Steven W. Brostoff

Proteins of Myelin

The protein composition of CNS myelin is relatively simple, with two protein fractions comprising 60–80% of the total membrane protein. These are the highly basic, histonelike protein (a single protein in most animals, two in some species) and the hydrophobic proteolipid protein fraction (predominantly a single protein). The remainder of the proteins fall into a heterogeneous group composed of some enzymes, glycoproteins, and an as yet undetermined number of minor proteins of unknown structure and function, most of which have a higher molecular weight than the two major protein fractions. These protein classes are readily separated in several different polyacrylamide electrophoretic systems. The most commonly used procedure is one which employs sodium dodecyl sulfate (SDS) and a high concentration of acrylamide. A typical separation pattern is presented in Fig. 1. The relative proportions and absolute amounts of each protein class can be determined by a spectrophotometric scan of stained or of unstained gels (Greenfield et al., 1971; Poduslo and Braun, 1975).

Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.

T cell receptor peptide vaccination in rheumatoid arthritis : A placebo-controlled trial using a combination of Vβ3, Vβ14, and Vβ17 peptides

OBJECTIVE Restricted T cell receptor (TCR) gene usage has been demonstrated in animal models of autoimmune disease and has resulted in the successful use of TCR peptide therapy in animal studies. This clinical trial was undertaken to determine the safety and efficacy of a combination of Vbeta3, Vbeta14, and Vbeta17 TCR peptides in Freunds incomplete adjuvant (IFA) in patients with rheumatoid arthritis (RA). METHODS A double-blind, placebo-controlled, multicenter, phase II clinical trial was undertaken using IR501 therapeutic vaccine, which consists of a combination of 3 peptides derived from TCRs (Vbeta3, Vbeta14, and Vbeta17) in IFA. A total of 99 patients with active RA received either 90 microg (n = 31) or 300 microg (n = 35) of IR501 or IFA alone (n = 33) as a control. The study medication and placebo were administered as a single intramuscular injection (1 ml) at weeks 0, 4, 8, and 20. RESULTS Treatment with IR501 was safe and well tolerated. None of the patients discontinued the trial because of treatment-related adverse events. Efficacy was measured according to the American College of Rheumatology 20% improvement criteria. Using these criteria, patients in both IR501 dosage groups showed improvement in disease activity. In the most conservative analysis used to evaluate efficacy, an intent-to-treat analysis including all patients who enrolled, the 90-microg dosage group showed a statistically significant improvement compared with control patients at the 20-week time point after the third injection. Trends toward improvement were shown in both the 90-microg and the 300-microg dosage groups at week 24 after the fourth injection. CONCLUSION IR501 therapeutic vaccine therapy was safe and well tolerated, immunogenic, and demonstrated clinical improvement in RA patients. Additional clinical trials are planned to confirm and extend these observations.

Results of a phase I clinical trial of a T-cell receptor vaccine in patients with multiple sclerosis. II. Comparative analysis of TCR utilization in CSF T-cell populations before and after vaccination with a TCRVβ6 CDR2 peptide

We report here the results of a phase I trial of a T-cell receptor (TCR) V beta 6 CDR2 region peptide vaccine in 10 patients with multiple sclerosis who showed biased over-representations of V beta 6 mRNA among T-cells in their cerebrospinal fluids (CSF). One group of 5 patients was immunized twice during a four week period with 100 micrograms of the TCRV beta 6 peptide 39-LGQGPEF LTYFQNEAQLEKS-58 emulsified in incomplete Freunds adjuvant (IFA); the second group of 5 MS patients received 300 micrograms of the same peptide in IFA over a similar time period. Patients were monitored for adverse events, immunogenicity of the peptide and changes in their CSF T-cell populations. The results indicate that this peptide was immunogenic (T-cell proliferation assays and recall DTH responses) in some of the patients, although none of the immunized patients produced detectable anti-peptide antibodies. More importantly, we show that the 5 patients treated with higher doses of the vaccine displayed a slight decrease in CSF cellularity, a lack of growth of CSF cells in cytokine supplemented expansion cultures that implies a significant absence of a subset of activated CD4 T-cells and a marked diminution in V beta 6 mRNA levels among T-cells in these cultures. By comparison, in 5 patients receiving the lower dosage of the vaccine, CSF cellularity was the same or slightly increased over pre-vaccination levels, CSF cells from 1 patient failed to grow in expansion cultures and cultured CSF cells from 2 patients underwent a change from an oligoclonal V beta 6 pattern to one that was more polyclonal. These results justify a more through exploration of the use of TCR peptide vaccines as a possible therapeutic treatment for MS.

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins.

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity‐purified anti‐P2 and anti‐myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate‐polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P2 protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P2 protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high‐molecular‐weight proteins were also detected with anti‐myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high‐molecular‐weight P2 cross‐reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.

Results of a phase I clinical trial of a T-cell receptor peptide vaccine in patients with multiple sclerosis. I. Analysis of T-cell receptor utilization in CSF cell populations

To identify a panel of multiple sclerosis patients (MS) for a phase I clinical trial of a T-cell receptor (TCR) peptide vaccine we characterized the T-cell populations present in the cerebrospinal fluids (CSF) of a large group of patients with respect to surface phenotype and state of activation, TCR beta chain utilization, features of the CDR3 junctional region, the extent of clonality and persistence of selected clonotypes over time. These CSF cell populations consist of approximately 60% CD4+ T-cells, half of which bear IL-2 receptors, indicating these activated T-cells may be part of the pathogenic process in MS. When these activated CD4+ T-cells were selectively expanded in IL-2/IL-4 supplemented cultures, an over-representation of several TCRV beta families was noted in 39/47 patients, the most frequent being V beta 6.5, V beta 6.7, V beta 2, V beta 5 and V beta 4. Biased expression of various members of the V beta 6 family was seen in 21 of this group of 39 patients. Clonal analysis of TCR beta 6 CDR3 sequences, revealed two notable features: clonal dominance and clonal persistence. CSF cells from two-thirds of MS patients contained a dominant clone comprising 50% or more of sequences and the same patient-specific clone could be shown to persist for up to 18 months. This clonal prevalence and over representation of V beta 6+TCR raises the possibility that immunization with a V beta 6 peptide vaccine may produce a regulatory immune response leading to a clinical benefit.

A T cell epitope for experimental allergic neuritis

A synthetic peptide representing residues 57-81 of the bovine P2 protein produced severe paralytic experimental allergic neuritis (EAN) in Lewis rats. Peptide 57-81 could also be used to stimulate a P2 protein-specific T cell line to transfer paralytic EAN to naive recipient rats. A smaller peptide representing residues 60-81 produced a milder form of clinical disease. Residues 60-81 are the shortest peptide sequence thus far described which can produce clinical EAN. The structural predictions for the sequence represented by these peptides support the contention that T cell antigenic sites tend to be amphipathic alpha-helical structures.

Experimental allergic neuritis. I. Rat strain differences in the response to bovine myelin antigens.

Strain differences among rats to the induction and severity of experimental allergic neuritis (EAN) in response to whole PNS myelin were observed. Lewis rats were highly susceptible and developed severe EAN without central nervous system lesions (EAE), while Brown Norway rats were most resistant. Wistar, Sprague-Dawley, and Buffalo rats were susceptible but developed less severe disease than Lewis rats. Only Lewis rats consistantly developed EAN in response to isolated P2 protein. The severity of EAN was enhanced by treatment of the P2 with mercaptoethanol prior to injection. None of the strains developed EAN in response to galactocerebroside and none developed the lesions of EAE in response to any of the bovine myelin antigens tested. Myelin protein profiles from these rat strains were similar which suggests that factors other than target tissue differences, such as genetically determined immune responses to bovine myelin antigens, must be involved in these differing responses.

Immunological Responses to Myelin and Myelin Components

The majority of the data available on the immunology of central nervous system (CNS) myelin relate to the specificity of myelin, and in particular its basic protein component, in bringing about the clinical signs and histopathologically defined lesions characteristic of experimental allergic encephalomyelitis (EAE) via a primarily cell-mediated response. The remainder of the available data concern the detection of humoral antibody responses to myelin basic protein (MPB), cerebroside, and ganglioside. In the latter case, antibody to these components has been detected not only in experimental animals but also in normal human serum and in the serum of patients with a variety of neurological disorders.

The differentiation of synthesis from incorporation of basic protein in Quaking mutant mouse myelin.

The incorporation of radioactive label into the myelin basic protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. four Quaking mice (32 days) and 4 littermate controls were injected intracranially with 150 muCi [2-3H]glycine and 25 muCi of [2-14C]glycine, respectively. One hour later, the 8 mice were sacrificed and their brains combined for common homogenization. The 3H/14C ratios of the small and large basic protiens in whole brain were 3.44 and 2.48 respectively, while the 3H/14C ratios for these proteins in myelin were 0.79 and 1.00, respectively. In the same experiment, the microsomal fraction had a 3H/14C ratio of 2.98 which is the expected ratio for normal incorporation. The results indicate that the synthesis of basic protein in whole brain of Quaking mouse proceeds at a normal rate, but specifically, the incorporation of basic protein into myelin is depressed suggesting a defect at the step of assembly of myelin components into a final membrane product.