Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.

Background: The role of antimyelin antibodies as biomarker in multiple sclerosis is subject of debate. Here antimyelin antibody reactivity against native myelin is studied in CSF and serum.Objective: To compare antimyelin antibody reactivity between patients with multiple sclerosis (MS) and patients with other neurological diseases in CSF and serum. In addition, MRI measures were studied in relation to antimyelin antibody reactivity.Methods: 77 MS patients (13 primary progressive, 27 secondary progressive and 37 relapsing remitting), 26 patients with other non-inflammatory neurological diseases and 9 patients with inflammatory neurological diseases other than MS were included. A myelin flow cytometry assay was used to detect anti-myelin antibody levels which were expressed as mean fluorescence intensity (myelin-MFI). MRI outcome measures were new or persistent T2 lesions, gadolinium enhancing T1 lesions and brain atrophy which were assessed by normalized brain volumes.Results: There was no significant difference between myelin-MFI values in serum and CSF between MS patients and controls (Mann-Whitney test p = 0.19 and p = 0.51). Myelin-MFI values in CSF were not correlated with number of 12 lesions (Spearman r = -0.023, p = 0.85), number of gadolinium enhancing T1 lesions (Spearman r = -0.066, p = 0.588) or normalized brain volume (Spearman r = -0.065, p = 0.594).Conclusions: These results do not confirm an association between anti-myelin antibody reactivity and the presence of MS or MRI measures of disease activity. (C) 2012 Elsevier B.V. All rights reserved.

Background: Oxidative stress plays an important role in multiple sclerosis (MS). Isoprostanes are biomarkers for oxidative stress and have been related to neurological disease progression. Objective: To study whether plasma isoprostane levels were related to disease progression in MS. Methods: Plasma levels of 8,12-iso-iPF2alpha-VI were determined in 17 patients with clinically isolated syndrome (CIS), 41 relapsing–remitting MS (RRMS) patients and 5 primary progressive MS (PPMS) patients and related to MRI and clinical disease parameters. Results: Isoprostane levels were similar in CIS (60.9, interquartile range (IQR): 47.7–77.7 pg/ml) and RRMS patients (65.3, IQR: 51.9–82.8 pg/ml). The plasma levels were lower in PPMS patients (42.5, IQR: 37.1–49.9) pg/ml, p<0.05) compared to CIS and RRMS patients in this cohort, which was not confirmed in a second cohort. Baseline isoprostane levels were not related to clinical progression defined by conversion form CIS to RRMS or change in Expanded Disability Status Scale (EDSS) or MS Functional Composite (MSFC) scores during six years of follow-up (CIS + RRMS), nor to change in volume of gadolinium enhancing lesions, T2 lesion load or T1 hypointense lesion load during 2.8 years of follow-up (CIS + RRMS). Conclusion: These results do not support a strong role of 8,12-iso-iPF2alpha-VI in the prediction of disease progression in MS.

Reactive oxygen species (ROS) contribute to the formation and persistence of multiple sclerosis (MS) lesions by acting on distinct pathological processes. In the initial phase of MS lesion development, locally produced ROS may induce blood-brain barrier disruption and enhance leukocyte migration and myelin phagocytosis. In advanced stages, ROS may contribute to lesion persistence by mediating oligodendroglial damage and axonal injury. Generally, high levels of ROS cause oxidative stress, which induces transcription of antioxidant response element (ARE)-regulated genes. These genes encode various endogenous antioxidant enzymes that confer protection against oxidative damage. Here, we studied the expression of endogenous antioxidant enzymes in control brain tissue and various stages of MS lesions. ARE-regulated antioxidant enzymes, including superoxide dismutase-1 and -2, catalase, peroxiredoxin-1 and heme oxygenase-1 are expressed by glial cells in white matter from control brains and normal appearing white matter. Interestingly, these cytoprotective antioxidant enzymes are markedly upregulated in active demyelinating MS lesions. Particularly, foamy macrophages containing myelin and cellular debris and hypertrophic astrocytes abundantly expressed endogenous antioxidant enzymes. We speculate that enhanced antioxidant enzyme production in inflammatory MS lesions indicates the occurrence of oxidative stress and may reflect an adaptive defense mechanism to counteract ROS-induced cellular damage.

Migration of monocytes across the blood-brain barrier (BBB), a common hallmark of neuroinflammatory disorders, requires the active participation of brain endothelial cells, to rearrange their cytoskeleton and tight junctions. We previously showed that reactive oxygen species (ROS) play a pivotal role during transendothelial migration of monocytes, by altering BBB integrity. However, long-term exposure to ROS may induce adaptive responses in brain ECs to counteract an oxidative attack. To identify these cellular adaptations, we studied the effect of ROS on gene and protein expression in brain endothelial cells. Using real-time quantitative PCR and proteomic techniques we show that superoxide induces differential gene and protein expression in brain endothelium, including molecules involved in redox regulation, among which peroxiredoxin-1 (Prx1). To further study the role of Prx1 in transendothelial monocyte migration, we established a brain endothelial cell line overexpressing Prx1. Our data show that Prx-1 overexpression protects brain endothelial cells from ROS-induced cell death and enhances the integrity of the brain endothelial layer. In addition, Prx1 overexpression reduces monocyte adhesion and subsequent transendothelial migration by decreasing ICAM-1 expression on brain endothelium. Our data implicate that Prx1 may be an important regulator of cellular protection against oxidative stress and suggest that induction of antioxidant enzymes may be a tool to prevent monocyte migration into the central nervous system and thus be of relevance for the treatment of neuroinflammation.

Acute experimental allergic encephalomyelitis (EAE) is widely used as an animal model for multiple sclerosis (MS). EAE reflects the inflammatory phase of the disease, such as blood-brain barrier damage and infiltration of T cells and monocyte-derived macrophages in the central nervous system. To identify novel pathways involved in the pathogenesis of EAE we studied alterations in gene transcription and protein expression in the course of acute EAE induced in the Lewis rat. Longitudinal microarray analysis of the cerebellum and brainstem revealed strong transcriptional regulation of three novel groups of genes participating in neuroinflammatory processes, which followed distinct kinetics in the course of EAE. Expression of the first group was highly increased before disease onset and declined while EAE progressed, whereas expression of the second group strongly correlated with clinical signs of EAE. Expression of the third group of genes initially followed clinical scores, but remained elevated in the recovery phase. In addition, we found enhanced expression of endogenous antioxidant enzyme systems, which correlated with disease severity. Immunohistochemical verification showed increased expression levels of antioxidant enzymes in the vicinity of EAE-associated leukocyte infiltrates, predominantly in reactive astrocytes and infiltrated macrophages. More importantly, similar areas were found positive for nitrotyrosine, indicating the occurrence of local oxidative stress associated with macrophages and astrocytes. Treatment with the antioxidant α-lipoic acid, which inhibits monocyte infiltration and reduces the development of clinical signs in acute EAE, prevents the expression of endogenous antioxidant enzymes in brains of EAE animals. Activation of the antioxidant enzyme pathway may reflect an adaptive defense mechanism to control an oxidative attack. Our data identified novel gene clusters and proteins regulated in the course of EAE, which may provide future targets for treatment strategies in neuroinflammatory diseases.

Introduction: Body fluid biomarkers for clinical subtyping and monitoring of disease progression are of considerable interest in multiple sclerosis (MS). Proteomics tools are optimal for the unbiased simultaneous detection of large series of peptides and proteins.Objectives: To identify novel candidate biomarkers discriminating patients with MS from patients with other neurological diseases (OND), and for subtyping of relapsing-remitting (RR), secondary progressive (SP) and primary progressive (PP) MS patients using a high-throughput MALDI-TOF-based mass spectrometry method.Methods: Paired cerebrospinal fluid (CSF) and serum samples of 41 RRMS, 30 SPMS, 13 PPMS patients and 25 patients with OND were analysed.Results: Out of a total of 100 detected peptides in CSF and 200 peptides in serum, 11 peptides were differentially regulated in serum and two in CSF between patients with MS and the OND control group. Eleven peptides were differentially regulated in both serum and CSF between relapse-onset MS and PPMS patients. Lastly, four peptides were differentially regulated in serum and two in CSF between RRMS and SPMS patients. Specific peaks regulated in MS were tentatively identified as fragments of secretogranin III and complement C3. The peak intensity of the CSF peptide ion with m/z value 8607.7 correlated to atrophy (r = -0.27, p < 0.005), black hole volumes (r = 0.31, p < 0.008) and total lesion load (r = 0.34, p < 0.003). A serum peptide with m/z value of 872.4 elevated in SPMS correlated to Expanded Disability Status Scale (r = 0.341, p < 0.005) and atrophy (r = -0.286, p < 0.028).Conclusions: Using high-throughput body fluid profiling by MALDI-TOF mass spectrometry, small proteins and peptides were detected as promising candidate biomarkers for diagnosis and disease progression of MS.

OBJECTIVE:Axonal degeneration is the likely cause of disease progression in multiple sclerosis (MS). Our previous results indicated that neuron-specific N-acetylaspartate (NAA) is a candidate CSF biomarker for disease progression in MS. The aim of this study was to explore the potential of NAA as an early biomarker of axonal damage in MS. Next, we wanted to know the additional value of measurement of NAA compared to other candidate markers for axonal damage, such as neurofilament subunits and tau protein.METHODS:Levels of NAA, neurofilament light, neurofilament heavy, and tau were determined in CSF of patients with clinically isolated syndrome (CIS, n = 38), relapsing-remitting MS (RRMS, n = 42), secondary progressive MS (SPMS, n = 28), and primary progressive MS (PPMS, n = 6); patients without neurologic disease (ND, n = 28); noninflammatory neurologic controls (n = 18); and inflammatory neurologic controls (n = 39).RESULTS:CSF NAA levels were decreased in patients with SPMS compared to ND controls, patients with CIS, and patients with RRMS. CSF NAA levels in patients with CIS and RRMS were similar to those in ND subjects. All axonal damage proteins showed specific patterns of changes and relations with disease activity measures. The neurofilament light chain levels were already increased in patients with CIS, especially in patients who converted to MS. The neurofilament heavy chain levels were highest in the patients with SPMS. Tau levels were similar in MS and ND.CONCLUSIONS:CSF N-acetylaspartate (NAA) levels were not different from patients without neurologic disease in early stages of multiple sclerosis, though decreased as the disease progressed. Combining CSF NAA and neurofilament levels yields information on different phases of axonal pathology.

The process of demyelination occurring in diseases as multiple sclerosis is usually investigated in animal models. A major drawback of animal models is that only one condition can be tested per animal, necessitating many animals and systemic effects are factors to be considered. The aim of the study was to develop a reproducible in vitro model for de‐ and remyelination using whole brain spheroid cultures and lysophosphatidyl choline (LPC). In spheroid cultures, single cell suspensions of embryonic day 15 rodent brain cells reaggregate under constant rotation. Three‐dimensional contacts form between the central nervous system cell types present. Multilayered myelin is maximal in four‐week old cultures. A week of repeated exposure to LPC led to 30% loss of MBP protein concentration and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase activity measurements in both rat and mouse spheroids and 56% loss in the number of myelin sheets, with partial remyelination after a week of recovery. The number of dividing cells was increased after LPC exposure and oligodendrocytes were shown to be among the dividing cells. Microglia and astrocytes were not affected and neurons were relatively spared. This suggests that LPC toxicity is specific for myelin and oligodendrocytes. LPC toxicity could be decreased using cholesterol and simvastatin, suggesting that LPC works through altering membrane composition. Thus, in different rodent species and using different read‐outs, we could reproducibly show de‐ and remyelination in spheroid cultures after LPC exposure. This model for demyelination with potential for remyelination offers possibilities for testing novel therapies and studying mechanisms of remyelination. © 2009 Wiley‐Liss, Inc.

BACKGROUND:In multiple sclerosis (MS) myelin debris has been observed within MS lesions, in cerebrospinal fluid and cervical lymph nodes, but the route of myelin transport out of the brain is unknown. Drainage of interstitial fluid from the brain parenchyma involves the perivascular spaces and leptomeninges, but the presence of myelin debris in these compartments has not been described. AIMS:To determine whether myelin products are present in the meninges and perivascular spaces of MS patients. METHODS:Formalin-fixed brain tissue containing meninges from 29 MS patients, 9 non-neurological controls, 6 Alzheimer's disease, 5 stroke, 5 meningitis and 7 leucodystrophy patients was investigated, and immunohistochemically stained for several myelin proteins [proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)]. On brain material from MS patients and (non)neurological controls, PLP immunostaining was used to systematically investigate the presence of myelin debris in the meninges, using a semiquantitative scale. RESULTS:Extensive extracellular presence of myelin particles, positive for PLP, MBP, MOG and CNPase in the leptomeninges of MS patients, was observed. Myelin particles were also observed in perivascular spaces of MS patients. Immunohistochemical double-labelling for macrophage and dendritic cell markers and PLP confirmed that the vast majority of myelin particles were located extracellularly. Extracellular myelin particles were virtually absent in meningeal tissue of non-neurological controls, Alzheimer's disease, stroke, meningitis and leucodystrophy cases. CONCLUSIONS:In MS leptomeninges and perivascular spaces, abundant extracellular myelin can be found, whereas this is not the case for controls and other neurological disease. This may be relevant for understanding sustained immunogenicity or, alternatively, tolerogenicity in MS.

Reactive oxygen species (ROS) and subsequent oxidative damage may contribute to the formation and persistence of multiple sclerosis (MS) lesions by acting on distinct pathological processes. ROS initiate lesion formation by inducing blood–brain barrier disruption, enhance leukocyte migration and myelin phagocytosis, and contribute to lesion persistence by mediating cellular damage to essential biological macromolecules of vulnerable CNS cells. Relatively little is known about which CNS cell types are affected by oxidative injury in MS lesions. Here, we show the presence of extensive oxidative damage to proteins, lipids, and nucleotides occurring in active demyelinating MS lesions, predominantly in reactive astrocytes and myelin-laden macrophages. Oxidative stress can be counteracted by endogenous antioxidant enzymes that confer protection against oxidative damage. Here, we show that antioxidant enzymes, including superoxide dismutase 1 and 2, catalase, and heme oxygenase 1, are markedly upregulated in active demyelinating MS lesions compared to normal-appearing white matter and white matter tissue from nonneurological control brains. Particularly, hypertrophic astrocytes and myelin-laden macrophages expressed an array of antioxidant enzymes. Enhanced antioxidant enzyme production in inflammatory MS lesions may reflect an adaptive defense mechanism to reduce ROS-induced cellular damage.

Background: Elevated homocysteine levels are associated with various neurodegenerative diseases and have even been identified as a risk factor for some of these. Homocysteine levels may be elevated in patients with multiple sclerosis (MS) but large studies are lacking and the relation with disease progression remains to be determined. Aim: The aim of the study was to investigate homocysteine levels in patients with MS and in controls, and to study the relationship between homocysteine levels and clinical progression in MS. Methods: Serum homocysteine levels were compared between MS subtypes (n = 219) and controls (n = 152). Homocysteine levels were associated with baseline and follow-up clinical severity scores. Results: The results showed that serum homocysteine values were similar in patients with MS and controls. Baseline scores on the Expanded Disability Status Scale were higher in patients with secondary progressive MS (SPMS) in the top compared with the bottom quartile of homocysteine levels (p = 0.02). The baseline scores on the Multiple Sclerosis Functional Composite (MSFC) and Paced Auditory Serial Addition Test (PASAT), which measures cognitive functioning, were lower in patients with SPMS in the top compared with the bottom quartile of homocysteine levels (MSFC, p = 0.02; PASAT, p = 0.02). High homocysteine levels were associated with a decline in PASAT scores during follow-up in patients with primary progressive MS (p = 0.009). Conclusion: Serum total homocysteine levels are associated with several measures of disease progression in MS but are not elevated in patients with MS compared with controls. The association of homocysteine levels with cognition in patients with progressive MS raises the question of whether homocysteine directly impacts on MS or reflects a more general neurodegenerative process.

Leukocyte infiltration is a key step in the development of demyelinating lesions in multiple sclerosis (MS), and molecules mediating leukocyte-endothelial interactions represent prime candidates for the development of therapeutic strategies. Here we studied the effects of blocking the integrin-associated tetraspanin CD81 in in vitro and in vivo models for MS. In an in vitro setting mAb against CD81 significantly reduced monocyte transmigration across brain endothelial cell monolayers, both in rodent and human models. Interestingly, leukocyte as well as endothelial CD81 was involved in this inhibitory effect. To assess their therapeutic potential, CD81 mAb were administered to mice suffering from experimental autoimmune encephalomyelitis (EAE). We found that Eat2, but not 2F7 mAb directed against mouse CD81 significantly reduced the development of neurological symptoms of EAE when using a preventive approach. Concomitantly, Eat2 treated animals showed reduced inflammation in the spinal cord. We conclude that CD81 represents a potential therapeutic target to interfere with leukocyte infiltration and ameliorate inflammatory neurological damage in MS.

Levels of the brain‐specific cholesterol metabolite 24S‐hydroxycholesterol are proposed as possible biomarkers for multiple sclerosis (MS). It is not yet clear for which aspect of the MS disease manifestations 24S‐hydroxycholesterol is a reflection. We studied the relation of serum levels of 24S‐hydroxycholesterol and other sterols to the disease characteristics of acute experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Serum was analyzed for cholesterol precursors, oxysterols, and plant sterols during the course of disease development. Significantly increased levels of the cholesterol metabolites 24S‐hydroxycholesterol and 27‐hydroxycholesterol were observed on day 9, before the onset of clinical signs. The serum levels of these oxysterols gradually increased up to 193% and 415%, respectively, at day 17, when clinical symptoms had recovered. Total cholesterol levels were slightly but significantly decreased on day 9 and day 17 in treated animals. Serum levels of cholesterol precursors and plant sterols decreased gradually from day 11 and day 14, respectively. Immunostaining of the 24S‐hydroxycholesterol‐forming enzyme Cyp46 was shown in macrophage infiltrates. In vitro experiments confirmed the presence of Cyp46 in macrophages and showed a decreased expression after LPS treatment. The data indicate that changes in serum oxysterols occur early in EAE and can be formed by macrophages. These early changes indicate an important role for oxysterols in the development of EAE. © 2007 Wiley‐Liss, Inc.