Holly Armstrong

Prevention of experimental allergic encephalitis in Lewis rats by rat and bovine spinal cord proteins

EXPERIMENTAL allergic encephalitis (EAE) is an inflammatory autoimmunedisease of the central nervous system (CNS) that is mediated by T lymphocytes sensitised to the organ-specific basic protein (BP) of CNS myelin2. Lesions of delayed hypersensitivity develop in the brain and spinal cord when sensitised lymphocytes traverse the walls of CNS blood vessels and react with the target antigen in myelin3. EAE can be prevented by injecting BP in Freunds incomplete adjuvant (FIA) before challenge, suppressed by injecting BP soon after challenge and treated by injections of BP after clinical symptoms appear4. Prevention, suppression and treatment are immunologically specific for BP and can be dissociated from the production of humoral antibody against BP5. The demonstration that pretreatment of guinea pigs with bovine spinal cord protein (BSCP)6,7 prevented the development of EAE8,9 is thus of considerable importance because BSCP is chemically and antigenically distinct from bovine BP and is not encephalitogenic. As pretreatment with SCP induced a state of nonspecific unresponsiveness to bovine BP in the guinea pig, we investigated whether SCP had anti-encephalitogenic activity in the Lewis rat, a highly inbred rat strain used extensively for the study of EAE. We report here that Lewis rats pre-treated with rat SCP (ref. 10) were protected against EAE, as they did not develop the disease when they were subsequently immunised with rat myelin BP in complete Freunds adjuvant (CFA).

Preparative isoelectric focusing in agarose

A method is described for preparative isoelectric focusing in agarose using low electroendosmotic agarose. Resolution comparable to that seen on analytical polyacrylamide gels is attainable as demonstrated by the isolation of bands with identical idiotypes from the serum of a patient with a monoclonal gammopathy.

Isolation and further characterization of the anti-encephalitogenic rat spinal cord protein from brain, spinal cord and peripheral nerves.

Abstract After fresh rat nervous tissue was delipidated with acetone, all the immunoreactive rat spinal cord protein (RSCP) in brain (RB-SCP), spinal cord (RSCP) and peripheral nerve (RSCP-PN) could be extracted with 0.05 M sodium acetate buffer, pH 5.0. The extracts were purified by absorption on CM-52 cellulose at pH 5.0 and by gel filtration on Sephadex G-50. At this stage, RSCP-PN formed one band when analyzed by SDS-polyacrylamide gel electrophoresis. In contrast, preparations of RB-SCP and RSCP contained two components of similar molecular size. Only the larger component was present in the specific precipitates formed by mixing anti-RSCP antibody with RB-SCP or RSCP, clearly indicating that the smaller component was a contaminant. Thus, the final purification step for RB-SCP and RSCP consisted of absorption on cyanogen bromide-activated Sepharose-4B coupled to anti-RSCP antibody followed by elution of immunoreactive RSCP at pH 2.5. RB-SCP, RSCP and RSCP-PN isolated from delipidated nervous tissue were identical by immunodiffusion analysis. RSCPs have a molecular weight of 11 000 ± 500, as estimated by gel exclusion chromatography and by SDS-polyacrylamide gel electrophoresis. They have the electrophoretic mobility in agarose at pH 8.6 of a serum β-globulin and an isoelectric point of pH 4.5. RB-SCP and RSCP contain respectively about 17, 24 and 42% of basic, acidic and hydrophobic amino acids. RSCP-PN has a somewhat higher content of acidic amino acids, (31%) and a lower proportion of hydrophobic amino acids (32%). All RSCPs lack cystine and contain only 0.2% of tyrosine. Immunodiffusion analyses showed that at the antibody level there is no immunochemical relation between bovine SCP (BSCP) and purified RB-SCP, RSCP or RSCP-PN. However, there is a protein in rat peripheral nerve that is similar but not identical to BSCP. This protein is not present in extracts of brain or in extracts of spinal cord from which the spinal nerves have been removed.