Comprehensive phenotyping in multiple sclerosis: discovery based proteomics and the current understanding of putative biomarkers

Currently, there is no single test for multiple sclerosis (MS). Diagnosis is confirmed through clinical evaluation, abnormalities revealed by magnetic resonance imaging (MRI), and analysis of cerebrospinal fluid (CSF) chemistry. The early and accurate diagnosis of the disease, monitoring of progression, and gauging of therapeutic intervention are important but elusive elements of patient care. Moreover, a deeper understanding of the disease pathology is needed, including discovery of accurate biomarkers for MS. Herein we review putative biomarkers of MS relating to neurodegeneration and contributions to neuropathology, with particular focus on autoimmunity. In addition, novel assessments of biomarkers not driven by hypotheses are discussed, featuring our application of advanced proteomics and metabolomics for comprehensive phenotyping of CSF and blood. This strategy allows comparison of component expression levels in CSF and serum between MS and control groups. Examination of these preliminary data suggests that several CSF proteins in MS are differentially expressed, and thus, represent putative biomarkers deserving of further evaluation.     

Emerging imaging and liquid biomarkers in multiple sclerosis

The advent of highly effective disease modifying therapy has transformed the landscape of multiple sclerosis (MS) care over the last two decades. However, there remains a critical, unmet need for sensitive and specific biomarkers to aid in diagnosis, prognosis, treatment monitoring, and the development of new interventions, particularly for people with progressive disease. This review evaluates the current data for several emerging imaging and liquid biomarkers in people with MS. MRI findings such as the central vein sign and paramagnetic rim lesions may improve MS diagnostic accuracy and evaluation of therapy efficacy in progressive disease. Serum and cerebrospinal fluid levels of several neuroglial proteins, such as neurofilament light chain and glial fibrillary acidic protein, show potential to be sensitive biomarkers of pathologic processes such as neuro-axonal injury or glial-inflammation. Additional promising biomarkers, including optical coherence tomography, cytokines and chemokines, microRNAs, and extracellular vesicles/exosomes, are also reviewed, among others. Beyond their potential integration into MS clinical care and interventional trials, several of these biomarkers may be informative of MS pathogenesis and help elucidate novel targets for treatment strategies.     

Effect of 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-1-benzazepin-8-yl)-1-propanone fumarate,a novel acetylcholinesterase inhibitor,on spatial cognitive impairment induced by chronic cerebral hypoperfusion in rats

Reversible block of sodium channels by endogenous substances was claimed to account for the fast relapses and remissions seen in demyelinating autoimmune disorders. The pentapeptide QYNAD, isolated from the cerebrospinal fluid from patients with multiple sclerosis (MS), blocked Na+ channels in various types of cultured cells. We show that 100 microM QYNAD bath-applied to isolated rat sciatic nerve causes the amplitude and area of the compound nerve action potential to decrease by 30-40%, while the latency increases. Wash-out reverses the changes in part. This suggests that QYNAD may indeed contribute to the fast symptom changes in MS and related diseases.     

Peptide Specificity of Anti-Myelin Basic Protein Antibodies in Patients with Acute Optic Neuritis and the HLA System

Multiple sclerosis (MS) may be an autoimmune disease, partially caused by autoreactivity to myelin basic protein (MBP) and other central nervous system proteins. Acute optic neuritis (ON) is a frequent first symptom of MS. The role of the HLA system in anti-MBP antibody production in ON was investigated employing a restriction fragment length polymorphism system for genomic HLA-DQ and -DR typing and an immunospot assay for the detection of individual cells secreting antibodies to three different synthetic MBP peptides. Thirty-two out of 40 patients (80%) with ON had cells in cerebrospinal fluid secreting anti-MBP peptide antibodies while this occurred in 10/19 patients with other neurological diseases (53%; mainly in patients with inflammatory diseases in the central nervous system). This difference was statistically significant ( P = 0.03). None of the three examined peptides were immunodominant in any patient group. It was found, however, that presence of HLA DR15, which is associated with MS, may be associated further with predominant production of antibodies to the MBP amino acids 63–88 in patients with ON ( P = 0.002, corrected for multiple comparisons).     

In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis

We found an increase in peripheral-blood lymphocytes bearing the T-cell-specific activation antigen Ta1 in 20 of 35 patients with progressive multiple sclerosis, 4 of 18 patients with stable or improving multiple sclerosis, 1 of 17 patients with other neurologic diseases, and 1 of 14 normal controls (P less than 0.0002, Fisher's exact test). No increases in two other markers of T-cell activation, T113 and the interleukin-2 receptor, were found. In the cerebrospinal fluid, patients with progressive multiple sclerosis (pleocytosis, 3.9 +/- 1.6 cells per cubic millimeter) had 42 +/- 3.0 per cent Ta1+ cells. In contrast, patients with other inflammatory central nervous system diseases (36 +/- 13 cells per cubic millimeter) had 9.6 +/- 1.8 per cent Ta1+ cells (P less than 0.01). In patients with other neurologic diseases without inflammation (0.7 +/- 0.16 cells per cubic millimeter), the percentage of Ta1+ cells was equivalent to that in patients with multiple sclerosis (39 +/- 5.4 per cent), although the absolute number was lower. There was a positive correlation between the presence of Ta1+ cells in the spinal fluid and blood of patients with other neurologic diseases, but not patients with multiple sclerosis. Less than 1 per cent of lymphocytes from the spinal fluid of patients with multiple sclerosis expressed interleukin-2 receptors, as compared with 9.8 per cent of cells from subjects with other inflammatory neurologic diseases (P less than 0.01). These results suggest that the T cells in the spinal fluid of patients with multiple sclerosis may be activated by a different mechanism or in a different temporal sequence from that in patients with other nervous system diseases. Furthermore, the increase in Ta1+ cells in the peripheral blood of patients with multiple sclerosis demonstrates systemic immune activation in the disease; monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Body fluid biomarkers in multiple sclerosis: how far we have come and how they could affect the clinic now and in the future

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system, which affects over 2.5 million people worldwide. Although MS has been extensively studied, many challenges still remain in regards to treatment, diagnosis and prognosis. Typically, prognosis and individual responses to treatment are evaluated by clinical tests such as the expanded disability status scale, MRI and presence of oligoclonal bands in the cerebrospinal fluid. However, none of these measures correlates strongly with treatment efficacy or disease progression across heterogeneous patient populations and subtypes of MS. Numerous studies over the past decades have attempted to identify sensitive and specific biomarkers for diagnosis, prognosis and treatment efficacy of MS. The objective of this article is to review and discuss the current literature on body fluid biomarkers in MS, including research on potential biomarker candidates in the areas of miRNA, mRNA, lipids and proteins.     

Biological markers for axonal degeneration in CSF and blood of patients with the first event indicative for multiple sclerosis

Axonal degeneration is now recognized as an important pathological feature of multiple sclerosis (MS). Acute axonal damage happens early in the disease course, and therefore early changes might occur in markers in body fluids, such as cerebrospinal fluid (CSF) and blood. In our study we investigated the relevance of serum and CSF markers for axonal damage in patients with clinically isolated syndrome indicative for MS. We measured the concentration of tau, phospho-tau, S100B, Amyloid beta and neuron specific enolase (NSE) in CSF and serum. Interestingly, the NSE concentration in CSF and serum was decreased in clinically isolated syndrome (CIS)-patients in comparison to the control group indicating reduced neuronal metabolic activity in the early stage of the disease. Concerning other biomarkers, we did not observe any changes in the concentrations between groups. Moreover, we did not detect any correlation between Expanded Disability Status Scale (EDSS) and the concentration of investigated proteins.     

Expansion of CD5 - B cells in multiple sclerosis correlates with CD80 (B7-1) expression

The pathogenetic role of autoantibodies in multiple sclerosis (MS) is uncertain. CD5+ B cells commonly produce autoantibodies, but CD5 expression has also been implicated in B-cell tolerance. We studied B-cell subsets, anti-myelin protein antibody-secreting cells in cerebrospinal fluid (CSF) and a panel of serum autoantibodies in patients with clinically isolated syndromes (CIS), suggestive of MS and patients with clinically definite MS (CDMS). Patients with CDMS had a higher percentage of CD5- B cells in CSF than did control subjects (P = 0.02). CIS patients with immunoglobulin G (IgG) oligoclonal bands in CSF or multiple lesions on magnetic resonance imaging (MRI) had a higher percentage of CD5- B cells in CSF than did the remaining CIS patients (P = 0.03). The percentage of CD5- and CD80+ B cells correlated positively and the percentage of CD5+ B cells correlated negatively with the number of CSF cells secreting anti-myelin basic protein (anti-MBP) antibodies. The prevalence of serum autoantibodies was comparable in the three patient groups. We conclude that intrathecal expansion of CD5- B cells appears to be more characteristic in MS patients, and CD5+ B cells may be associated with a lower prevalence of anti-myelin antibody production.     

Viral antibodies in cerebrospinal fluid of multiple sclerosis and control patients: comparison between radioimmunoassay and conventional techniques.

Cerebrospinal fluid antibodies to measles, rubella, vaccinia, herpes simplex, and varicella-zoster viruses in four patient study groups (clinically definite multiple sclerosis [MS], early probable MS, optic neuritis, and control patients with other neurological diseases) were assayed by radioimmunoassay, complement fixation, hemagglutination-inhibition, or complement-enhanced plaque reduction methods. Antibodies were more frequently found and at higher dilutions by radioimmunoassay than by other techniques. Measles virus antibody, the most frequently found antibody, was present in the cerebrospinal fluid of 72% of MS patients and 5% of control patients. The differences between the numbers of MS patients and control patients with antibodies to other viruses were not as marked. Thus, 58% of MS patients versus 21% of control patients had antibody to rubella virus, 20 versus 3% had antibody to vaccinia virus, 50 versus 33% had antibody to herpes simplex virus, and 25 versus 8% had antibody to varicella virus. Sixty-seven percent of MS patients and 26% of control patients had antibodies to two or more viruses in their cerebrospinal fluid.     

多发性硬化患者血清和脑脊液中S-100b蛋白测定及临床意义

为探讨S 10 0b蛋白与多发性硬化 (MS )临床的关系 ,采用ELISA方法检测 6 0例急性活动期MS ,2 6例对照组血清及脑脊液中S 10 0b含量 ,并进行比较分析。结果发现 ,血清及脑脊液中S 10 0b水平 ,急性活动期MS高于对照组 (P均 <0 0 1) ,MS组血清和脑脊液中S 10 0b含量异常增高者分别为 2 0 %和 96 6 % ;脑脊液S 10 0b水平MS急性活动 7d内高于急性活动 7d以上者 (P <0 0 5 ) ,两者又均高于对照组 (P均 <0 0 1) ;血清S 10 0b水平MS急性活动 7d内高于急性活动 7d以上者及对照组 (P均 <0 0 1) ,急性活动 7d后与对照组比较无差异 (P >0 0 5 ) ;脑脊液S 10 0b水平不同病残程度之间比较无显著性差异 (P >0 0 5 ) ,血清S 10 0b水平在中、重度病残者高于轻度病残者 (P均 <0 0 1) ,重度病残者虽高于中度病残者 ,但差异无显著性意义 (P >0 0 5 )。提示S 10 0b蛋白可作为判断MS疾病活动性及病情严重程度的重要生化标志物之一。     

Fatigue in Multiple Sclerosis: Mechanisms,Evaluation, and Treatment

Among patients with multiple sclerosis (MS), fatigue is the most commonly reported symptom, and one of the most debilitating. Despite its high prevalence and significant impact, fatigue is still poorly understood and often under-emphasized because of its complexity and subjective nature. In recent years, an abundance of literature from specialists in sleep medicine, neurology, psychiatry, psychology, physical medicine and rehabilitation, and radiology have shed light on the potential causes, impact, and treatment of MS-related fatigue. Though such a diversity of contributions clearly has advantages, few recent articles have attempted to synthesize this literature, and existing overviews have focused primarily on potential causes of fatigue rather than clinical evaluation or treatment. The aims of this review are to examine, in particular for sleep specialists, the most commonly proposed primary and secondary mechanisms of fatigue in MS, tools for assessment of fatigue in this setting, and available treatment approaches to a most common and challenging problem.

Citation:

Braley TJ; Chervin RD. Fatigue in multiple sclerosis: mechanisms, evaluation, and treatment. SLEEP 2010;33(8):1061-1067.     

多发性硬化患者淋巴细胞激活的临床意义

目的：探讨多发性硬化(MS)患者淋巴细胞的激活状态及临床意义。方法：流式细胞仪测定28例缓解复发型MS患者(复发期20例，缓解期8例)外周血(PB)、12例复发期脑脊液(CSF)及11例复发期MS患者予糖皮质激素治疗后淋巴细胞CD69和HLA-DR表达的阳性百分率。结果：复发期MS患者阳淋巴细胞HLA-DR 和CD3 ／HAL-DR 的百分率高于对照组和缓解期MS，缓解期MS患者CD3 /HLA-DR 的百分率高于对照组3复发期MS患者CSF中CD69 、HLA-DR 和CD3 ／HLA-DR 的百分率均高于PB；复发期MS患者CSF中CD69 的百分率与血脑屏障受损呈正相关；激素治疗降低复发期MS PB淋巴细胞HLA-DR 的百分率。结论：MS患者淋巴细胞激活涉及MS的发病机制并可作为MS活动期的一个指标。     

Association between tumor necrosis factor-alpha and disease progression in patients with multiple sclerosis.

BACKGROUND. Cachectin, or tumor necrosis factor-alpha (TNF-alpha), is a principal mediator of the inflammatory response and may be important in the pathogenesis and progression of multiple sclerosis, an inflammatory disease of the central nervous system. METHODS. In a 24-month prospective study, we used a sensitive enzyme-linked immunosorbent assay to determine levels of TNF-alpha in cerebrospinal fluid and serum in 32 patients with chronic progressive multiple sclerosis and in 20 with stable multiple sclerosis and 85 with other neurologic diseases. An attempt was made to relate TNF-alpha levels with the degree of disability of the patients with multiple sclerosis and with their neurologic deterioration during the 24 months of observation. RESULTS. High levels of TNF-alpha were found in the cerebrospinal fluid of 53 percent of the patients with chronic progressive multiple sclerosis and in none of those with stable multiple sclerosis (P less than 0.001). TNF-alpha was detected in the cerebrospinal fluid of 7 percent of the controls (P less than 0.01) with other neurologic disease. In patients with chronic progressive multiple sclerosis, mean TNF-alpha levels were significantly higher in the cerebrospinal fluid than in corresponding serum samples (52.41 vs. 11.88 U per milliliter; range, 2 to 178 vs. 2 to 39; P less than 0.001). In these patients, cerebrospinal fluid levels of TNF-alpha correlated with the degree of disability (r = 0.834, P less than 0.001) and the rate of neurologic deterioration (r = 0.741, P less than 0.001) before the start of the study. Cerebrospinal fluid levels also correlated with the increase in neurologic disability after 24 months of observation (r = 0.873, P less than 0.001). CONCLUSIONS. These data provide evidence of intrathecal synthesis of TNF-alpha in multiple sclerosis and suggest that the level of TNF-alpha in cerebrospinal fluid correlates with the severity and progression of the disease. Our results suggest that TNF-alpha may reflect histologic disease activity in multiple sclerosis and could be used to monitor outcomes or responses to therapy.     

多发性硬化患者抗髓鞘碱性蛋白抗体的检测及其意义

目的 检测多发性硬化(MS)患者抗髓鞘碱性蛋白(MBP)抗体并探讨其意义.方法 采用ELISA方法测定56例多发性硬化患者急性期血清和脑脊液配对标本的抗髓鞘碱性蛋白抗体,30例其它神经疾病(OND)患者及36例正常人(NC)为对照.结果 以OND组患者OD值为标准,测得MS患者脑脊液抗MBP抗体,其阳性率为26.8%,与OND组比较有显著性意义;以NC组OD值为对照,MS患者血清抗MBP抗体阳性率为78.6%,OND组抗MBP抗体阳性率为50%,与NC组均有显著性意义,MS患者与OND组患者抗MBP抗体阳性率比较亦有显著性意义.结论 多发性硬化患者脑脊液和血液中均可检测到抗MBP抗体,可为临床诊断和治疗提供指导.     

Relative increase of inflammatory CD4+ T cells in the cerebrospinal fluid of multiple sclerosis patients and control individuals.

Of three patients with multiple sclerosis (MS) and two non-MS individuals a large number of CD4+ T cell clones was obtained from the cerebrospinal fluid and peripheral blood by direct limiting dilution. The CD4+ T cell clones from cerebrospinal fluid and peripheral blood lymphocytes were compared for their cytotoxic activity and lymphokine production. Cytotoxic capacity of cloned T cells was analysed with the use of anti-CD3 antibodies and target cells bearing Fc receptors for murine IgG. Recently, we demonstrated the existence of two different subsets of human CD4+ T cell clones by phenotypic and functional criteria. One type of CD4+ T cell with anti-CD3 mediated cytotoxic activity, in analogy with murine studies, is the inflammatory or TH1 subtype, the main producer of interleukin (IL-2), interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)-alpha, -beta, whereas the other type of CD4+ T cell clone lacked anti-CD3 mediated cytotoxicity and produced minimal amounts of IL-2 concomitant with reduced levels of IFN-gamma and TNF-alpha, -beta. The present study demonstrates that among three MS patients, relatively more inflammatory CD4+ T cell clones with cytotoxic activity and IFN-gamma and TNF-alpha, -beta production were derived from the cerebrospinal fluid as compared with peripheral blood lymphocytes. Also among control individuals more inflammatory CD4+ T cell clones could be obtained from the cerebrospinal fluid as from the peripheral blood. The enrichment of inflammatory CD4+ T cells, therefore, appears to be physiological rather than associated with the disease.     

Analysis of CD4+CD8+ double‐positive T cells in blood,cerebrospinal fluid and multiple sclerosis lesions

T cells with a CD4⁺ CD8⁺ double‐positive (DP) phenotype are present in small numbers in the peripheral blood of healthy humans and may have anti‐viral capacities. Here we investigate numbers and function of DP T cells in patients with relapsing–remitting multiple sclerosis (MS), either treatment‐naive or under therapy with natalizumab. Flow cytometry analysis revealed that frequencies of circulating DP T cells in treatment‐naive and natalizumab‐treated MS patients are comparable to healthy controls. These cells have a memory phenotype with cytotoxic potential, express high levels of CD49d and are similarly functional in treatment‐naive as well as natalizumab‐treated MS patients. DP T cells were enriched in the cerebrospinal fluid, but do not invade acutely inflamed MS lesions. In conclusion, DP T cells are functional in MS and may play a role in the immune surveillance of the central nervous system, but do not display functional impairment under natalizumab therapy.     

Immunoassay of P2 protein in cerebrospinal fluid in neurological disorders.

Cerebrospinal fluid samples were obtained at lumbar puncture from 53 patients with a wide variety of neurological disorders. Cerebrospinal fluid samples were tested for the presence of P2 protein, a constituent of myelin, with an enzyme linked immunosorbent assay technique using a specific polyclonal antibody. High concentrations of P2 in the cerebrospinal fluid paralleled a raised IgG index (clearance ratio), the presence of oligoclonal bands, as well as raised white cell counts or depressed albumin:IgG ratios. Twenty one patients had been diagnosed as having definite or probable multiple sclerosis and the remaining 32 had other conditions. Of the 13 patients with high positive P2, 12 (92%) were in the multiple sclerosis category; of the 40 patients with low (12) or undetectable (28) P2 concentrations, only nine (23%) were diagnosed as having multiple sclerosis. In this patient population the presence of high immunoreactive P2 concentrations in cerebrospinal fluid was closely associated with evidence of intrathecal immunoglobulin synthesis and with the clinical diagnosis of multiple sclerosis. On this basis it is suggested that immunoassay of P2 concentration in the cerebrospinal fluid may be of potential value in the investigation of patients with demyelinating disorders.     

Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

We studied more than 300 cerebrospinal fluid proteins from 21 patients with Creutzfeldt-Jakob disease. We also examined cerebrospinal fluid from 100 normal controls and more than 400 patients with various neurologic disorders other than Creutzfeldt-Jakob disease. Four abnormal proteins that were identified in the patients with Creutzfeldt-Jakob disease were absent in the normal persons. Two of these proteins (Mr [relative molecular mass], 40,000; pl [isoelectric point], 5.7 and Mr 40,000; pl 5.9) were also present in some patients with multiple sclerosis, herpes simplex encephalitis, schizophrenia, Parkinson's disease, or Guillain-Barré or Beh?et's syndrome. Two proteins (Mr 26,000; pl 5.2 and Mr 29,000; pl 5.1) were present in all patients with Creutzfeldt-Jakob disease and in 5 of 10 patients with herpes simplex encephalitis, but in none of the other control groups. A subsequent blinded study of these cerebrospinal fluid proteins from patients with Creutzfeldt-Jakob disease, Alzheimer's disease, Huntington's disease, multi-infarct dementia, parkinsonism dementia of Guam, or the specific dementia of the acquired immunodeficiency syndrome resulted in the ability to distinguish all cases of Creutzfeldt-Jakob disease from the other types of dementia. Although the identity and origin of the abnormal spinal fluid proteins are not yet known, these preliminary results suggest that their presence may help in the diagnosis of Creutzfeldt-Jakob disease.     

CSF hypocretin-1 concentrations correlate with the level of fatigue in multiple sclerosis patients

Considering the multiplicity of symptoms associated with multiple sclerosis (MS), there is possibility that hypocretin system function might be involved in the pathogenesis of the disease. The current study aimed to investigate the hypocretin-1 levels in cerebrospinal fluid (CSF) of MS patients in relation to different neurological deficit measures including: Ambulation Index (AI), Expanded Disability Status Scale (EDSS), Fatigue Severity Scale (FSS), and Epworth Sleepiness Scale (ESS) in relapse-onset MS patients. 53 subjects were included into the study: 38 patients with a diagnosis of MS and 15 healthy controls. Among MS patients, 25 had relapsing-remitting and 13 secondary progressive MS. CSF hypocretin-1 levels did not differ between MS patients and healthy controls (p > 0.05). A positive correlation between hypocretin-1 level and fatigue level was found in MS patients (p < 0.05) and this effect was even stronger in the MS subgroup suffering from fatigue (p = 0.01). Hypocretin system seems to be generally unchanged in MS but a positive correlation between hypocretin-1 level and fatigue may indicate involvement of some compensatory mechanisms stimulating the production of the neuropeptide in MS patients.     

Gene-expression signatures: biomarkers toward diagnosing multiple sclerosis

Identification of biomarkers contributing to disease diagnosis, classification or prognosis could be of considerable utility. For example, primary methods to diagnose multiple sclerosis (MS) include magnetic resonance imaging and detection of immunological abnormalities in cerebrospinal fluid. We determined whether gene-expression differences in blood discriminated MS subjects from comparator groups, and identified panels of ratios that performed with varying degrees of accuracy depending upon complexity of comparator groups. High levels of overall accuracy were achieved by comparing MS with homogeneous comparator groups. Overall accuracy was compromised when MS was compared with a heterogeneous comparator group. Results, validated in independent cohorts, indicate that gene-expression differences in blood accurately exclude or include a diagnosis of MS and suggest that these approaches may provide clinically useful prediction of MS.