjuvant. After recovery from experimental allergic encephalomyelitis (EAE) the animals were given a booster immunization, bled, and the specificity of the individual anti-fragment antisera was examined by direct binding assays by using radioiodinated fragment 43-88 and peptides 43-67, 68-88, and 79-88. Competitive-binding experiments with these peptides in competition with labeled fragment 43-88 were also done. The results of these experiments indicated that the immunodominant region of fragments 43-88 was the carboxy-terminal half of the molecule. Individual antisera recognized different antigenic determinants within this region.     

Effect of levamisole and etimisole on the development of experimental allergic encephalomeylitis

A single subcutaneous inoculation with 0.02 mg of heterologous myelin basic protein (MBP) in combination with Freund's complete adjuvant resulted in clinical and histological manifestations of experimental allergic encephalomyelitis (EAE) in 80-90% of treated guinea pigs. Daily parenteral administration of levamisole and etimisole during the latent period produced a suppressive effect on EAE development, reducing morbidity and mortality rates and preventing pathomorphological changes in the CNS. Animals receiving the drugs had decreased delayed hypersensitivity reactions to MBP in vitro. Etimisole brought about a moderate decline in the formation of circulating anti-MBP antibodies, while levamisole did not affect the strength of the humoral response, something which confirmed the primary role of cell-mediated immune reactions in the CNS demyelinization process. The reported findings may be significant in developing therapeutic strategies with respect to demyelinization diseases.