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Oral administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with Torpedo AChR+complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG) and the suppression of AChR-specific B cell responses and counteracts the development of AChR-reactive interferon-gamma (IFN-gamma) secreting T cells. To study the involvement of the T helper type 1 (Th1) cell-related lymphokine IFN-gamma, the Th2 cell-related interleukin-4 (IL-4), and transforming growth factor beta (TGF-beta) that suppresses the synthesis of IFN-gamma and IL-4, we used in situ hybridization with complementary DNA oligonucleotide probes to enumerate mononuclear cells (MNC) expressing mRNA for the cytokines IFN-gamma, IL-4, and TGF-beta. Upon in vivo recognition of AChR, popliteal, inguinal, and mesenteric lymph nodes, spleen and thymus of rats with EAMG contained higher levels of IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells compared to CFA-injected control rats, implicating the involvement in EAMG of AChR-reactive Th1 and Th2 cells in parallel. TGF-beta was also upregulated in EAMG. Oral tolerance to EAMG was characterized by suppression of the levels of MNC expressing IFN-gamma and IL-4, but augmentation of cells expressing TGF-beta. The results suggest that IFN-gamma, IL-4, and TGF-beta are involved in the development of EAMG, and that TGF-beta is important in the induction of oral tolerance to EAMG.

To study whether nervous tissue trauma provokes myelin antigen autoreactive T and B cell responses in humans we examined consecutive blood samples from 7 patients with polyneuropathy undergoing diagnostic sural nerve biopsy and 8 control patients undergoing other types of minor surgery. The antigen-specific T cells were assessed by enumerating cells secreting interferon-gamma (IFN-gamma) in response to the myelin components P0, P2, myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to 4 selected MBP peptides. B cell mediated immunity was assessed by counting numbers of cells secreting antibodies directed against the myelin proteins. On day 7 after biopsy, there were 3-10-fold increased numbers of T and B cells reactive with P0, P2, MBP and MAG in blood of polyneuropathy patients compared to controls, while levels of cells recognizing purified protein derivate or responding to phytohemagglutinin (PHA) did not differ significantly. Comparison of prebiopsy levels on day 0 with post-biopsy levels on day 7 in the polyneuropathy patients revealed a significant increase in T cells recognizing P0, P2 and MAG, and in B cells secreting IgG antibodies against P0 and P2. On day 14 after nerve biopsy these differences were no longer seen. We suggest that in patients with polyneuropathy, sural nerve biopsy with the ensuing wallerian degeneration and myelin breakdown causes transiently increased levels of circulating myelin autoreactive T and B cells. It remains to be determined if this has a physiological role in nerve trauma responses and/or affects the clinicopathological course of the peripheral neuropathy.

Immune mechanisms of possible importance for the development and maintenance of peripheral nerve myelin breakdown in HMSN I were analysed by measuring B- and T-cell activation in blood, bone marrow and cerebrospinal fluid. Patients with polyneuropathies of other etiologies served as one control group and patients with tension headache as another. Flow cytometry of blood and bone marrow mononuclear cells revealed that an increased number of CD3+, CD4+ and CD4- CD8- T-cells expressed a late stage activation marker (Ta1). Analysis of T-cells primed for myelin antigens, by studies of IFN-gamma secretion in response to antigen in vitro, showed that both HMSN I and other polyneuropathy patients had low (but significant) numbers of T-cells recognizing whole PNS-myelin. Increased numbers of IgG- and IgM-producing cells were found in blood and bone marrow in the HMSN I patients. Patients with both HMSN I and the other polyneuropathies had few cells in peripheral blood and in bone marrow producing antibodies binding to P2, MAG and MBP in a solid phase immunospot assay. Many cells in the cerebrospinal fluid produced antibodies against MAG. Thus, there was a strong general activation of B- and T-cells in HMSN I while the immunity directed toward peripheral nerve was only slightly elevated. It is an open question if this immune activation is related to the primary gene defect or a secondary event to the nerve damage. The pathogenetic importance of the immune response in maintaining the nerve damage in HMSN I is unclear.

Antibodies against acetylcholine receptor (AChR) can be detected in most patients with myasthenia gravis (MG) and are considered to be involved in the immunopathogenesis of this disease. AChR are isolated from crude receptor preparations by binding to alpha-bungarotoxin (alpha-BuTx). Patients with MG have also antibodies against a second protein tentatively named presynaptic membrane receptor (PsmR), which has been isolated from crude receptor utilizing beta-bungarotoxin (beta-BuTx). PsmR could represent another antigen besides AChR relevant for the development of MG. We have now evaluated the T cell reactivity to PsmR in MG and controls by analysing the frequencies of cells which in response to PsmR in short-term cultures secreted interferon-gamma (IFN-gamma). The B cell response to PsmR was analysed in parallel by counting cells secreting anti-PsmR antibodies. Most patients with MG had PsmR reactive T cell in blood with a median number of about 1 per 44,000 mononuclear cells. Cells secreting anti-PsmR antibodies belonging to the IgG and IgA isotypes, less frequently of the IgM isotype were detected in most MG patients. A positive correlation was found between T cells reactive with PsmR and anti-PsmR IgG antibody secreting cells. PsmR reactive T and B cells were also detected in control patients, but at much lower numbers. Our results indicate that MG is accompanied by T as well as B cell responses to PsmR, in addition to the previously recognized responses to AChR. It remains to be shown whether these PsmR reactivities are of pathogenetic importance in MG.

An epitope on peripheral nerve myelin was detected by the use of a mouse monoclonal antibody directed against the 38 kDa subunit of herpes simplex virus (HSV) type 1 ribonucleotide reductase. Immunohistochemistry showed reactivity solely in PNS myelin. In nerve roots there was a sharp border in transitional zones to the negative CNS myelin. The immunoreactivity was found in rat, guinea pig, bovine and human peripheral nerves. Western blot analysis of peripheral nerve myelin as well as purified P0 revealed a distinctly stained band corresponding to a molecular weight of approximately 29 kDa. The present finding of a shared antigenic determinant between HSV ribonucleotide reductase and peripheral nerve P0 may be of pathogenetic relevance in virus induced demyelinating diseases in the peripheral nervous system.

Antibodies against acetylcholine receptor (AChR), a protein that binds to alpha-bungarotoxin (alpha-BuTx), are characteristic for myasthenia gravis (MG) and are considered to be of importance in the pathogenesis of the disease. Antibodies against a beta-BuTx binding protein, presynaptic membrane receptor (PsmR), have also been reported in most MG patients. We have analysed the specificity and cross-reactivity of antibodies to bovine AChR and PsmR in sera from 11 patients with MG. More than 90% of antibodies to PsmR were adsorbed specifically by PsmR conjugated affinity chromatography. Similarly, more than 90% of anti-AChR antibodies were absorbed by AChR conjugated affinity chromatography. Specificities of antibodies from affinity chromatography were also confirmed by ELISA and agarose isoelectric focusing. However, the antibodies to PsmR and AChR from MG patients' sera showed about 45-55% cross-reactivity, and there was a high correlation between serum levels of both antibodies. The demonstration of anti-PsmR antibodies could be important in documenting presynaptic damage and understanding the pathogenetic process in MG.

When the B-cell response was examined by enumeration of immunoglobulin (Ig)-secreting cells, normal cerebrospinal fluid (CSF)--in contrast to previous beliefs--contained IgG-secreting cells, indeed at an 8-fold higher proportion per 10(4) mononuclear cells (MNC) than blood. As expected, the proportion of IgG-producing cells was greatly increased in MS CSF. Evaluation of antibody (Ab) responses at the cellular level, thereby bypassing draw-backs inherent in determinations of circulating Ab levels, such as Ab binding to target, revealed that in one MS patient group, 57% had, in CSF, cells secreting IgG Ab against myelin basic protein (MBP) and, in another MS group, 55% had, in CSF, cells producing IgG Ab against myelin-associated glycoprotein (MAG); both MBP and MAG are possible targets for immune attack in MS. Anti-MBP and anti-MAG IgG antibody-secreting cells could occur in parallel or independently. They were rarely detected in blood, reflecting strong sequestration in CNS CSF. Their possible role in MS pathogenesis is envisaged in light of recently suggested coupling between polyclonal B-cell hyperresponsiveness and antigen-driven specific responses in autoimmune-prone individuals.

The B cell response to central nervous system (CNS) myelin and myelin basic protein, as well as total numbers of IgG secreting cells, was studied in acute experimental allergic encephalomyelitis using a nitrocellulose immunospot assay. The method was able to detect single plasma cells secreting antibodies. Cells secreting antibodies against myelin antigens were detected in regional lymph node cell suspension by day 5 post-immunization (p.i.). At that time no anti-myelin antibodies were detected free in serum. Later, at day 15 p.i., specific antibody secreting cells were found in bone marrow and spleen indicating a generalization of the immune response. The B cell response became partly sequestered to the target of immune attack since an increased number of IgG secreting cells was detected among mononuclear cells recovered from the CNS. Studies of cellular secretion of antibodies rather than free levels in body fluids may be a more accurate reflection of the in vivo B cell response. These findings may be generally considered in studies of B cell mediated immunity in neuroinflammatory diseases.