Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.

Mutations in the human gene for the myelin recognition molecule protein zero (P0) give rise to severe and progressive forms of dominantly inherited peripheral neuropathies. We have previously reported that mice homozygous for a null mutation in P0 have severely hypomyelinated nerves ten weeks after birth. Here we show hypomyelination already exists at day four with subsequent demyelination and impaired nerve conduction. Furthermore, heterozygous mutants show normal myelination, but develop progressive demyelination after four months of age. Thus, the pathology of homo- and heterozygous P0 mutants resembles that of the severely affected Déjérine-Sottas and the more mildly affected Charcot-Marie-Tooth type 1B patients, respectively.

Hypermyelination and demyelinating peripheral neuropathy in Pmp22-deficient mice.

Peripheral myelin protein PMP22 has been suggested to have a role in peripheral nerve myelination and cell proliferation. Defects at the PMP22 locus are associated with peripheral neuropathies such as Charcot-Marie-Tooth disease type 1A. We now demonstrate that mice devoid of Pmp22 are retarded in the onset of myelination and develop abundant sausage-like hypermyelination structures (tomacula) at a young age followed by severe demyelination, axonal loss and functional impairment. Mice carrying one functional copy of Pmp22 are less affected but they also exhibit focal tomacula comparable to the morphological features in hereditary neuropathy with liability to pressure palsies (HNPP). We conclude that Pmp22 is required for the correct development of peripheral nerves, the maintenance of axons and the determination of myelin thickness and stability.

Serum antibodies to conformational and linear epitopes of myelin oligodendrocyte glycoprotein are not elevated in the preclinical phase of multiple sclerosis.

BACKGROUND: The proposed predictive value of serum anti-myelin antibodies for the development of multiple sclerosis after a first clinically isolated syndrome was recently challenged. OBJECTIVE: To investigate myelin autoantibodies before first disease manifestation using different detection methods. METHODS: Patients with multiple sclerosis who had donated blood at a time prior to development of clinically isolated syndrome were identified via the German National Multiple Sclerosis Society. Control sera were obtained from age- and gender-matched blood donors. IgG-/IgM-antibodies against the extracellular part of native, cell surface-expressed myelin oligodendrocyte glycoprotein were detected by flow cytometry. Antibodies against linear epitopes were identified by immunoblot using recombinant myelin oligodendrocyte glycoprotein (aa1-125) and human myelin basic protein preparations. RESULTS: Fifty eight serum samples from 25 patients covering an interval of 7.3 years-2 months prior to disease onset were available. Longitudinal investigations were performed in 19 patients (2-14 samples per patient, 7 years-2 months prior to disease onset). No significant differences in the prevalence or titres of anti-myelin antibodies were detected between sera of preclinical individuals and healthy donors by either flow cytometry or immunoblot. There was no correlation between interval before clinically isolated syndrome and autoantibody status. Occurrence of antibodies was not associated with symptomatology/severity of clinically isolated syndrome. CONCLUSION: Neither anti-myelin autoantibodies against cell surface-expressed native myelin oligodendrocyte glycoprotein nor against linear epitopes have a predictive or discriminative role during the preclinical disease phase for developing clinically isolated syndrome or multiple sclerosis later in life.

[Amyotrophic lateral sclerosis: management of bulbar symptoms]. / Amyotrophe Lateralsklerose: Symptomatische Therapie bulbärer Symptome.

Symptomatic treatment of amyotrophic lateral sclerosis (ALS) is relevant in preventing complications and improving quality of life as long as curative therapies are still out of sight. About one third of ALS patients show disabling problems associated with dysarthria, dysphagia, sialorrhea, and a pseudobulbar affective disorder already in the early stages of ALS. A multidisciplinary approach is the cornerstone of symptomatic treatment of bulbar and pseudobulbar ALS features. Except for riluzole randomized controlled trials are lacking. Here, we review the current views with regard to epidemiology, pathophysiology, diagnosis, and practical aspects of treating bulbar and pseudobulbar symptoms.

The PTPN22gain-of-function+1858T(+) genotypes correlate with low IL-2 expression in thymomas and predispose to myasthenia gravis.

Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) inhibits T-cell activation and interleukin-2 (IL-2) production. The PTPN22(gain-of-function)+1858T(+) genotypes predispose to multiple autoimmune diseases, including early-onset (non-thymomatous) myasthenia gravis (MG). The disease association and the requirement of IL-2/IL-2 receptor signaling for intrathymic, negative T-cell selection have suggested that these genotypes may weaken T-cell receptor (TCR) signaling and impair the deletion of autoreactive T cells. Evidence for this hypothesis is missing. Thymoma-associated MG, which depends on intratumorous generation and export of mature autoreactive CD4(+) T cells, is a model of autoimmunity because of central tolerance failure. Here, we analyzed the PTPN22 +1858C/T single nucleotide polymorphism in 426 German Caucasian individuals, including 125 thymoma patients (79 with MG), and investigated intratumorous IL-2 expression levels. Unlike two previous studies on French and Swedish patients, we found strong association of PTPN22 +1858T(+) genotypes not only with early-onset MG (P=0.00034) but also with thymoma-associated MG (P=0.0028). IL-2 expression in thymomas with PTPN22 +1858T(+) genotypes (P=0.028) was lower, implying weaker TCR signaling. We conclude that the PTPN22(gain-of-function) variant biases towards MG in a subgroup of thymoma patients possibly by impeding central tolerance induction.

Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons.

Autoantibodies to the synaptic protein amphiphysin play a crucial pathogenic role in paraneoplastic stiff-person syndrome. Impairment of GABAergic inhibition is the presumed pathophysiological mechanism by which these autoantibodies become pathogenic. Here we used calcium imaging on rat embryonic motor neurons to investigate whether antibodies to amphiphysin directly hinder GABAergic signaling. We found that the immunoglobulin G fraction from a patient with stiff-person syndrome, containing high titer antibodies to amphiphysin and inducing stiffness in rats upon passive transfer, reduced GABA-induced calcium influx in embryonic motor neurons. Depletion of the anti-amphiphysin fraction from the patient's IgG by selective affinity chromatography abolished this effect, showing its specificity for amphiphysin. Quantification of the surface expression of the Na(+)/K(+)/2Cl(2-) cotransporter revealed a reduction after incubation with anti-amphiphysin IgG, which is concordant with a lower intracellular chloride concentration and thus impairment of GABA mediated calcium influx. Thus, anti-amphiphysin antibodies exert a direct effect on GABA signaling, which is likely to contribute to the pathogenesis of SPS.

Altered innate immune response of plasmacytoid dendritic cells in multiple sclerosis.

Plasmacytoid dendritic cells (pDCs) are of crucial importance in immune regulation and response to microbial factors. In multiple sclerosis (MS), pDCs from peripheral blood showed an immature phenotype, but its role in susceptibility to MS is not determined. Because infectious diseases are established triggers of exacerbations in MS, in this study we have characterized the expression of Toll-like receptors (TLR) and the maturation and functional properties of peripheral blood pDCs from clinically stable, untreated MS patients in response to signals of innate immunity. After stimulation of TLR-9, interferon (IFN)-alpha production by pDCs was significantly lower in MS (n = 12) compared to healthy controls (n = 9). In an allogenic two-step co-culture assay we found an impaired effect of TLR-9 stimulation on IFN-gamma expression of autologous naive T cells in MS patients (n = 4). In peripheral blood mononuclear cells, TLR-9 stimulation with type A CpG ODN resulted in a higher expression of TLR-1, -2, -4, -5 and -8 in MS patients (n = 7) compared with healthy controls (n = 11). These findings suggest an altered innate immune response to microbial stimuli in MS patients and may help understanding of why common infectious agents trigger MS attacks.

The role of macrophages in demyelinating peripheral nervous system of mice heterozygously deficient in p0.

Mice heterozygously deficient in the p0 gene (P0(+/-)) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder. In motor nerves of P0(+/)- mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.

Myasthenia gravis: passive transfer from man to mouse.

Daily injections into mice of an ammonium sulfate-precipitated immunoglobulin fraction of serum from patients with myasthenia gravis were carried out for up to 14 days. The mice showed reduced amplitudes of miniature endplate potentials and reduced numbers of acetylcholine receptors at the neuromuscular junctions. Some mice showed typical decremental responses on repetitive nerve stimulation, with reversal by neostigmine. This represents the first evidence of a circulating factor in the serum of patients with myasthenia gravis which on passive transfer reproduces features of the disease in experimental animals.

Myeloma neuropathy: passive transfer from man to mouse.

Mice were injected daily, for up to 10 weeks, with purified monoclonal immunoglobulin G from patients with myelomatous polyneuropathy or benign gammopathy. The animals developed a demyelinating polyneuropathy with slowed nerve conduction velocities. The putative antinerve factor may be an antibody since injection of Fab fragments from the monoclonal immunoglobulin G produced a similar demyelination. This provides evidence of a circulating factor in the serum of myeloma patients with polyneuropathy that reproduces typical features of the human disease on passive transfer. This disorder is thus distinguished from other neuropathies that occur as remote effects of malignant disease but have no identified pathogenic factors associated with them.

Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation.

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.

Basic and escalating immunomodulatory treatments in multiple sclerosis: current therapeutic recommendations.

This review updates and extends earlier Consensus Reports related to current basic and escalating immunomodulatory treatments in multiple sclerosis (MS). The recent literature has been extracted for new evidence from randomized controlled trials, open treatment studies and reported expert opinion, both in original articles and reviews, and evaluates indications and safety issues based on published data. After data extraction from published full length publications and critically weighing the evidence and potential impact of the data, the review has been drafted and circulated within the National MS Societies and the European MS Platform to reach consensus within a very large group of European experts, combining evidence-based criteria and expert opinion where evidence is still incomplete. The review also outlines a few areas of controversy and delineates the need for future research.

A role for BDNF in mechanosensation.

Brain-derived neurotrophic factor (BDNF) is a survival factor for certain sensory neurons during development. Using electrophysiology in BDNF-deficient mice, we show here that slowly adapting mechanoreceptors (SAM), but not other types of cutaneous afferents, require BDNF in postnatal life for normal mechanotransduction. Neurons lacking BDNF did not die, but instead showed a profound and specific reduction in their mechanical sensitivity, which was quantitatively the same in BDNF -/- and BDNF +/- animals. Postnatal treatment of BDNF +/- mice with recombinant BDNF completely rescued the mechanosensitivity deficit. Therefore BDNF is important for regulating SAM mechanosensitivity, independent of any survival-promoting function.

Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis.

Autoimmune helper T lymphocytes were selected from the blood of two myasthenic patients of different HLA-DR type, using acetylcholine receptor (AChR) from Torpedo californica. These polyclonal T cell lines were tested for reactivity with three synthetic peptides corresponding to the NH2-terminal region of the human AChR alpha subunit. This segment is a good candidate for T cell epitopes since it has a propensity to form an amphipathic alpha helix. The peptides elicited 10-30% of the response induced by native Torpedo AChR. Different peptides were recognized by the autoreactive T cells of the two patients. These results suggest that the NH2-terminal region of the AChR alpha chain contains T cell-stimulating epitopes, and that the T cell autoimmune response in myasthenia gravis, like the B cell response, is heterogeneous.

Motor neuropathy in porphobilinogen deaminase-deficient mice imitates the peripheral neuropathy of human acute porphyria.

Acute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias' enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD-/-) imitate acute porphyria through massive induction of hepatic delta-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD-/- mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD-/- mice exhibit a marked decrease in large-caliber (>8 microm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD-/- mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor delta-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy.

Immune deficiency in mouse models for inherited peripheral neuropathies leads to improved myelin maintenance.

The adhesive cell surface molecule P(0) is the most abundant glycoprotein in peripheral nerve myelin and fulfills pivotal functions during myelin formation and maintenance. Mutations in the corresponding gene cause hereditary demyelinating neuropathies. In mice heterozygously deficient in P(0) (P(0)(+/-) mice), an established animal model for a subtype of hereditary neuropathies, T-lymphocytes are present in the demyelinating nerves. To monitor the possible involvement of the immune system in myelin pathology, we cross-bred P(0)(+/-) mice with null mutants for the recombination activating gene 1 (RAG-1) or with mice deficient in the T-cell receptor alpha-subunit. We found that in P(0)(+/-) mice myelin degeneration and impairment of nerve conduction properties is less severe when the immune system is deficient. Moreover, isolated T-lymphocytes from P(0)(+/-) mice show enhanced reactivity to myelin components of the peripheral nerve, such as P(0), P(2), and myelin basic protein. We hypothesize that autoreactive immune cells can significantly foster the demyelinating phenotype of mice with a primarily genetically based peripheral neuropathy.

Ultrastructural findings in endomyocardial biopsy of patients with Kearns-Sayre syndrome.

Kearns-Sayre syndrome is clinically defined by progressive external ophthalmoplegia, atypical retinitis pigmentosa and the potential occurrence of complete atrioventricular (AV) block. Right septal endomyocardial biopsy specimens from nine patients (four men and five women with a mean [+/- SD] [corrected] age of 36.3 +/- 14.4 years) with chronic progressive external ophthalmoplegia and mitochondrial skeletal myopathy were studied. Three patients had atypical retinal pigmentation. An atrioventricular or intraventricular conduction defect was observed in five patients. A pacemaker was prophylactically implanted in one patient because of abnormal conduction distal to the His bundle. Ultrastructural investigations revealed mitochondriosis in many heart muscle cells and an increased variability of mitochondrial form and size in all patients. In seven patients, 0.4 to 2.1% of all examined myocytes contained exclusively abnormal mitochondria. Three main types were observed: huge, mainly round mitochondria with concentric cristae; large, round or oval mitochondria with transverse or curved cristae; and small, vacuolated mitochondria. The volume density of myofibrils was reduced (41.9 +/- 11.1 compared with the normal value of 56.5 +/- 2.5 volume density [in percent], p less than 0.01) in these myocytes. Increasing numbers of vacuolated mitochondria correlated significantly with a reduction of myofibrils (r = -0.64, p less than 0.01). The data suggest that the ventricular myocardium of most patients with complete and even incomplete Kearns-Sayre syndrome is affected by disseminated mitochondrial cytopathy.

Impact of the Asp299Gly polymorphism in the toll-like receptor 4 (tlr-4) gene on disease course of multiple sclerosis.

In multiple sclerosis patients, infection is often associated with disease deterioration. Lipopolysaccharide (LPS) from gram-negative bacteria signals via the toll-like receptor 4 (TLR-4) pathway. Therefore, we investigated the role of an Asp299Gly mutation in the TLR-4 receptor in 890 MS patients with multiple sclerosis and 350 healthy controls. No association of different genotypes with MS susceptibility, MS subtypes, or disease severity was found. In vitro LPS stimulation studies showed a significantly lower proliferation of PBMCs from donors heterozygous for the Asp299Gly mutation in comparison to PBMCs from individuals with the wild-type genotype (p=0.01). However, these functional changes seem not to have any impact on the clinical presentation of MS patients with different TLR-4 genotypes.

Soluble vascular cell adhesion molecule (VCAM) is associated with treatment effects of interferon beta-1b in patients with secondary progressive multiple sclerosis.

BACKGROUND: Subcutaneous IFNbeta-1b (Betaferon) is an established immunomodulatory treatment for relapsing remitting MS and active secondary progressive multiple sclerosis (SPMS). It modulates cytokine and adhesion molecule expression but long term in vivo effects of IFNbeta-1b on the immune system are not known in multiple sclerosis. OBJECTIVE: To address the effects of IFNbeta-1b on serum levels for soluble adhesion molecules and cytokine receptors from MS patients. METHODS: Serial blood samples were obtained from 40 patients of the frequent MRI subgroup (20 patients each from the placebo and the IFNbeta-1b treatment group), participating in the European multi-center clinical trial with IFNbeta-1b for secondary progressive MS, at regular intervals for up to 36 months. Soluble adhesion molecules (sVCAM, sICAM-1, sL-Selectin) as well as TNF-receptor I and II were analysed in the serum of patients by enzyme linked immunosorbent assays (ELISAs). Monthly brain MRI was performed in 34 of these patients (16 patients from the placebo and 18 from the IFNbeta-1b group) during months 1-6 and 19-24 to monitor disease activity as assessed by newly occurring gadolinium (Gd) enhancing lesions. RESULTS: An early and significant increase in sVCAM and sTNF-RII serum levels was detected in 16 out of 20 patients (80 %) treated with subcutaneous IFNbeta-1b already at month 1 but was absent in all but one patient during placebo treatment (p < 0.01). Raised sVCAM and TNF RII serum levels during months 1-6 inversely correlated with less MRI activity in the 19-24 months treatment interval in the IFNa-1b treatment group ( p = 0.0093 for TNF-RII; p = 0.047 for VCAM). CONCLUSIONS: sVCAM and sTNF RII levels in the serum of SPMS patients are increased during IFNbeta-1b therapy and may at least in part explain some of the treatment effects, like reduced immune cell transmigration.