Encephalitogenic Basic Protein Research Articles

Effect of splenectomy on the development of experimental autoimmune encephalomyelitis in rats with different genetic background.

We studied the effect of total or partial splenectomy on experimental autoimmune encephalomyelitis (EAE) development in rats with different genetic background after immunization with guinea pig basic encephalitogenic protein in Freund's complete adjuvant. The operations did not affect some strains with a high susceptibility to EAE but may have reduced the EAE development in the Fischer strain, which shows only a weak susceptibility. In some hybrids and backcrosses, but not in others, splenectomy resulted in a clear-cut reduction of neurological signs of EAE. This effect may be ascribed to the elimination of contrasuppressive cell populations.

Cellular and genetic basis of the relative resistance to the induction of experimental allergic encephalomyelitis (EAE) in albino oxford (AO) rats.

EAE has been widely studied as a possible model for human demyelinative disease. It can be readily induced in several strains of rats by a single injection of spinal cord or encephalitogenic basic protein emulsified in complete Freund’s adjuvant (CFA). We have previously reported that AO rats always exhibited significantly lower susceptibility to the induction of EAE when compared with Lewis rats (1). In an attempt to delineate the mechanisms of the relative resistance to the induction of EAE in AO rats we studied comparatively the cellular requirements for the induction of EAE in Lewis, AO and Dark August (DA) rats. Further, we have undertaken a study of susceptibility to EAE in AO and DA rats, their F1 hybrids and animals of backcross generations in order to establish possible genetic determinants of the resistance to EAE in AO rats.KeywordsBasic ProteinExperimental Allergic EncephalomyelitisRelative ResistanceBackcross GenerationSuppressor Cell ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The immunologic response in mice unresponsive to experimental allergic encephalomyelitis.

The suppressor cells that are involved in antigen-induced protection against EAE in mice were investigated with respect to their effect on the immune response. The cellular immune response to the basic encephalitogenic protein (BE) and to PPD were studied in mice with either actively induced or adoptively transferred unresponsiveness to EAE. The results demonstrate that the DTH response to BE, as assayed in the radiometric ear skin test, was suppressed in mice protected against EAE. Moreover, the passive transfer of DTH response to BE by effector lymphocytes was also inhibited by the preinjection of suppressor cells. On the other hand, the suppressor cells did not affect the response to PPD in all these experiments. The results indicate that suppressor cells that mediate unresponsiveness to EAE regulate also the cellular immune response to BE in a specific manner. These suppressor cells are probably active both at the induction and the effector phase of the immune response.

Induction of experimental allergic encephalomyelitis in genetically resistant strains of mice

Experimental allergic encephalomyelitis (EAE) is an acute autoimmune disease of the central nervous system. It is elicited in various laboratory animal species by a single injection of either whole central nervous system tissue homogenate, or the purified basic encephalitogenic protein isolated therefrom. Clinical manifestations of hind leg paralysis usually occur 11–21 days after challenge, preceded by histological lesions of perivascular infiltration in the brain1. EAE has been intensively investigated both as a model for human demyelinating diseases and as a prototypic organ-specific autoimmune disease1. EAE is under genetic control in guinea pigs, rats and mice, and strains that are genetically susceptible or resistant to EAE have been demonstrated2–4. We have previously5,6 investigated the genetic control of susceptibility to EAE in mice and demonstrated that the disease could be induced only in the SJL/J strain of mice, or in its crosses with various resistant strains. The resistance to EAE is not associated with genetic differences in the response of the various strains to pertussis vaccine, which is used as adjuvant in the induction of EAE in mice, as both the sensitive SJL/J strain and the resistant strains BALB/c, DBA/2 and C57BL/6J are responsive to pertussis7. A great difference was observed, however, in the sensitivity to EAE of the various F1 hybrids of SJL/J. Thus, crossing the sensitive SJL/J strain with part of the resistant strains, such as NZB, BALB/c or its congenic strains BALB . B10 and BALB. C3H, led to fully susceptible hybrids, with even higher incidence of EAE than that observed in the parental SJL/J strain. On the other hand, crossing SJL/J with other strains, such as C57BL/6J or DBA/2, led to hybrids with very low susceptibility to EAE. These results suggested that in the crosses with the various resistant BALB strains and NZB strain, there is probably complementation with another gene. It was therefore postulated that in these resistant strains, the influence of the gene determining susceptibility to EAE is masked by the presence of naturally occurring suppressor cells. We present here experimental evidence which corroborates this assumption.

Allergic encephalomyelitis induced in guinea pigs by a peptide from the NH2-terminus of bovine P2 protein.

Experimental allergic neuritis (EAN) is an inflammatory demyelinating disease of the peripheral nervous system (PNS) first described by Waksman and Adams1,2 in rabbit, guinea pig and mouse after injection of PNS antigen in Freund's complete adjuvant. In rabbit and mouse, the histological lesions were confined to the PNS but the guinea pig reacted curiously to PNS antigens by developing lesions in the central nervous system (CNS) as well as the PNS. Experimental allergic encephalomyelitis (EAE), the CNS counterpart of EAN, is known to be a response to CNS myelin basic protein. In early experiments3,4, basic proteins from PNS myelin also produced a disease in guinea pigs characterised by CNS lesions. Characterisation of the proteins of PNS myelin demonstrated the presence of a basic protein (P1) which was similar to the encephalitogenic basic protein of CNS myelin5. At first it seemed that the CNS lesions were being caused by this (P1) protein. However, immunological studies from our laboratories suggested that when guinea pigs were sensitised to PNS myelin, the resulting lesions were a response to the small PNS myelin basic protein (P2) and not to the P1 protein6. In a preliminary report7, we indicated that a peptide fraction derived from the P2 protein could produce a disease in guinea pigs characterised by CNS lesions. Sequence studies under way in our laboratory now enable us to report that this response is being caused by a tryptophan-containing 20-residue peptide (CN3) derived from the NH2-terminal segment of the bovine P2 protein.

STUDIES ON AUTOIMMUNE ENCEPHALOMYELITIS IN THE GUINEA PIG—TIT. A COMPARATIVE STUDY OF TWO AUTOANTIGENS OF CENTRAL NERVOUS SYSTEM MYELIN1

Abstract— A new CNS myelin autoantigen(s) (referred to as M2), different from the encephalitogenic basic protein (BP), can be detected with guinea‐pig demyelinating and complement fixing (CF) sera raised against guinea pig CNS tissue or myelin (Lebaret al., 1976). M2 and BP were present in mouse, rat, rabbit, bovine and human CNS tissues when tested with guinea‐pig homologous specific antisera; they were not present in non‐CNS tissues. Both autoantigens were also detected in newborn guinea‐pig myelin and myelin‐like fractions.The CF activity of myelin with demyelinating (anti‐M2) sera was not altered by trypsin; however, absorption experiments showed that M2 was partly trypsin sensitive. Both antibodies against the trypsin sensitive and the trypsin resistant determinants of M2 were demyelinating. Both determinants of M2 were preselit in mouse, rat, rabbit, bovine‘and human CNS tissues and in guinea‐pig newborn myelin. CF BP activity of myelin was partially or even totally abolished by trypsin, but the persistent encephali‐togenicity of trypsin‐treated myelin could be attributed to non‐CF encephalitogenic peptides from BP.In accordance with recent work our results tend to support an inner localization of BP in myelin; M2, on the other hand, would be a surface antigen(s).

DEVELOPMENTAL CHANGES OF MYELIN PROTEINS IN THE CHICK PERIPHERAL NERVE

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)‐polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR‐P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF‐P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR‐PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR‐P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.

Sensitization to myelin basic protein in attacks of multiple sclerosis: A preliminary report

Recent attempts to detect the presence of antibody to encephalitogenic basic protein in multiple sclerosis have generally been unsuccessful. The utilization of a new system of double immunodiffusion in detecting such antibody in EAE serum prompted us to apply this method in the search of such antibody MS sera. In our preliminary investigation of 67 sera, antibody was detectable in MS sera, but it was most often found during convalescence rather than an acute exacerbation of illness. Antibody was also found in several myasthenic patients, and occasionally in subjects with other neurological disease.

Cellular and humoral responses to myelin basic protein in multiple sclerosis: a dichotomy.

The macrophage migration inhibition factor (MIF) assay and a counterimmunodiffusion assay were utilized to measure immune responses to human myelin basic protein in 75 patients with multiple sclerosis (MS) and in 120 control subjects. Eight out of ten MS patients in acute exacerbation and one out of seventeen convalescent, but none of chronically ill MS patients gave positive results in the MIF test. Forty-six percent of the patients with negative MIF assays but only 22% of those with positive assays had positive antibody results. In the counterimmunodiffusion assay, myelin basic protein antibody was demonstrated in almost 2/3 of patients during convalescence but it was not present in those whose illness had been stable for 6 months or longer. While no correlation with the stage or duration of the illness was present in other disorders, in MS an inverse correlation with clinical activity and in vitro evidence of cellular sensitization to encephalitogenic basic protein was apparent.

Enzyme and protein studies of demyelination in diabetes

Neuropathological evidence of demyelination was found in the brain and sciatic nerve of diabetic patients at autopsy. The activity of acid proteinase was somewhat increased in the white matter but decreased in the gray matter of diabetic patients. No increase was observed in the activity of neutral proteinase in diabetic white and gray matter. The activities of β-glucuronidase and 2′,3′-cyclic nucleotide-3′-phosphohydrolase (CNP) were of the same level as those of the controls. The activities of all 4 enzymes appeared to be increased in the diabetic nerve, with the possible exception of CNP which was measured from only 1 nerve. Furthermore, the amount of total protein was markedly decreased in diabetic peripheral myelin. The encephalitogenic basic protein of diabetic brain myelin was normal in the disc gel electrophoretic patterns of brain myelin proteins. However, the basic proteins of peripheral myelin were reduced in a number of diabetic patients. The present biochemical findings for diabetic white and gray matter were largely normal. Instead, the increased activities of at least the proteinases and β-glucuronidase in diabetic peripheral nerve, together with the loss of basic proteins, indicate extensive biochemical damage of the peripheral nervous system in diabetes. They suggest that demyelination and other phenomena observed in diabetic peripheral nerve are not caused only by angiopathy and impaired circulation.

The autoimmune features of acute transverse myelopathy.

Lymphocytes from patients with acute transverse myelopathy (ATM) were shown to undergo a specific and significant transformation when cultured in vitro in the presence of either the central nervous myelin basic encephalitogenic protein (BE) or the peripheral nerve myelin P2 protein. A similar pattern of response was demonstrated in acute disseminated encephalomyelitis and in acute myeloradiculitis. Lymphocytes from patients suffering from other autoimmune neurological disorders or other neurological diseases affecting the spinal cord showed no response to there immunologically related antigens, which have previously been found to have the capacity of inducing experimental allergic encephalomyelitis, either alone or with experimental allergic neuritis, when injected into animals. The specific in vitro response to BE and P2 suggests that in vivo sensitization of lymphocytes to such self-antigens occurs in ATM and than a cell-mediated, probably postinfecious autoimmune mechanism may be an important factor in the pathogenesis of the disease.

Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.

Oligodendroglia from human autopsied brain: bulk isolation and some chemical properties.

Abstract— Oligodendroglia were isolated from fresh and frozen human autopsied brains by a modification of our technique for isolation of neuronal perikarya and astroglia (Iqbal & Tellez‐Nagel, 1972). Cerebral white matter was minced in a hypertonic hexose‐Ficoll buffered medium, passed through successive screens of decreasing pore size, and the cells were then separated on a discontinuous sucrose density gradient using low speed centrifugation. Cells could also be isolated by substituting sucrose for hexoses in the cell isolation medium. About 95% of the isolated cells had morphology characteristic of oligodendroglia. The usual contaminants in this fraction were capillary fragments, red blood cells, and a few astrocytes. Free myelin was only sparsely seen. About 5–10% of the isolated oligodendroglia had one or more loops of loose membranes extending from the cell plasma membrane. These membraneous loops resembled myelin. The ultrastructural preservation of the cells was poor. The average yield per gram of wet tissue was 52 million cells amounting to 2.4 mg protein, 283 μg DNA, and 94 μg RNA. The protein, DNA, and RNA contents per average cell were 47, 5.3, and 1.8 pg respectively. About 50% of the tissue DNA was recovered as isolated cells, suggesting a larger proportion of oligodendroglia to astroglia, the other principal cell type in human white matter.Comparison of the total protein profiles of the isolated oligodendroglia with those of the purified human myelin on sodium dodecyl sulfate‐polyacrylamide gels revealed the presence of all human myelin constituent proteins in the isolated cells except the intermediate (or DM‐20) myelin proteins. Densitometry of the gels stained with Fast Green showed that the ratio of protein bands corresponding to myelin encephalitogenic basic protein (BP) to myelin proteolipid protein (PLP) in the isolated cells was three‐fold as high as in purified human myelin. The oligodendroglial BP which constituted about 14% of the total cell protein contents, also coelectrophoresed with the myelin BP on pH 4.3 polyacrylamide gels. Furthermore, the two dimensional peptide maps of the tryptic digest of the cell BP labelled with 125I were also identical to the similarly treated myelin BP. The data suggest that the BP can be used as a chemical marker for oligodendroglia.

Unprimed spleen cell populations recognize macrophage-bound antigen with opposite net electric charge.

Previous studies have demonstrated an inverse charge relationship between the net electrical charge of antigen and the responding cells. In the present study we attempted to establish whether this phenomenon holds also for the primary recognition phase of cell-mediated immunity, when the involvement of the macrophage in presenting antigen is obligatory. Normal spleen cells were fractionated over negatively and positively charged columns. The fractionated cell populations, as well as the original cells, were sensitized in vitro on macrophage monolayers that were pulsed either with the basic encephalitogenic protein of myelin or with the acidic copolymer poly(Glu50,Tyr50). Cells eluted from glass bead columns(i.e., the more negative cells) could be sensitized only with the basic antigen, while cells eluted from poly(L-lysine)-glass bead columns (i.e., more positive cells) could be sensitized only to the acidic antigen. Thus, in delayed type hypersensitivity the inverse charge relationship prevails also for the primary immunological recognition of antigen bound to macrophages.

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F 1 hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.

Experimental allergic encephalomyelitis. — Water, sodium and potassium concentration in spinal cord and hemispheres and their changes after dexamethasone treatment

Water content, Na+, K+ and Cl- concentration were measured in rats with experimentally produced allergic encephalomyelitis. Increase of water content by 10%, accumulation of Na+ by 13%, of Cl- by 20% and decrease of K+ by 10% was observed in the spinal cord, but no substantial changes were found in the brain tissue. The hydration of spinal cord is accompanied by perivascular infiltrates. Dexamethasone, administered from day 5, after injecting sensitising encephalitogenic basic protein, effects normal levels of water content and Na+, K+ and Cl- concentration.

Multiple sclerosis, Guillain-Barré syndrome and myelin basic protein-specific cellular antibody.

MULTIPLE sclerosis (MS) and the Guillain–Barre syndrome (GBS) are demyelinating diseases of improven aetiology and pathogenesis, in which infectious and immunological factors have been suggested to have a role1–3. The notion that sensitisation to basic proteins from myelin might be involved has arisen from certain similarities between these human diseases and the autoimmune demyelinating diseases of animals, experimental allergic encephalomyelitis (EAE) and experimental allergic neuritis (EAN)4. EAE is caused by sensitisation to the encephalitogenic basic protein from central myelin (CNS-BP) and EAN by sensitisation to peripheral myelin. One of the two basic proteins of peripheral myelin, P2 protein5, is considered a likely candidate for the neuritogenic factor6. To help determine whether sensitisation to myelin basic proteins is involved in the pathogenesis of MS and GBS, we have used horseradish peroxidase (HRP)-labelled myelin basic proteins7,8 to seek evidence of an antibody of this specificity in nervous system plasma cells in these diseases. Our negative findings do not support a pathogenetic role for myelin basic proteins in MS or GBS.

Studies on Autoimmune Encephalomyelitis in the Guinea Pig

Abstract 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.

Cell‐mediated immunity to neural antigens in idiopathic polyneuritis and myeloradiculitis

Patients with peripheral nervous system disorders were tested for the presence of cellular hypersensitivity to peripheral and central nervous system antigens by means of the in vitro lymphocyte transformation technique. Lymphocytes sensitized to the neuritogenic peripheral nervous system P1L basic protein were found in pure polyradiculitis of the Guillain-Barré syndrome type. Lymphocytes from patients with myeloradiculitis underwent transformation by peripheral P2 basic protein and by central nervous system basic encephalitogenic protein. In cases of chronic relapsing polyneuropathy response was shown to the central nervous system basic encephalitogen and to both of the peripheral nerve basic proteins. Lymphocytes from patients with other neurologic conditions showed no response to any oth these antigens. These findings suggest that cell mediated immunity to specific basic proteins of the myelin plays a rolw in the pathogenesis of the above-mentioned demyelinating disorders and may lead to a new approach in their classification and diagnosis.

The intrinsic fluorescence characteristics of the myelin basic protein.

Abstract—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m‐urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head‐groups. The fluorescence results suggest that the basic protein can be used as a natural, non‐perturbing probe which will report on its environment after it has reacted with other membrane components.