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The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme ("genuine casein kinase", G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn2+ concentration (>1 mM), and is negligible if Mn2+ is replaced by physiological Mg2+ concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2+, but also confers a comparable activity to Fam20C assayed with Mg2+. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased KmATP, as judged from kinetics run with the β(28-40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) and neuromyelitis optica (NMO) are chronic demyelinating diseases of the central nervous system (CNS). Recently, variants of vitamin D metabolizing genes, including rs12368653, rs10876994, rs118204009 and rs703842 in CYP27B1, and rs2248359 in CYP24A1 have been identified to be associated with the pathogenicity of MS in Caucasian populations. However, these results have not been replicated in Han Chinese population. Here we investigated the association of these variants with MS and NMO susceptibility in 149 MS patients, 110 NMO patients and 294 healthy controls using MassARRAY system and Sanger sequencing. We found that the frequencies of the A allele of rs703842 were higher in MS patients than controls (p=0.032), and statistical differences were observed in the genotypes of both rs703842 (p=0.013) and rs10876994 (p=0.001) between NMO patients and controls. In addition, we found difference in the genotype of rs12368653 between MS patients and controls (p=0.008). However, no difference was found in rs2248359 among these three groups. The reported rare mutation p.R389H (rs118204009) was not found in our study. In conclusion, our study suggested that variants of CYP27B1 were associated with both MS and NMO patients in Han Chinese population., (Copyright © 2014. Published by Elsevier B.V.)

Mitogen-activated protein kinase phosphatase (MKP)-1 provides a negative feedback mechanism for regulating mitogen-activated protein kinase (MAPK) activity and thus a variety of cellular processes such as proliferation, differentiation, growth and apoptosis. MKP-1 is established as a central regulator of a variety of functions in the immune, metabolic and cardiovascular systems, and it is now increasingly acknowledged as having a role to play in the nervous system. It has been implicated in regulating processes of neuronal cell development and death as well as in glial cell function. Reduced MKP-1 levels have been observed in models of neurological conditions including Huntington's disease, multiple sclerosis, ischemia and cerebral hypoxia. It has also been suggested to have a role to play in psychiatric disorders such as major depressive disorder. Here, we discuss the role of MKP-1 in nervous system development and disease and examine current evidence providing insight into MKP-1 as a potential therapeutic target for various diseases of the central nervous system.

Neurodegeneration is a chronic progressive loss of neuronal cells leading to deterioration of central nervous system (CNS) functionality. It has been shown that neuroinflammation precedes neurodegeneration in various neurodegenerative diseases. Matrix metalloproteinases (MMPs), a protein family of zinc-containing endopeptidases, are essential in (neuro)inflammation and might be involved in neurodegeneration. Although MMPs are indispensable for physiological development and functioning of the organism, they are often referred to as double-edged swords due to their ability to also inflict substantial damage in various pathological conditions. MMP activity is strictly controlled, and its dysregulation leads to a variety of pathologies. Investigation of their potential use as therapeutic targets requires a better understanding of their contributions to the development of neurodegenerative diseases. Here, we review MMPs and their roles in neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and multiple sclerosis (MS). We also discuss MMP inhibition as a possible therapeutic strategy to treat neurodegenerative diseases.

Background and Aim: Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease of the central nervous system. Genetic risk factors are known to contribute to the etiology of MS. Methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism has been associated with susceptibility to various autoimmune diseases. The aim of this study was to investigate a possible association between the MTHFR gene C677T polymorphism and MS in Turkish patients., Methods: The study included 130 MS patients and 150 group-matched controls. Genomic DNA was isolated and genotyped using polymerase chain reaction-based restriction fragment length polymorphism assay for the MTHFR gene exon C677T polymorphism., Results: The genotype and allele frequencies of C677T polymorphism showed statistically significant differences between MS patients and controls (P = 0.002 and P = 0.002; odds ratio, 1.79; 95% confidence interval, 1.23-2.63, respectively). A significant association was observed when the patients were compared with the controls according to CC genotype versus CT + TT genotypes (P = 0.0005; odds ratio, 2.35; 95% confidence interval, 1.45-3.82). There were no statistically significant association between MTHFR gene C677T polymorphism and baseline clinical and demographical characteristics of MS patients., Conclusions: These results showed that T allele of C677T polymorphism was associated with MS susceptibility in Turkish population.

Background: It is unknown whether diets with a high dietary total antioxidant capacity (TAC) can modify oxidative stress, low-grade inflammation, or liver dysfunction, all of which are risk factors for multiple sclerosis disease. This study assesses alanine amino-transferase (ALT), aspartate-aminotransferase (AST) and gamma-glutamyl transferase (GGT) activities in MS patients treated with co-supplemented hemp seed and evening primrose oils as well as Hot-nature diet and the therapeutic potential this intervention., Methods and Materials: In this double blind, randomized trial, 100 MS patients with EDSS<6 were allocated into 3 groups: "group A", who received co-supplemented hemp seed and evening primrose oils with advised Hot-nature diet; "group B",who received olive oil; and "group C", who received the co-supplemented oils. Clinically, EDSS as well as serum level of liver enzymes (GGT, AST, and ALT) were assessed at baseline and after 6 months., Results: Mean follow-up was 180±2.9 SD days (N=65, 23 M and 42 F aged 34.25±8.07 years with disease duration of 6.80±4.33 years). There was no significant difference in the study parameters at baseline. Serum levels of liver enzymes (GGT, AST, and ALT) were serially monitored. Intervention was associated with liver function alteration in three groups. Significance decreased in EDSS score and the levels of liver enzymes were found in groups A and C, whereas elevated serum liver enzymes and EDSS score were observed in group B after the intervention., Conclusion: Selecting foods according to their Total antioxidant capacity such as co-supplemented hemp seed and evening primrose oils with Hot-nature diet affects antioxidant intake and can have beneficial effects on improving EDSS score and activity of liver enzymes in RRMS patients., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sphingosine 1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitrile 5 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the cocrystal structure of derivative 31 with the homodimeric human S1P lyase. 31 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis. In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases.

Infiltration of leukocytes is a major pathological event in white matter lesion formation in the brain of multiple sclerosis (MS) patients. In grey matter lesions, less infiltration of these cells occur, but microglial activation is present. Thus far, the interaction of β-integrins with extracellular matrix proteins, e.g. fibronectin, is considered to be of importance for the influx of immune cells. Recent in vitro studies indicate a possible role for the enzyme tissue Transglutaminase (TG2) in mediating cell adhesion and migration. In the present study we questioned whether TG2 is present in white and grey matter lesions observed in the marmoset model for MS. To this end, immunohistochemical studies were performed. We observed that TG2, expressed by infiltrating monocytes in white matter lesions co-expressed β1-integrin and is located in close apposition to deposited fibronectin. These data suggest an important role for TG2 in the adhesion and migration of infiltrating monocytes during white matter lesion formation. Moreover, in grey matter lesions, TG2 is mainly present in microglial cells together with some β1-integrin, whereas fibronectin is absent in these lesions. These data imply an alternative role for microglial-derived TG2 in grey matter lesions, e.g. cell proliferation. Further research should clarify the functional role of TG2 in monocytes or microglial cells in MS lesion formation.

Matrix metalloproteinases (MMPs) play a critical role in the blood-brain barrier permeability and in invasion of the leukocytes into the central nervous system during multiple sclerosis (MS). In this respect, in the present study, we have evaluated the possible role of MMP-9 and MMP-2 on the expression of soluble CD154 (sCD154) and membrane-bound isoform of the CD154 in Iranian MS patients. The expressions of the aforementioned protein-related genes were analyzed at the levels of messenger RNA and proteins by real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. The results showed a high expression of CD154 isoforms, MMP-9 and MMP-2, in MS patients in contrast to controls (p < 0.001). We found an increase in sCD154 concentration (i.e., >3-fold) in patients with a higher MMPs/tissue inhibitor of metalloproteinase 1 (TIMP-1) ratio. Furthermore, secondary-progressive MS patients with exacerbate period showed higher positive correlation between increasing sCD154 concentration and overexpression of MMP-2 (p < 0.001). Our data demonstrate that following the exacerbation period, sCD154 concentration is increased in patients, which is mutually related to the MMPs/TIMP-1 ratio. This relationship may represent a new link between sCD154 concentration and the MMPs/TIMP-1 ratio with prognostic implications., (Copyright © 2014. Published by Elsevier B.V.)

Chronic demyelination, on-going inflammation, axonal loss and grey matter neuronal injury are likely pathological processes that contribute to disease progression in multiple sclerosis (MS). Although the precise contribution of each process and their aetiological substrates is not fully known, recent evidence has implicated oxidative damage as a major cause of tissue injury in MS. The degree of tissue injury caused by oxidative molecules, such as reactive oxygen species (ROS), is balanced by endogenous anti-oxidant enzymes which detoxify ROS. Understanding endogenous mechanisms which protect the brain against oxidative injury in MS is important, since enhancing anti-oxidant responses is a major therapeutic strategy for preventing irreversible tissue injury in the disease. Our aims were to determine expression and activity levels of the hydrogen peroxide-reducing enzyme catalase in MS grey matter (GM). In MS GM, a catalase enzyme activity was elevated compared to control GM. We measured catalase protein expression by immune dot-blotting and catalase mRNA by a real-time polymerase chain reaction (RT-PCR). Protein analysis studies showed a strong positive correlation between catalase and microglial marker IBA-1 in MS GM. In addition, calibration of catalase mRNA level with reference to the microglial-specific transcript AIF-1 revealed an increase in this transcript in MS. This was reflected by the extent of HLA-DR immunolabeling in MS GM which was significantly elevated compared to control GM. Collectively, these observations provide evidence that microglial catalase activity is elevated in MS grey matter and may be an important endogenous anti-oxidant defence mechanism in MS., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Blood platelets have been widely proposed as biomarkers in studies of mitochondrial function and aging-related and neurodegenerative diseases. Defects in mitochondrial function were found not only in the substantia nigra of Parkinson's disease patients but also in their blood platelets. Similarly, it has also been described in the blood platelet mitochondria of Alzheimer's disease patients. To study mitochondrial aerobic metabolism function and protein expression in platelets of multiple sclerosis (MS) patients and control subjects, mitochondrial aconitase, mitochondrial superoxide dismutases 1 and 2 (SOD1 and SOD2), and respiratory complex enzyme activities in platelets of MS patients and control subjects were determined. Likewise, mitochondrial lipid peroxidation and mitochondrial SOD1 and cytochrome c expressions were investigated. Mitochondrial aconitase activity was higher in MS patients than in controls (P < 0.05). A significant increase on all respiratory complex activities in MS patients was observed (P < 0.05). Mitochondrial lipid peroxidation was significantly higher in MS patients than in controls (P < 0.05). Significant changes of cytochrome c and mitochondrial SOD1 expressions were detected (P < 0.05), with a decrease of 44 ± 5 % and an increase of 46 ± 6 %, respectively. Our study reveals that significant changes in mitochondrial aerobic metabolism function and mitochondrial SOD1 and cytochrome c expressions are produced in platelets of MS patients.

The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the NGAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest., (Copyright © 2013 Elsevier Inc. All rights reserved.)

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson's disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well-understood. Thus, to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic, and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer's disease for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT(+) motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cord of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of μ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor - calpastatin) in spinal cord of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction. We proposed calpain over-activation and calpain-calpastatin dysregulation driving in a cascade of inflammatory responses (microglial activation and T cell infiltration) and degenerative pathways culminating in axonal degeneration and neuronal death in spinal cord of Parkinson's disease patients. This may be one of the crucial mechanisms in the degenerative process., (© 2013 International Society for Neurochemistry.)

Introduction: Multiple sclerosis (MS) is a chronic immunological disease of the central nervous system characterized by early inflammatory demyelination and subsequent neurodegeneration. Although major progress has occurred, MS is still an incurable disease. Further, parenteral application and/or safety issues of the currently licensed drugs are associated with low patient compliance. Thus, there remains an unmet need for the development of more effective and well-tolerated oral therapies for the treatment of MS. At this point in time, different oral available substances are under investigation and hold promise in the treatment of relapsing-remitting MS (RRMS)., Areas Covered: The physical, chemical and pharmacological properties of laquinimod , as well as its suggested mechanisms of action, clinical efficacy and side-effect profile are reviewed., Expert Opinion: Laquinimod is a new orally administered synthetic drug designed as an immunomodulator. Its mechanisms of action are not yet fully elucidated. Studies in mice and humans revealed different mechanisms of action, including anti-inflammatory and neuroprotective effects. So far, Phase II and Phase III clinical trials have shown its efficacy on magnetic resonance imaging based measures of disease activity, annualized relapse rate and disability progression in RRMS patients. Current data suggest a relatively modest efficacy by measures of relapse rate and there seems to be no superiority in comparison to established disease-modifying agents in relapsing-remitting MS. Further studies are necessary to evaluate both neuroprotective efficacy and optimal dosage of laquinimod in more detail.

Unlabelled: Alterations in the rs4680 Val158Met polymorphism are associated with the presence of pain. No study has investigated the role of Val158Met polymorphism in the susceptibility to exhibit pain in multiple sclerosis (MS). Our aim was to investigate the relationship between Val158Met polymorphism (rs4680) and the presence of pain in MS. One hundred eight (n = 108) patients (mean age: 44 ± 8 years) with a definitive diagnosis of MS and 108 matched controls participated. Fifty-eight patients (54%) had pain and 50 (46%) did not report pain. After amplifying Val158Met polymorphisms by polymerase chain reactions, rs4680 genotype frequencies and allele distributions were calculated. We classified individuals according to their Val158Met polymorphism: Val/Val, Val/Met, and Met/Met. The results showed that distribution of rs4680 Val158Met genotypes was not significantly different between individuals with MS in general and healthy people (χ2 = 2.212, P = .331). When we differentiate MS patients with pain and those without pain, the prevalence of Val158Met genotypes was significantly different (χ2 = 9,610, P = .046): Patients experiencing pain exhibited higher prevalence of Met/Met genotype than those without pain and healthy controls. Current results suggest that the Met allele of Val158Met polymorphism could be a potential risk factor for the development of pain in MS but not for the predisposition of MS itself., Perspective: This study suggests that the Val158Met polymorphism is associated with the presence of pain in MS, but it is not a risk factor for MS itself because the presence of the Met/Met genotype was more prevalent in those patients with pain. This study provides further evidence of potential genetic factors that predispose patients with MS to develop pain., (Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMPs) are a family of extracellular proteases associated with extracellular matrix remodeling. They are involved in many physiological and reparative processes. MMPs can break down all extracellular constituents; therefore, their expression is very tightly regulated and their abnormal activity or over production has been linked to many diseases including multiple sclerosis (MS) which is a leading cause of non-traumatic disability in young adults in North America. Recently many studies, both in animals and humans, have been conducted to better elucidate the underlying causes, mechanisms and pathophysiology of MS. In this review, we discuss the potential role of pathological upregulation of MMPs in MS and future challenges which if properly addressed might help in development of potential cure for this disease.

Multiple sclerosis (MS), the most common disabling neurologic disease of young adults, is considered a classical T cell-mediated disease and is characterized by demyelination, axonal damage, and progressive neurological dysfunction. The currently available disease-modifying therapies are limited in their efficacy, and improved understanding of new pathways contributing to disease pathogenesis could reveal additional novel therapeutic targets. The p38 mitogen-activated protein kinase (MAPK) signaling pathway is known to be triggered by stress stimuli and to contribute to inflammatory responses. Importantly, a number of recent studies have identified this signaling pathway as a central player in MS and its principal animal model, experimental allergic encephalomyelitis. Here, we review the evidence from mouse and human studies supporting the role of p38 MAPK in regulating key immunopathogenic mechanisms underlying autoimmune inflammatory disease of the central nervous system and the potential of targeting this pathway as a disease-modifying therapy in MS.

The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized.

Multiple sclerosis (MS) is an immunoinflammatory disease that affects the central nervous system. Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. We show in this study that HO-1 expression is reduced in PBMCs of MS patients and that during exacerbation of the disease there is a significant downregulation of this enzyme. We conclude that HO-1 may play a significant role in the maintenance of immune homeostasis which is disrupted in autoimmune disorders, such as MS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS., Methods: XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies., Results: We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination., Conclusion: These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.

Leukocytes are major cellular effectors of the immune response. To accomplish this task, these cells display a vast arsenal of proteinases, among which, members of the MMP family are especially important. Leukocytes express several members of the MMP family, including secreted- and membrane-anchored MT- MMPs, which synergistically orchestrate an appropriate proteolytic reaction that ultimately modulates immunological responses. The MT-MMP subfamily comprises TM- and GPI-anchored proteinases, which are targeted to well-defined membrane microdomains and exhibit different substrate specificities. Whereas much information exists on the biological roles of secreted MMPs in leukocytes, the roles of MT-MMPs remain relatively obscure. This review summarizes the current knowledge on the expression of MT-MMPs in leukocyte and their contribution to the immune responses and to pathological conditions.

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.

Inflammatory perivascular cuffs are comprised of leucocytes that accumulate in the perivascular space around post-capillary venules before their infiltration into the parenchyma of the central nervous system. Inflammatory perivascular cuffs are commonly found in the central nervous system of patients with multiple sclerosis and in the animal model experimental autoimmune encephalomyelitis. Leucocytes that accumulate in the perivascular space secrete matrix metalloproteinases that aid their transmigration into the neural parenchyma. We described previously that the upstream inducer of matrix metalloproteinase expression, extracellular matrix metalloproteinase inducer (CD147), was elevated in experimental autoimmune encephalomyelitis, and that its inhibition reduced leucocyte entry into the central nervous system. Here we investigated whether the expression of extracellular matrix metalloproteinase inducer varies with the temporal evolution of lesions in murine experimental autoimmune encephalomyelitis, whether it was uniformly upregulated across multiple sclerosis specimens, and whether it was a feature of inflammatory perivascular cuffs in multiple sclerosis lesions. In experimental autoimmune encephalomyelitis, elevation of extracellular matrix metalloproteinase inducer was correlated with the appearance and persistence of clinical signs of disease. In both murine and human samples, extracellular matrix metalloproteinase inducer was detected on endothelium in healthy and disease states but was dramatically increased in and around inflammatory perivascular cuffs on leucocytes, associated with matrix metalloproteinase expression, and on resident cells including microglia. Leucocyte populations that express extracellular matrix metalloproteinase inducer in multiple sclerosis lesions included CD4+ and CD8+ T lymphocytes, B lymphocytes and monocyte/macrophages. The extra-endothelial expression of extracellular matrix metalloproteinase inducer was a marker of the activity of lesions in multiple sclerosis, being present on leucocyte-containing perivascular cuffs but not in inactive lesions. By using a function-blocking antibody, we implicate extracellular matrix metalloproteinase inducer in the adhesion of leucocytes to endothelial cells and determined that its activity was more crucial on leucocytes than on endothelium in leucocyte-endothelial cell engagement in vitro. Extracellular matrix metalloproteinase inducer activity regulated the level of alpha 4 integrin on leucocytes through a mechanism associated with nuclear factor κB signalling. Blocking extracellular matrix metalloproteinase inducer attenuated the transmigration of monocytes and B lymphocytes across a model of the blood-brain barrier in culture. In summary, we describe the prominence of extracellular matrix metalloproteinase inducer in central nervous system inflammatory perivascular cuffs, emphasize its dual role in matrix metalloproteinase induction and leucocyte adhesion, and highlight the elevation of extracellular matrix metalloproteinase inducer as an orchestrator of the infiltration of leucocytes into the central nervous system parenchyma.

Objectives: It has been implicated in many studies that reactive oxygen species play a role in the development of demyelination in multiple sclerosis (MS). Paraoxonase 1 (PON1) is an antioxidant enzyme that protects cell membranes against oxidative modification. Mitoxantrone is a cytotoxic drug approved for the treatment of MS with adverse effects associated potentially with an increased level of oxidative stress. The aim of this study was to assess the influence of mitoxantrone therapy on PON1 activity in patients with MS., Methods: A studied group included 26 patients with secondary progressive MS, 16 women and 10 men. The blood was collected before the beginning of the therapy as well as after 6 and 12 months. Patients were receiving mitoxantrone every 12 weeks. Serum PON1 activity was assayed using two synthetic substrates: paraoxon and phenyl acetate., Results: Paraoxonase 1 activity toward paraoxon and phenyl acetate and lipid profile did not change significantly in patients receiving mitoxantrone., Conclusions: Mitoxantrone therapy does not influence PON1 activity., (© 2012 John Wiley & Sons A/S.)

Multiple sclerosis (MS) is the most common CNS-demyelinating disease of humans, showing clinical and pathological heterogeneity and a general resistance to therapy. We first discovered that abnormal myelin hypercitrullination, even in normal-appearing white matter, by peptidylarginine deiminases (PADs) correlates strongly with disease severity and might have an important role in MS progression. Hypercitrullination is known to promote focal demyelination through reduced myelin compaction. Here we report that 2-chloroacetamidine (2CA), a small-molecule, PAD active-site inhibitor, dramatically attenuates disease at any stage in independent neurodegenerative as well as autoimmune MS mouse models. 2CA reduced PAD activity and protein citrullination to pre-disease status. In the autoimmune models, disease induction uniformly induced spontaneous hypercitrullination with citrulline+ epitopes targeted frequently. 2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord infiltrates, through selective removal of newly activated T cells. 2CA essentially prevented disease when administered before disease onset or before autoimmune induction, making hypercitrullination, and specifically PAD enzymes, a therapeutic target in MS models and thus possibly in MS.

Vitamin D deficiency has been implicated as a risk factor for multiple sclerosis (MS), but how vitamin D metabolism affects MS pathophysiology is not understood. We studied the expression of vitamin D receptor (VDR) and related enzymes, including 1,25(OH)(2)D-24-hydroxylase (24-OHase; CYP24A1) and 25(OH)D-1α-hydroxylase (CYP27B1), in CNS tissues of 39 MS patients and 20 controls and in primary human glial cells in vitro. In control and MS normal-appearing white matter (NAWM), nuclear VDR immunostaining was observed in oligodendrocyte-like cells, human leukocyte antigen (HLA)-positive microglia, and glial fibrillary acidic protein-positive astrocytes. There was a 2-fold increase in VDR transcripts in MS NAWM versus control white matter (p = 0.03). In chronic active MS lesions, HLA-positive microglia/macrophages showed nuclear VDR staining; astrocytes showed nuclear and cytoplasmic VDR staining. Staining for 24-OHase was restricted to astrocytes.VDR and CYP27B1 mRNA expressions were increased in active MS lesions versus NAWM (p < 0.01, p = 0.04, respectively). In primary human astrocytes in vitro, the active form of vitamin D, 1,25(OH)(2)D(3), induced upregulation of VDR and CYP24A1. Tumor necrosis factor and interferon-γ upregulated CYP27B1 mRNA in primary human microglia and astrocytes. Increased VDR expression in MS NAWM and inflammatory cytokine-induced amplified expression of VDR and CYP27B1 in chronic active MS lesions suggest increased sensitivity to vitamin D in NAWM and a possible endogenous role for vitamin D metabolism in the suppression of active MS lesions.

Serine proteases, a sub-category of the protease family, participate in various physiologic and pathologic conditions. Serine proteases are involved in different arms of the immune system and play an important role in inflammation. They have been evaluated as therapeutic targets in several inflammatory diseases. The Bowman-Birk protease inhibitor (BBI), a soybean-derived serine protease inhibitor, is resistant to temperature and acidic conditions. These characteristics make it a good candidate for oral administration, with no major side effects. In addition, the therapeutic effect of BBI has been shown in inflammatory diseases and cancer. We have demonstrated the immunoregulatory and anti-inflammatory effects of BBI in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Here we review the role of serine proteases in inflammatory diseases, with emphasis on the potential of BBI as a novel oral therapy for multiple sclerosis., (Copyright © 2012. Published by Elsevier Inc.)

Mitoxantrone (MX) is approved for the treatment of aggressive relapsing-remitting, secondary-progressive and progressive-relapsing form of multiple sclerosis (MS). The mechanism of its action is multiaxial, however, it is not free from side effects. The causes of the side effects are still unknown and require further investigation. The aim of this study was to investigate the influence of MX therapy on enzymatic parameters of endogenous antioxidative status: manganese and copper/zinc superoxide dismutase (MnSOD, Cu/ZnSOD), catalase (CAT), glutathione peroxidase (GSH-Px) and lipid peroxidation marker--malondialdehyde (MDA) in blood serum and cerebrospinal fluid (CSF) in patients suffering from MS. After the MX therapy serum and the CSF MDA concentrations increased significantly. We reported that MnSOD activities decrease in serum and the CSF, while, surprisingly, the serum Cu/ZnSOD activity increases after the MX therapy. We also noted a marked decrease in CSF CAT and GSH-Px activity after the MX treatment. Our results strongly suggest the influence of MX therapy on oxidation/antioxidation status of serum and the CSF. These findings open up new opportunities for a better understanding of underlying physiopathological events in MS and provide a new insight into MX's mechanisms of action, especially its potent side effects.

Introduction: The JAK family comprises of the four non-receptor tyrosine kinases JAK1, JAK2, JAK3 and Tyk2, which play key, but differing, roles in cytokine receptor signal transduction. A non-selective JAK inhibitor, ruxolitinib, has recently been approved to treat myelofibrosis whereas tofacitinib is poised for approval to treat rheumatoid arthritis. Selective inhibition of JAK3, JAK1 or Tyk2 provides the opportunity to achieve clinical efficacy in the treatment of inflammatory diseases while reducing the risk of dose-limiting effects attributable to JAK2 inhibition., Areas Covered: This review considers the small number of published patent filings that claim either selective JAK1 or selective Tyk2 inhibitors. These are considered in the context of the considerably larger number of disclosures and patent filings claiming selective JAK2 or JAK3 inhibitors., Expert Opinion: The recent disclosure of the clinical efficacy of a selective JAK1 inhibitor (GLPG-0634) in rheumatoid arthritis and detailed disclosure of the some potent and highly selective JAK1 inhibitors provide a clear stimulus for further activity in this area. The availability of a selective Tyk2 inhibitor will provide the opportunity for better understanding of the physiological role of this kinase. Recent patent applications indicate that Tyk2 selectivity is achievable and Tyk2 inhibitors have potential in the treatment of multiple sclerosis.

We explore the nitric oxide synthase modulation by methylated arginines, asymmetric (ADMA) and symmetric (SDMA) dimethyl-l-arginine and arginase, in early phase of experimental autoimmune encephalomyelitis (EAE), the most frequently used animal model for studying the multiple sclerosis (MS), during the treatment with selective inducibile nitric oxide synthase inhibitor - aminoguanidine (AG) and oxidative scavenger N-acetyl-l-cysteine (NAC), compared to the clinical signs, continual to our previous research. The given results showed that the arginase activity was significantly increased in EAE rats compared to the healthy and AG treated EAE animals (p<0.05), and it was significantly decreased compared to the NAC treated EAE animals (p<0.05) in examined tissues. The ADMA and SDMA levels were significantly decreased in EAE untreated animals compared to the AG and NAC treated EAE animals (p<0.05). As we have reported in our previous papers, nitric oxide (NO) production, was significantly increased in examined tissues of EAE rats compared to the control group (p<0.05). In AG and NAC treated EAE group NO production was decreased in all tissues compared to untreated EAE animals (p<0.05). Also, the AG and NAC treatment of EAE rats during the development of the disease, significantly decreased the clinical score of EAE treated animals compared to EAE group. Arginase and methylated arginine derivatives, involving also NO, appear to be essential modulators of the inflammatory response in acute phase of MS. The continued research of these findings may provide a new area in the treatment of multiple sclerosis acute phase., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Given the significant socioeconomic impact of progressive multiple sclerosis (MS) and the paucity of treatment options, there is an urgent need to develop new and effective therapies for this disabling condition. The relatively recent appreciation that progressive disability is largely driven by neuronal loss has focused considerable research attention on neuroprotective strategies. This has coincided with the emergence of oxidative damage as a prominent effector mechanism of axonal damage in studies of MS pathogenesis, which has opened up a new range of putative targets for neuroprotective therapy in MS. Mitochondrial sirtuins are NAD(+)-dependent protein deacetylases associated with the control of metabolism, aging, and stem cell proliferation and differentiation. Their role in inflammatory demyelinating disease has not been fully characterized, and is the subject of ongoing research. Here, we expound the rationale behind selecting mitochondrial sirtuins as a therapeutic target in demyelinating disease, and report preliminary data that warrant further investigation., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis.

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling., Objectives: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP., Methods: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method., Results: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation., Conclusion: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS.

Background: The vitamin D receptor (VDR) polymorphisms have been reported to be associated with multiple sclerosis (MS), however, evidence remains conflicting. A meta-analysis was conducted to investigate this association., Methods: We searched Pubmed, Medline and Embase databases for case-control studies evaluating the association between the VDR Apa-I, Bsm-I, Fok-I, Taq-I polymorphisms and MS risk. Data were extracted using standardized forms and odds ratios (OR) with 95% confidence intervals (CI) were calculated., Results: 11 case-control studies involving a total of 2599 cases and 2816 controls were included in this meta-analysis. Available data did not suggest an association between any of the VDR polymorphisms and the risk for MS. For Taq-I, which is the most investigated VDR polymorphism with 8 studies (2472 cases and 2446 controls), the combined OR was 1.12 (95% CI: 1.00-1.26) for the dominant model (tt+Tt vs. TT), 1.03(95% CI: 0.88-1.20) for the recessive model (tt vs. Tt+TT), and 1.04 (95% CI: 0.78-1.38) for the homozygote model (tt vs. TT). ORs for other VDR polymorphisms were similar., Conclusion: The VDR Apa-I, Bsm-I, Fok-I and Taq-I polymorphisms are not associated with MS risk., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Despite significant advancement in developing therapies for multiple sclerosis (MS), drugs that cure this devastating disorder are an unmet need. Among the remedies showing efficacy in preclinical MS models, inhibitors of poly(ADP-ribose) polymerase (PARP)-1 have gained great momentum. Emerging evidence demonstrates that PARP-1 inhibitors epigenetically regulate gene expression and finely tune transcriptional activation in immune and neural cells. In this review, we present an appraisal of the effects of PARP-1 and its inhibitors on immune activation, with particular emphasis on the processes taking place during the autoimmune attack directed against the central nervous system. One explanation is that drugs inhibiting PARP-1 activity protect from neuroinflammation in MS models via immunomodulation and direct neuroprotection. PARP-1 inhibitors have already reached the clinical arena as cancer treatments, and observations made in treating these patients could help advance treatments for MS., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Benign multiple sclerosis (BMS) occurs in about 15% of patients with relapsing-remitting multiple sclerosis (RRMS) that over time do not develop significant neurological disability. The molecular events associated with BMS are not clearly understood. This study sought to underlie the biological mechanisms associated with BMS. Blood samples obtained from a cohort of 31 patients with BMS and 36 patients with RRMS were applied for gene expression microarray analysis using HG-U133A-2 array (Affymetrix). Data were analyzed by Partek and pathway reconstruction was performed by Ingenuity for the most informative genes (MIGs). We identified a differing gene expression signature of 406 MIGs between BMS patients, mean±SE age 44.5±1.5 years, 24 females, 7 males, EDSS 1.9±0.2, disease duration 17.0±1.3 years, and RRMS patients, age 40.3±1.8 years, 24 females, 12 males, EDSS 3.5±0.2, disease duration 10.9±1.4 years. The signature was enriched by genes related RNA polymerase I (POL-1) transcription, general inflammatory response and activation of cell death. The most significant under-expressed pathway operating in BMS was the POL-1 pathway (p = 4.0*10(-5)) known while suppressed to activate P53 dependent apoptosis and to suppress NFκB induced inflammation. In accordance, of the 30 P53 target genes presented within the BMS signature, 19 had expression direction consistent with P53 activation. The transcripts within the pathway include POL-1 transcription factor 3 (RRN3, p = 4.8*10(-5)), POL-1 polypeptide D (POLR1D, p = 2.2*10(-4)), leucine-rich PPR-motif containing protein (LRPPRC p = 2.3*10(-5)), followed by suppression of the downstream family of ribosomal genes like RPL3, 6,13,22 and RPS6. In accordance POL-1 transcript and release factor PTRF that terminates POL-1 transcription, was over-expressed (p = 4.4*10(-3)). Verification of POL-1 pathway key genes was confirmed by qRT-PCR, and RRN3 silencing resulted in significant increase in the apoptosis level of PBMC sub-populations in RRMS patients. Our findings demonstrate that suppression of POL-1 pathway induce the low disease activity of BMS.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by a progressive loss of myelin and a failure of oligodendrocyte (OL)-mediated remyelination, particularly in the progressive phases of the disease. An improved understanding of the signaling mechanisms that control differentiation of OL precursors may lead to the identification of new therapeutic targets for remyelination in MS. About 100 mammalian Protein Tyrosine Phosphatases (PTPs) are known, many of which are involved in signaling both in health and disease. We have undertaken a systematic genomic approach to evaluate PTP gene activity in multiple sclerosis autopsies and in related in vivo and in vitro models of the disease. This effort led to the identification of Dusp15/VHY, a PTP previously believed to be expressed only in testis, as being transcriptionally regulated during OL differentiation and in MS lesions. Subsequent RNA interference studies revealed that Dusp15/VHY is a key regulator of OL differentiation. Finally, we identified PDGFR-beta and SNX6 as novel and specific Dusp15 substrates, providing an indication as to how this PTP might exert control over OL differentiation.

Cladribine (2-chloro-2'-deoxyadenosine) is a purine nucleoside analogue (PNA) which causes targeted and sustained reduction of peripheral lymphocyte counts. Cladribine tablets produced significant treatment benefit for patients with relapsing-remitting multiple sclerosis in the phase 3 CLARITY study. In addition to the well-characterised cell-specific phosphorylation of PNAs responsible for lymphocyte reduction, the mode of action of cladribine may encompass distinct activities contributing to its overall effects on the immune system. Here we demonstrate that clinically relevant concentrations of cladribine also inhibit cytokine secretion by human peripheral blood T cells in vitro through mechanisms independent of the induction of lymphocyte death., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The presence of the blood-brain barrier (BBB) restricts the movement of soluble mediators and leukocytes from the periphery to the central nervous system (CNS). Leukocyte entry into the CNS is nonetheless an early event in multiple sclerosis (MS), an inflammatory disorder of the CNS. Whether BBB dysfunction precedes immune cell infiltration or is the consequence of perivascular leukocyte accumulation remains enigmatic, but leukocyte migration modifies BBB permeability. Immune cells of MS subjects express inflammatory cytokines, reactive oxygen species (ROS) and enzymes that can facilitate their migration to the CNS by influencing BBB function, either directly or indirectly. In this review, we describe how immune cells from the peripheral blood overcome the BBB and promote CNS inflammation in MS through BBB disruption., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Activity of erytrocyte glutationperoxidase (GP), glutationreductase (GR), glutationtransferase (GT), glucose-6-phosphatdehydrogenase (G6PDH), catalase and superoxiddysmutase (SOD), and also, the level of malonic dialdehyde (MDA) and total antioxidant activity of blood serum were studied in patients with different types of multiple sclerosis. Investigation of peripherical blood was carried out on first day of treatment and after standard therapy of copaxone. All MS patients had high level of MDA and activity of GP in erythrocytes in comparison with a control group. Other antioxidant enzymes of erythrocytes and total antioxidant activity of blood serum exhibited weak positive dynamics in patients with a relapsing remittance of multiple sclerosis (RRMS). Decrease of activity of antioxidant system in patients with secondary progression multiple sclerosis (SPMS) was more pronounced and remained unchanged after the treatment. This is consistent with the more severe clinical course of thise disease.

Background: Increased activity of angiotensin-converting enzyme (ACE) in the blood and cerebrospinal fluid of patients with multiple sclerosis (MS), and the inhibition of ACE in experimental autoimmune encephalomyelitis, suggested that ACE may play a role in the pathogenesis and progression of MS. We recently published the first report on the potential association of MS and ACE I/D polymorphism in Slovenian and Croatian patients with MS, in which it was shown that the DD genotype might contribute to a higher risk of developing MS in men. To confirm these findings in a similar ethnic population, we analyzed ACE I/D gene polymorphism in patients with MS from Bosnia and Herzegovina., Subjects and Methods: One hundred and seventy patients with MS and 170 healthy controls were genotyped by the polymerase chain reaction method., Results: There was no significant difference in the distribution of ACE I/D genotypes (p=0.783) or in the allelic frequencies (p=0.538) between patients with MS and control subjects. When patients with MS were stratified by sex, no statistically significant differences in allele or genotype distributions were observed. Finally, there was no indication of an impact of the ACE I/D genotype on disease course or severity., Conclusion: The ACE I/D polymorphism is not a risk factor for development of MS, nor does it contribute to disease severity in this Bosnia and Herzegovina population.

Oxidative stress is an important factor which contribute to the pathogenesis of lesions in multiple sclerosis (MS). Whole body cryotherapy (WBCT) is often used in treatment neurological and orthopedic diseases., The Aim, Material and Methods: The aim of this study was to determinate the level of total antioxidative status (TAS) in plasma and activity of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes of MS patients (n = 28) before and after 10 exposures of WBCT (-120 degrees C/3 minutes/day). 16 MS patients during 10 exposures of WBCT additionally were supplemented by 10 mg of melatonin., Results: Increasing of TAS level in plasma as well as supplemented with melatonin and non-supplemented MS patients was observed after 10 exposures of WBCT Melatonin statistically significant increased activity of SOD and CAT in erythrocytes of MS patients treated with WBCT., Conclusions: Results of our study indicate significant increase of TAS level in plasma of MS patients of WBCT treatment. This indicate that WBCT might be a therapy which suppress oxidative stress in MS patients.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

Background: Neuropsychological studies have extensively described the presence of cognitive dysfunction in MS patients. One possible pharmacological treatment of the impairment could be based on acetylcholinesterase inhibitors (AChEIs), which have shown efficacy in alleviating cognitive impairment in many other disorders. The findings on the efficacy of AChEI medication in MS associated cognitive symptoms are preliminary and no studies concerning cerebral acetylcholinesterase (AChE) activity in these patients have been published., Objective: The objective of the study was to examine cerebral AChE activity in cognitively deteriorated MS patients. Cerebral AChE activity of 10 MS patients with secondary progressive disease and marked cognitive impairment, and 10 healthy controls, was studied with positron emission tomography using tracer (11)C-MP4A., Methods: The cognitive profile of the patients was assessed with CERAD (Consortium to Establish a Registry for Alzheimer's Disease)., Results: No differences in cortical AChE activity between MS patients and controls were seen., Conclusions: In the patient group regional AChE activities had inverse correlations with Word learning and MMSE (Mini-Mental State Examination) scores. In the group of cognitively deteriorated MS patients no change in cerebral AChE activity, compared with controls, was observed, but within the patient group more pronounced cognitive symptoms were associated with higher cerebral AChE activity.

Functional NK cell deficiencies are associated with autoimmune diseases, including multiple sclerosis. NK cells can promote or inhibit adaptive immunity via either cytokine production or cytotoxicity toward immature dendritic cells and activated T cells. In humans, this immunoregulatory role resides in the CD56(bright) NK cell subset, which is selectively expanded by daclizumab, a CD25-blocking Ab that suppresses multiple sclerosis-associated inflammation. The objective of this study was to investigate the molecular mechanisms underlying the cytotoxicity of NK cells toward activated T cells. We demonstrated that NK cells induce caspase-independent apoptosis that requires NK cell degranulation and causes mitochondrial dysfunction in activated T cells. Although both granzyme A and granzyme K (GrK) can mediate this form of apoptosis, quantitatively we observed preferential transfer of GrK to target cells. Consequently, gene silencing of GrK in the NK-92 cell line, which retains functional characteristics of CD56(bright) NK cells, profoundly inhibited the ability of NK-92 cells to kill activated syngeneic T cells. Finally, we demonstrated that daclizumab treatment significantly enhanced this newly defined mechanism of cytotoxicity by CD56(bright) NK cells. Our study describes the important physiological role that GrK plays in immunoregulation of adaptive immunity in humans and indicates that therapeutic exploitation of this pathway is beneficial in controlling autoimmunity.

Both genetic and inflammatory factors are suspected in the etiology of multiple sclerosis (MS). Of genetic factors, the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been associated with increased levels of plasma homocysteine, a neuronal excitotoxic amino acid. Sclerotic patients also have elevated levels of plasma and CSF homocysteine. In this study, the association between C677T polymorphism and MS was tested by recruiting 230 healthy and 194 multiple sclerotic age- and gender-matched patients. The MTHFR C677T polymorphism and the serum levels of inflammatory mediators IL-1β, TNFα, and CRP were measured. TNFα, CRP, and IL-1β levels were significantly higher in sclerotic patients. T allele was 1.7 times more present in this group. In patient's group, the levels of all inflammatory mediators were higher in T/T compared to two other genotypes. Evaluation of the age of onset of disease revealed that subjects with T allele developed the MS disease, almost 4 years sooner than other genotype. We concluded that having T allele of C677T in MS might be accompanied with higher levels of serum inflammatory mediators and a vulnerability to earlier age of onset of disease. Further studies are needed to elucidate the underlying mechanisms.