Michael J. Walsh

Immunoglobulin G, A, and M—Clonal restriction in multiple sclerosis cerebrospinal fluid and serum—Analysis by two-dimensional electrophoresis

Immunoglobulin G, A, and M (IgG, IgA, and IgM) were purified from multiple sclerosis (MS) cerebrospinal fluid (CSF) and sera and analyzed by two-dimensional electrophoresis. The light (L)-chains of MS CSF IgG were of limited diversity and dominated by about 30 major spots and many less intense spots. CSF IgG of patients with subacute sclerosing panencephalitis (SSPE) was dominated by 10 to 20 intense L-chain spots. L-chains from patients with fungal, syphilitic, and tuberculous infections of the central nervous system (CNS) ranged from 70 to in excess of 300 spots. The patterns of L-chains in MS CSF and autologous sera were different, indicating amplified synthesis of particular L-chains in MS CNS. Cathodic IgG fractions differing in isoelectric point by 0.1 to 0.3 pH units were isolated. SSPE CSF IgG fractions were dominated by a few L-chain spots, MS CSF fractions contained less than 20 spots, and MS serum fractions contained about 200 spots. MS CSF mu-chains were similar in isoelectric point range to serum but fractionated into discrete spots compared with a diffuse pattern in serum. MS CSF IgA alpha-chain spot overlapped in mobility with serum alpha-chains except a portion of the alpha-chain complex in CSF was shifted cathodically forming discrete spots. The CSF L-chains of both IgM and IgA were completely dissimilar to serum. These studies indicate major limited diversity and compartmentalized CNS synthesis of IgG, IgA, and IgM in MS. The restricted clonal pattern is proposed as consequent to B-cell stimulation by disease-related antigen(s) with limited epitope complexity. The dominance of a small number of L-chains supports results from idiotypic analysis suggesting finite clonal complexity of MS CSF IgG and indicates the feasibility of structural analysis of MS CSF L-chains, particularly amino acid sequence determinations.

The current status of multiple sclerosis intra-blood-brain-barrier IgG synthesis.

The status of the central nervous system as an immunologic organ synthesizing IgG in several neurological diseases has been established convincingly in the past two decades. The measurement of intra-BBB IgG synthesis has been accomplished. It has been demonstrated that this synthesis is a potential marker for an abnormal immunologic process within the CNS which if manipulated may be a quantifiable aspect of disease modulation. Modulation of intra-BBB IgG synthesis is feasible, but there is no evidence yet that modulation of this synthesis is an index of effective disease control in MS. Only ACTH and corticosteroids lead to a significant and lasting depression of intra-BBB IgG synthesis, even though clonal eradication is not achieved. Based on our immunopharmacologic studies in MS we have proposed an immunokinetic model. The qualitative analysis of the specificity of IgG synthesized intra-BBB in MS has not led to the etiology of MS, but utilization of quantitative methods may. Questions raised by this review with suggested experiments to advance further our understanding of intra-BBB IgG synthesis as it relates to the etiopathogenesis of MS are included.

Isotachophoresis quantitation of subtractions of multiple sclerosis intra‐blood‐brain barrier IgG synthesis modulated by ACTH and/or steroids

We combined the IgG is otachophoresis (ITP) method and a formula to quantitate IgG synthesis rate inside the blood-brain barrier (intra-BBB) in multiple sclerosis (MS) patients. In MS, most IgG synthesized was cathodic, but synthesis occurred in both anodic and cathodic regions. In addition, ACTH and/or steroids were found to reduce cathodic IgG synthesis more than anodic.

Immunoglobulin heavy chain associated protein in multiple sclerosis cerebrospinal fluid.

Immunoglobulins G, A, and M (IgG, IgA and IgM) were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and sera by Protein A-Sepharose (PAS) affinity chromatography or using solid-phase immunoabsorbents. An isoelectric point heterogeneous protein with apparent mol. wt of 74,000-80,000 was seen consistently when CSF Igs were immunoaffinity purified but was never seen when PAS was used for Ig purification. Control experiments exclude the binding of this protein non-specifically either to Sepharose or to Igs or to other CSF proteins. Analysis of purified Igs under non-reducing conditions leads to some reduction in staining pattern indicating that a portion of the protein may be disulfide linked to itself or to other CSF proteins. Immunoblot analysis of CSF Igs separated on 2-DE gels is consistent with a portion of the total CSF Ig protein existing as free heavy (H)-chains. PAS requires dimeric Fc fragment of Ig for binding; the differences in 2-DE gels of PAS and immunoaffinity purified Igs may be due to interaction of the 74,000-80,000 protein with free H-chain, which nevertheless, still leaves this complex available for binding by anti-H chain antisera. It has been previously suggested that the cytotoxicity of free H-chains is abrogated in the absence of the complementary L-chains by H-chain binding proteins; the protein reported here has features similar to these proteins reported previously and may be involved in some way in regulating Ig production in the MS central nervous system.

Isoelectric point restriction of cerebrospinal fluid and serum IgG antibodies to measles virus polypeptides in multiple sclerosis

Thirty consecutive isoelectric point (pI)-discrete IgG fractions were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and used to immune precipitate measles virus (MV) polypeptides. Most basic fractions were enriched in activity against nucleocapsid protein (NP), and to a lesser extent against hemagglutinin (H) protein; intermediate fractions were enriched in activity against H and fusion (F) proteins; and more anodic pI fractions were almost exclusively enriched in activity against the large (L) protein of MV. In MS there are marked differences between CSF and autologous serum in regard to antibody activity to MV. In contrast, there were similar profiles of antibody response to MV proteins in SSPE CSF and serum.

The Science of Intra-Blood-Brain-Barrier IgG Synthesis

Abstract The scientific basis for the detection and quantitation of intra-blood-brain-barrier (BBB) IgG synthesis is reviewed. The intra-BBB IgG synthesis rate formula is based upon experimental data and also the BBB permeability to proteins such as IgG and albumin. This formula is a valid quantitative estimate of IgG synthesis as shown using a radiolabeled IgG exchange test in multiple sclerosis (MS) patients. The advantage of this formula as a quantitative estimate of IgG synthesis over dimensionless ratios and the application of this formula to IgG subfractions quantified by isotachophoresis are discussed. In addition, qualitative evidence of intra-BBB IgG synthesis is provided by electrophoretic procedures which detect oligoclonal IgG bands present in cerebrospinal fluid (CSF) but not in serum. Both quantitative and qualitative detection of intra-BBB IgG synthesis correlate with each other and discriminate well between MS patients, and controls and we recommend routine use of both methods as diagnostic aids and as outcome measures in clinical trials of putative therapies.

New Proteins Unique to Cerebrospinal Fluid (CSF) Identified by Two-Dimensional Electrophoresis (2DE)

Abstract The proteins of CSF, ventricular fluid, and serum are analyzed by 2DE. Proteins of CSF and serum with high and low affinity for Protein A, Cibacron Blue, and Concanavalin A are also characterized by 2DE. A large number of CSF-enriched or CSF-unique proteins are also identified. Two protein complexes, very heterogenous in charge and molecular weight, are characterized as constituents of normal CSF but are markedly increased in several inflammatory brain diseases. Both proteins and several other proteins enriched in CSF bind to Cibacron Blue-Sepharose. The use of 2DE in conjunction with affinity chromatography and sensitive protein stains greatly enlarges the number of proteins previously identified as unique to CSF. By minor modifications of the 2DE procedure and silver staining, most of these proteins are visualized in CSF even without prior concentration.