Conduction block in immune-mediated neuropathy: paranodopathy versus axonopathy.

BACKGROUND AND PURPOSE: Conduction block is a pathognomonic feature of immune-mediated neuropathies. The aim of this study was to advance understanding of pathophysiology and conduction block in chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). METHODS: A multimodal approach was used, incorporating clinical phenotyping, neurophysiology, immunohistochemistry and structural assessments. RESULTS: Of 49 CIDP and 14 MMN patients, 25% and 79% had median nerve forearm block, respectively. Clinical scores were similar in CIDP patients with and without block. CIDP patients with median nerve block demonstrated markedly elevated thresholds and greater threshold changes in threshold electrotonus, whilst those without did not differ from healthy controls in electrotonus parameters. In contrast, MMN patients exhibited marked increases in superexcitability. Nerve size was similar in both CIDP groups at the site of axonal excitability. However, CIDP patients with block demonstrated more frequent paranodal serum binding to teased rat nerve fibres. In keeping with these findings, mathematical modelling of nerve excitability recordings in CIDP patients with block support the role of paranodal dysfunction and enhanced leakage of current between the node and internode. In contrast, changes in MMN probably resulted from a reduction in ion channel density along axons. CONCLUSIONS: The underlying pathologies in CIDP and MMN are distinct. Conduction block in CIDP is associated with paranodal dysfunction which may be antibody-mediated in a subset of patients. In contrast, MMN is characterized by channel dysfunction downstream from the site of block.

A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) has been a associated with a deletion of 1.5 megabases of chromosome 17p. One of four biopsy proven HNPP families that we have studied did not possess this deletion. As the deleted DNA region includes the coding region for a peripheral myelin gene (PMP22), we used single strand conformation analysis to examine this gene for mutations in the non-deleted HNPP family. An abnormal fragment in exon 1 was identified, and sequencing revealed a two base pair deletion in all affected family members. The deletion results in a frame shift, providing strong evidence that this gene has an important role in the pathogenesis of the disease.

Massive astrocyte destruction in neuromyelitis optica despite natalizumab therapy.

Auto-antibody mediated astrocyte injury is implicated as a primary event in neuromyelitis optica (NMO) by biomarker, post-mortem and experimental studies that differentiate the condition from multiple sclerosis. We describe the clinical, radiological and neuropathological features of a severe cerebral attack in a natalizumab-treated patient with relapsing myelitis and serum aquaporin-4 antibodies. Our findings support autopsy evidence that abrupt astrocyte destruction precedes demyelination in NMO, and emphasize the importance of serological testing in patients with limited disease. Adherence to current NMO diagnostic criteria may delay treatment, or lead to inappropriate therapy with beta-interferon or natalizumab.

Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.

Longitudinal assessment of anxiety, depression, and fatigue in people with multiple sclerosis.

OBJECTIVES: No longitudinal studies have concurrently evaluated predictors of anxiety, depression, and fatigue in people with multiple sclerosis (PwMS). This study determined factors that best predicted anxiety, depression, and fatigue in MS patients from a large pool of disease, cognitive, life-event stressor (LES), psychosocial, life-style, and demographic factors. DESIGN: A 2-year prospective longitudinal study evaluated predictors of psychological distress and fatigue in PwMS. METHODS: One hundred and one consecutive participants with MS were recruited from two MS clinics in Sydney, Australia. LES, anxiety, depression, and fatigue were assessed at baseline and at 3-monthly intervals for 2-years. Disease, cognitive, demographic, psychosocial, and life-style factors were assessed at baseline. Patient-reported relapses were recorded and corroborated by neurologists or evaluated against accepted relapse criteria. RESULTS: Depression strongly predicted anxiety and fatigue, and anxiety and fatigue strongly predicted later depression. Psychological distress (i.e. anxiety, depression) was also predicted by a combination of unhealthy behaviours (e.g. drug use, smoking, no exercise, or relaxation) and psychological factors (e.g. low optimism, avoidance coping), similar to the results of community-based studies. However, state-anxiety and fatigue were also predicted by immunotherapy status, and fatigue was also predicted by LES and demographics. CONCLUSIONS: These results suggest that similar factors might underpin psychological distress and fatigue in MS patients and community-well samples, although MS treatment factors may also be important. These results might assist clinicians in determining which MS patients are at greatest risk of developing anxiety, depression, or fatigue.

Autoantibody responses to nodal and paranodal antigens in chronic inflammatory neuropathies.

Autoantibodies to nodal/paranodal proteins have been reported in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). To determine the frequency of anti-paranodal antibodies in our cohort of CIDP patients and to validate the presence anti-nodal antibodies in MMN, sera were screened for IgG against human neurofascin 155, contactin-1, neurofascin 186 and gliomedin using ELISA. In CIDP patients, 7% were anti-NF155 IgG4 positive and 7% were anti-CNTN1 IgG4 positive. Positive results were confirmed using cell based assays and indirect immunofluorescence on teased nerve fibres. We did not detect IgG autoantibodies against these nodal/paranodal antigens in MMN patients.

Gene expression and genotyping studies implicate the interleukin 7 receptor in the pathogenesis of primary progressive multiple sclerosis.

Multiple sclerosis (MS) is an enigmatic disease of the central nervous system resulting in sclerotic plaques with the pathological hallmarks of demyelination and axonal damage, which can be directly or indirectly orchestrated by cells from the peripheral circulation. The majority of patients with MS follow a relapsing-remitting course in the early stages of the disease (RRMS) but most ultimately enter a secondary progressive phase (SPMS). About 10% of patients follow a primary progressive course from the onset (PPMS). We measured gene expression in whole blood of people with and without chronic progressive MS (CPMS), PPMS and SPMS, to discover genes which may be differentially expressed in peripheral blood in active disease, and so identify pathologically significant genes and pathways; and we investigated genetic differences in the promoters of dysregulated genes encoded in genomic regions associated with MS. If SPMS and PPMS were independently compared to the controls, there was little overlap in the set of most dysregulated genes. Ribosomal protein genes, whose expression is usually associated with cell proliferation and activation, were dramatically over-represented in the set of most down-regulated genes in PPMS compared to SPMS (P < 10(-4), chi(2)). The T cell proliferation gene IL7R (CD127) was also underexpressed in PPMS, but was up-regulated in SPMS compared to the controls. One interleukin 7 receptor (IL7R) promoter single nucleotide polymorphism (SNP), -504 C, was undertransmitted in PPMS trios (P = 0.05, TDT), and carriers of this allele were under-represented in PPMS cases from two independent patient cohorts (combined P = 0.006, FE). The four known IL7R promoter haplotypes were shown to have similar expression levels in healthy controls, but not in CPMS (P < 0.01, t test). These data support the hypothesis that PPMS has significant pathogenetic differences from SPMS, and that IL7R may be a useful therapeutic target in PPMS.

Ribozymes: aiming at RNA replication and protein synthesis.

The RNA world hypothesis is founded on the idea of an RNA replicase, or self-replicating RNA molecule, and presupposes the later emergence of ribozymes capable of catalyzing the synthesis of peptides. The recent demonstrations of ribozyme-catalyzed template-directed primer extension, and of ribozyme-catalyzed amide bond synthesis, confirm the plausibility of the RNA world, and highlight the steps that remain to be demonstrated in the laboratory.

Dominance of autoreactive T cell-mediated delayed-type hypersensitivity or antibody-mediated demyelination results in distinct forms of experimental autoimmune neuritis in the Lewis rat.

The role of anti-myelin antibodies in the pathogenesis of experimental autoimmune neuritis (EAN) induced in the Lewis rat by immunization with peripheral nerve myelin has been assessed. Passive transfer with lymph node cells (LNC) or purified serum immunoglobulin from rats with EAN was employed to directly measure the contribution of B cells and anti-myelin antibodies to demyelination and disease. Lewis rats with EAN transferred by LNC or purified serum immunoglobulin from EAN donors in conjunction with a low dose of P2-specific CD4+ T cells demonstrated profound histopathological and neurophysiological evidence of demyelination during disease. In contrast, the classical adoptive transfer model of EAN in the Lewis rat induced by the injection of P2-specific CD4+ T cells was characterized by histopathological and neurophysiological evidence of axonal dysfunction and degeneration with limited demyelination. These findings demonstrate that the synergistic action of T cells and anti-myelin antibodies mediating demyelination or purely T cell mediated axonal dysfunction and degeneration are distinct pathways by which a specific autoimmune response in the peripheral nervous system can cause neurological disease.

Chronic ophthalmoplegia with anti-GQ1b antibody.

Anti-GQ1b antibodies are typically found in patients with the Miller Fisher syndrome, all of whom will have, by definition, acute ophthalmoplegia. The authors describe three patients with chronic ophthalmoplegia in the presence of persistently high titers of immunoglobulin G anti-GQ1b antibody detected in an ELISA, one of whom improved with immunotherapy. Anti-GQ1b antibodies may be associated with some cases of chronic ophthalmoplegia of unknown cause.

Charcot-Marie-Tooth disease and Noonan syndrome with giant proximal nerve hypertrophy.

We report a family with Noonan syndrome (NS), giant proximal nerve hypertrophy, and hereditary motor sensory neuropathy type 1A (HMSN1A). Five members of a family were found to have clinical features of NS. In all cases, NS was associated with giant hypertrophy of proximal nerves and two individuals also exhibited café-au-lait spots. In one case, an 8-to-10-cm diameter pelvic mass was shown to be a grossly hypertrophied nerve, with histologic features of demyelination and remyelination. In addition, four of five family members affected with NS were found to have HMSN1A clinically and by demonstration of constitutional HMSN1A duplication on DNA testing. Linkage analysis for NS ruled out the involvement of the neurofibromatosis type 1 gene and the known NS locus in chromosome 12, supporting the existence of an additional NS locus.

Microwave fixation of whole peripheral nerve for rapid and efficient enzyme-linked immunosorbent assay (ELISA) screening of monoclonal antibodies.

Testing hybridoma supernatants for antibodies of interest involves extensive screening, particularly when the immunogen comprises whole cells. The number of different screening procedures is often large and unmanageable and depends on whether one is interested in, for example, cell surface or intracellular binding. This paper describes an initial screening technique using whole tissue homogenate rather than the individual tissue components. The tissue is fixed to the surface of 96-well microtitre plates by microwaves using a conventional microwave oven. This technique provides a rapid and cost-effective means of screening large numbers of monoclonal antibodies.

Neuroimmunological electron microscopy with microwave-accelerated fixation.

Immunoelectron microscopy is an important tool used to determine the precise location of immune complexes. Standard concentrations of glutaraldehyde destroy these complexes. This paper describes a method in which the period of glutaraldehyde fixation is shortened by concomitant microwave treatment. Using 1.25% glutaraldehyde and microwave fixation ideal preservation and demonstration of MHC class I antigen on Schwann cells was obtained by the peroxidase method.

Peripheral nervous system demyelination from systemic transfer of experimental allergic neuritis serum.

The ability of systemically transferred experimental allergic neuritis (EAN) serum to produce EAN lesions in recipient animals was studied. Seventeen Lewis rats received five daily 1-ml intraperitoneal (i.p.) injections of sera from rabbits with EAN induced with bovine myelin/complete Freund's adjuvant (CFA). Another 17 rats received similar injections of sera from rabbits inoculated with CFA alone. On day 0 (the first day of i.p. injections), all rats were injected in the proximal tibial branch of the right sciatic nerve with a single 10-microliters injection of 0.03 M 5-hydroxytryptamine (5-HT) in sterile 0.15 M saline. Proximal tibial branches of left sciatic nerves received similar single injections of saline alone. Animals were then studied using electrophysiological and histological techniques. In all animals, intraneural saline injection had no significant effect upon nerve conduction. In the presence of circulating CFA serum, 5-HT injection caused a mild gradual decrease in amplitude ratio becoming maximal by day 17 (P < 0.005) and partially resolving by day 28. In contrast, in the presence of circulating EAN serum, 5-HT injection caused a more rapid and severe decrease in amplitude ratio becoming maximal by days 6-10 (P < 0.001 day 6; P < 0.0001 day 10) and completely resolving by day 28. Histological analysis of nerves injected with 5-HT in CFA serum-treated animals showed areas of mild demyelination, axonal degeneration and some fibre loss consistent with needle trauma. In contrast, 5-HT-injected nerves in animals administered EAN serum showed areas of marked cellular infiltration and severe demyelination in association with numerous debris-filled infiltrating cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-gamma inhibition suppresses experimental allergic neuritis: modulation of major histocompatibility complex expression of Schwann cells in vitro.

This study examines the modulation of major histocompatibility complex (MHC) expression on Lewis rat Schwann cells (Scs) cultured in the presence of dorsal root ganglion neurons (DRG). MHC class I and II molecules were induced on Scs using recombinant murine interferon-gamma (IFN-gamma), lymph node cells (LNC), removed at day 9 from Lewis rats with experimental allergic neuritis (EAN) and syngeneic T-cell line cells responsive to P2 basic protein. EAN LNC induced MHC class I on Scs but only IFN-gamma or P2-responsive T-cells induced MHC class II. Control LNC from animals injected with Freund's adjuvant alone or naive spleen cells did not induce MHC class II. P2 T-cells clustered in aggregates to the Scs. Similar studies were performed with inhibitors of IFN-gamma; hydrocortisone, cyclosporin A, dibutyryl cyclic AMP, methyl-xanthine and prostaglandin E2. Each agent produced a dose-dependent inhibition of MHC expression and prevented clustering of P2-responsive T-cells to Scs.

Interactions between CD4+ T-cells and rat Schwann cells in vitro. 2. Cytotoxic effects of P2-specific CD4+ T-cell lines on Lewis rat Schwann cells.

The cytotoxic effects of CD4+ P2-specific T-cell lines on Schwann cells were examined in vitro with 51Cr-release cytotoxicity assays. Only those P2-specific T-cell lines capable of inducing EAN when injected back into adult Lewis rats were cytotoxic to the Schwann cells. The addition of exogenous P2 protein was not necessary for the cytotoxic effect. The monoclonal antibody (mAb) OX6 directed against Major histocompatibility complex (MHC) class II molecules blocked cytotoxicity, indicating an essential role for MHC class II molecules in this interaction between CD4+ T-cell lines and Schwann cells.

Synergy between antibody and P2-reactive T cells in experimental allergic neuritis.

Studies were conducted in experimental allergic neuritis (EAN) to evaluate the possible interaction of cellular and humoral immune mechanisms in the demyelinating process. EAN was induced in Lewis rats by passive transfer of T cells reactive to P2 myelin protein or by active immunisation with whole myelin. Animals were then given systemic antimyelin antibody or control serum and assessed clinically, electrophysiologically and with semiquantitative histological studies. Animals given intraperitoneal (i.p.) P2-reactive T cells and systemic antimyelin antibody developed much more severe disease than those given i.p. T cells alone (P < 0.001). In actively immunised animals, the addition of systemic antimyelin antibody did not significantly alter disease severity. We believe the more severe disease in animals receiving T cells and antimyelin antibody reflects synergy between cellular and humoral immune mechanisms whereby neural antigen-specific T cells breach the blood-nerve barrier, allowing demyelinating antibody access to the endoneurium. In EAN induced by active immunisation with whole myelin it is likely that both B and T cell activation occurs and that the more severe demyelination characteristic of this disease reflects the involvement of both humoral and cellular immunity.

Interactions between CD4+ T-cells and rat Schwann cells in vitro. 1. Antigen presentation by Lewis rat Schwann cells to P2-specific CD4+ T-cell lines.

Interactions between CD4+ P2-specific T-cell lines and Schwann cells were examined in vitro by scanning electron microscopy (SEM) and T-cell proliferation studies. CD4+ T-cell lines clustered around and attached to Schwann cells which expressed Major histocompatibility complex (MHC) class II molecules. Only those P2-specific T-cell lines capable of inducing experimental allergic neuritis (EAN) when injected into adult Lewis rats clustered around the Schwann cells. T-cell lines responsive to P2 but not able to induce EAN did not cluster around Schwann cells. The addition of exogenous P2 protein inhibited in a dose-dependent way clustering and proliferation of the P2-specific T-cell lines. Cytoplasmic P2 was detected in Schwann cells by immunofluorescent labelling and the results of proliferation assays in this study suggest that endogenous P2 protein was processed by the Schwann cells and presented to T-cell lines in association with MHC class II molecules. The clustering and proliferation of class II-restricted CD4+ P2-specific T-cell lines in the presence of Schwann cells provides evidence for a role for Schwann cells as facultative antigen presenting cells, processing and presenting 'self' endogenous antigen to CD4+ T-cell lines capable of inducing EAN.

Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy.

The inflammatory infiltrate within human sural nerve, biopsied from six patients with active chronic inflammatory demyelinating neuropathy (CIDP) was studied for T lymphocyte subsets and Class II antigen (Ia)-expressing cells. Immunohistochemical staining with mouse monoclonal antibodies, acid phosphatase staining, and electron microscopy were used to provide an alternative assessment of macrophage and other mononuclear cell numbers. In normal control nerves Class II antigen was present upon endothelial cells, very occasional mononuclear cells and sparsely within the perineurium. In CIDP nerve dense Class II antigen staining was prominent within nerve fascicles, in capillary endothelial cells and within the perineurium. T lymphocytes of suppressor and helper type were present in small numbers only. Moderate numbers of macrophage-monocytes were found in the patients within nerve fascicles but these cells accounted for only part of the dense Ia staining. Since two nerves with hypertrophic changes, Schwann cells forming 'onion bulbs', were clearly Ia positive, the dense and widespread staining in all nerves studied is best explained by Ia antigen expression upon mononuclear and some Schwann cells.