Arjan Malekzadeh

Body fluid biomarkers for multiple sclerosis—the long road to clinical application

There is a strong unmet clinical need for objective body fluid biomarkers to assist early diagnosis and estimate long-term prognosis, monitor treatment response and predict potential adverse effects in multiple sclerosis (MS). Here, we review recent studies (focusing on 2012 to early 2015) on body fluid markers in MS from the perspective of their clinical utility. Because the first step towards clinical implementation of a newly discovered biomarker is independent replication, we focus on biomarkers that have been validated in at least two independent cohorts. We also discuss recent data challenging earlier findings, and biomarkers for which new clinical uses are suggested. For early MS diagnosis and prediction of conversion from clinically isolated syndrome to MS, several new B-cell-associated candidate blood biomarkers have emerged. For prognosis, several novel axonal damage markers should be adopted to biomarker panels. The number of disease-modifying treatments for MS has increased sharply, but biomarkers for treatment response monitoring and adverse effect prediction are scarce, and markers for subtyping and staging of MS are still lacking. In view of the availability and implementation of several standardized protocols to optimize biomarker studies, we expect biomarker development for MS to be improved and accelerated, with clinical implementation in the near future.

Challenges in multi-plex and mono-plex platforms for the discovery of inflammatory profiles in neurodegenerative diseases

Pro and anti-inflammatory cytokines are involved in disease onset and pathophysiology of multiple sclerosis, Alzheimers disease and Parkinsons disease. It is likely that panels of multiple cytokines provide a good reflection of disease status and can be used as biological markers in body fluids. Different multi-plex platforms, Luminex-xMAP and Meso Scale Discovery, are able to detect multiple analytes in the same sample at the same time. In this literature based review, we offer an overview of the multi-plex platforms and compare them with the golden standard ELISA in their ability to accurately and sensitively detect cytokines in cerebrospinal fluid (CSF) and blood (serum/plasma). The detectability and levels of cytokines in multiple sclerosis, Alzheimers disease and Parkinsons disease are promising but also show discrepancies between studies. The current immuno-assays lack sensitivity for detection of various cytokines that have low concentrations of cytokines in CSF and blood, and therefore technical improvements are needed. With such improvements the use of large panels of cytokines as inflammatory profiles may offer additional value in diagnosis, prognosis and therapeutic response in neurodegenerative diseases.

Fatigue in Patients with Multiple Sclerosis: Is It Related to Pro- and Anti-Inflammatory Cytokines?

Objective. To investigate the pathophysiological role of pro- and anti-inflammatory cytokines in primary multiple sclerosis-related fatigue. Methods. Fatigued and non-fatigued patients with multiple sclerosis (MS) were recruited and their cytokine profiles compared. Patients with secondary fatigue were excluded. Fatigue was assessed with the self-reported Checklist Individual Strength (CIS20r), subscale fatigue. A CIS20r fatigue cut-off score of 35 was applied to differentiate between non-fatigued (CIS20r fatigue ≤34) and fatigued (CIS20r fatigue ≥35) patients with MS. Blood was collected to determine the serum concentrations of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IL-12p70, IL-17, TNFα, and IFN-γ) and anti-inflammatory cytokines (IL-4, IL-5, IL-10, and IL-13). We controlled for the confounding effect of age, gender, duration of MS, disease severity, type of MS, and use of immunomodulatory drugs. Results. Similar cytokine levels were observed between MS patients with (n = 21) and without fatigue (n = 14). Adjusted multiple regression analyses showed a single significant positive relationship, that of IL-6 with CIS20r fatigue score. The explained variance of the IL-6 model was 21.1%, once adjusted for the confounding effect of age. Conclusion. The pro-inflammatory cytokine interleukin-6 (IL-6) may play a role in the pathophysiology of primary fatigue in patients with MS. Trial Registrations. ISRCTN69520623, ISRCTN58583714, and ISRCTN82353628.

Novel diagnostic cerebrospinal fluid biomarkers for pathologic subtypes of frontotemporal dementia identified by proteomics

Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotemporal dementia (FTD) and its pathologic subtypes are lacking.

The effectiveness of aerobic training, cognitive behavioural therapy, and energy conservation management in treating MS-related fatigue: the design of the TREFAMS-ACE programme

BackgroundTREFAMS is an acronym for TReating FAtigue in Multiple Sclerosis, while ACE refers to the rehabilitation treatment methods under study, that is, Aerobic training, Cognitive behavioural therapy, and Energy conservation management. The TREFAMS-ACE research programme consists of four studies and has two main objectives: (1) to assess the effectiveness of three different rehabilitation treatment strategies in reducing fatigue and improving societal participation in patients with MS; and (2) to study the neurobiological mechanisms of action that underlie treatment effects and MS-related fatigue in general.Methods/DesignAmbulatory patients (n = 270) suffering from MS-related fatigue will be recruited to three single-blinded randomised clinical trials (RCTs). In each RCT, 90 patients will be randomly allocated to the trial-specific intervention or to a low-intensity intervention that is the same for all RCTs. This low-intensity intervention consists of three individual consultations with a specialised MS-nurse. The trial-specific interventions are Aerobic Training, Cognitive Behavioural Therapy, and Energy Conservation Management. These interventions consist of 12 individual therapist-supervised sessions with additional intervention-specific home exercises. The therapy period lasts 16 weeks. All RCTs have the same design and the same primary outcome measures: fatigue - measured with the Checklist Individual Strength, and participation - measured with the Impact on Participation and Autonomy questionnaire. Outcomes will be assessed 1 week prior to, and at 0, 8, 16, 26 and 52 weeks after randomisation. The assessors will be blinded to allocation. Pro- and anti-inflammatory cytokines in serum, salivary cortisol, physical fitness, physical activity, coping, self-efficacy, illness cognitions and other determinants will be longitudinally measured in order to study the neurobiological mechanisms of action.DiscussionThe TREFAMS-ACE programme is unique in its aim to assess the effectiveness of three rehabilitation treatments. The programme will provide important insights regarding the most effective treatment for MS-related fatigue and the mechanisms that underlie treatment response. A major strength of the programme is that the design involves three almost identical RCTs, enabling a close comparison of the treatment strategies and a strong overall meta-analysis. The results will also support clinical practice guidelines for the treatment of MS-related fatigue.Trial registrationsCurrent Controlled Trials ISRCTN69520623, ISRCTN58583714, and ISRCTN82353628

Comparison of multiplex platforms for cytokine assessments and their potential use for biomarker profiling in multiple sclerosis

BACKGROUND The levels of pro and anti‐inflammatory cytokines can be altered in different autoimmune pathologies, such as multiple sclerosis (MS). It is likely that cytokines in bodily fluids can provide a good reflection of ongoing disease patho‐physiology. In this study we aimed to validate multiplex cytokine platforms and evaluate whether these cytokines are differentially expressed in MS. METHODS Assay validation for simultaneous quantification of IL‐1&bgr;, IL‐6, IL‐8 and TNF‐&agr; in serum and CSF were performed using both the Luminex‐xMAP (Luminex) and Meso Scale Discovery (MSD) platforms. Next, the relation of the pro‐inflammatory cytokine 4‐plex with disease progression, symptoms and subtypes was studied in paired serum and CSF of MS patients (n = 56), and compared with healthy controls (n = 203), with the use of the MSD‐platform. RESULTS The MSD‐platform showed overall better assay characteristics such as, sensitivity, recovery and linearity compared to the Luminex for the 4‐plex cytokines in CSF and serum. IL‐6, IL‐8 and TNF‐&agr; (p < 0.001) levels were significantly increased in MS serum compared to healthy controls. Moreover, serum IL‐1&bgr; levels correlated with expanded disability status scale (EDSS) scores (r = −0.34, p < 0.05). Additionally, IL‐6 and IL‐8 CSF levels were both significantly decreased in MS patients compared to non‐inflammatory neurological disease controls. Noteworthy, higher IL‐8 CSF levels than IL‐8 serum levels were observed for MS patients, indicating intrathecal activation of macrophages in MS. CONCLUSION We have demonstrated that the pro‐inflammatory 4‐plex kit of the MSD‐platform shows better assay characteristics in comparison with Luminex kit for quantification of these cytokines in serum and CSF. Overall, the increased levels of IL‐6, IL‐8 and TNF‐&agr; in serum of MS patients compared to healthy controls, support the use of multiple cytokines for future MS biomarker and disease progression research. HIGHLIGHTSAssessment of multiplex immunoassays for IL‐1&bgr;, IL‐6, IL‐8 and TNF‐&agr; quantification.MSD platform overall showed better assay characteristics for IL‐6, IL‐8 and TNF‐&agr;.IL‐1&bgr;, IL‐6, IL‐8 serum levels were higher in MS patients compared to controls.IL‐6 and TNF‐&agr; CSF levels are lower in RRMS compared to NINDC.

Cerebrospinal fluid mtDNA concentration is elevated in multiple sclerosis disease and responds to treatment

Background: Mitochondrial dysfunction is increasingly recognized as an important feature of multiple sclerosis (MS) pathology and may be relevant for clinical disease progression. However, it is unknown whether mitochondrial DNA (mtDNA) levels in the cerebrospinal fluid (CSF) associate with disease progression and therapeutic response. Objectives: To evaluate whether CSF concentrations of mtDNA in MS patients can serve as a marker of ongoing neuropathology and may be helpful to differentiate between MS disease subtypes. To explore the effect of disease-modifying therapies on mtDNA levels in the CSF. Methods: CSF mtDNA was measured using a digital polymerase chain reaction (PCR) CSF mtDNA in two independent MS cohorts. The cohorts included 92 relapsing-remitting multiple sclerosis (RRMS) patients, 40 progressive multiple sclerosis (PMS) patients (27 secondary progressive and 13 primary progressive), 50 various neurologic disease controls, and 5 healthy controls. Results: Patients with PMS showed a significant increase in CSF mtDNA compared to non-inflammatory neurologic disease controls. Patients with higher T2 lesion volumes and lower normalized brain volumes showed increased concentration of mtDNA. Patients treated with fingolimod had significantly lower mtDNA copy levels at follow-up compared to baseline. Conclusion: Our results showed a non-specific elevation of concentration of mtDNA in PMS patients. mtDNA concentrations respond to fingolimod and may be used to monitor biological effect of this treatment.

Recent progress in omics-driven analysis of MS to unravel pathological mechanisms

At present, the pathophysiology and specific biological markers reflecting pathology of multiple sclerosis (MS) remain undetermined. The risk of developing MS is considered to depend on genetic susceptibility and environmental factors. The interaction of environmental factors with epigenetic mechanisms could affect the transcriptional level and therefore also the translational level. In the last decade, growing amount of hypothesis-free ‘omics’ studies have shed light on the potential MS mechanisms and raised potential biomarker targets. To understand MS pathophysiology and discover a subset of biomarkers, it is becoming essential to take a step forward and integrate the findings of the different fields of ‘omics’ into a systems biology network. In this review, we will discuss the recent findings of the genomic, transcriptomic and proteomic fields for MS and aim to make a unifying model.

Real-Time Assessment of Fatigue in Patients With Multiple Sclerosis: How Does It Relate to Commonly Used Self-Report Fatigue Questionnaires?

OBJECTIVES (1) To assess real-time patterns of fatigue; (2) to assess the association between a real-time fatigue score and 3 commonly used questionnaires (Checklist Individual Strength [CIS] fatigue subscale, Modified Fatigue Impact Scale (MFIS), and Fatigue Severity Scale [FSS]); and (3) to establish factors that confound the association between the real-time fatigue score and the conventional fatigue questionnaires in patients with multiple sclerosis (MS). DESIGN Cross-sectional study. SETTING MS-specialized outpatient facility. PARTICIPANTS Ambulant patients with MS (N=165) experiencing severe self-reported fatigue. INTERVENTIONS Not applicable. MAIN OUTCOME MEASURES A real-time fatigue score was assessed by sending participants 4 text messages on a particular day (How fatigued do you feel at this moment?; score range, 0-10). Latent class growth mixed modeling was used to determine diurnal patterns of fatigue. Regression analyses were used to assess the association between the mean real-time fatigue score and the CIS fatigue subscale, MFIS, and FSS. Significant associations were tested for candidate confounders (eg, disease severity, work status, sleepiness). RESULTS Four significantly different fatigue profiles were identified by the real-time fatigue score, namely a stable high (n=79), increasing (n=57), stable low (n=16), and decreasing (n=13). The conventional questionnaires correlated poorly (r<.300) with the real-time fatigue score. The Epworth Sleepiness Scale significantly reduced the regression coefficient between the real-time fatigue score and conventional questionnaires, ranging from 15.4% to 35%. CONCLUSIONS Perceived fatigue showed 4 different diurnal patterns in patients with MS. Severity of sleepiness is an important confounder to take into account in the assessment of fatigue.