Contribution of Metabolomics to Multiple Sclerosis Diagnosis, Prognosis and Treatment.

Metabolomics-based technologies map in vivo biochemical changes that may be used as early indicators of pathological abnormalities prior to the development of clinical symptoms in neurological conditions. Metabolomics may also reveal biochemical pathways implicated in tissue dysfunction and damage and thus assist in the development of novel targeted therapeutics for neuroinflammation and neurodegeneration. Metabolomics holds promise as a non-invasive, high-throughput and cost-effective tool for early diagnosis, follow-up and monitoring of treatment response in multiple sclerosis (MS), in combination with clinical and imaging measures. In this review, we offer evidence in support of the potential of metabolomics as a biomarker and drug discovery tool in MS. We also use pathway analysis of metabolites that are described as potential biomarkers in the literature of MS biofluids to identify the most promising molecules and upstream regulators, and show novel, still unexplored metabolic pathways, whose investigation may open novel avenues of research.

Integrated Lipidomics and Metabolomics Analysis of Tears in Multiple Sclerosis: An Insight into Diagnostic Potential of Lacrimal Fluid.

Metabolomics based on mass spectrometry represents an innovative approach to characterize multifactorial diseases, such as multiple sclerosis (MuS). To date, the most important biomarker source for MuS diagnosis is the cerebrospinal fluid. However, an important goal for research is to identify new molecules in more easily accessible biological fluids. A very interesting biofluid in MuS is represented by tears, considered as an intermediate fluid between the cerebrospinal fluid and serum. In this work, we developed a merged strategy for the analysis of lipids containing choline by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS), as well as for the targeted analysis of free carnitine, acylcarnitines and aminoacids by direct infusion mass spectrometry. Samples for both metabolomics and lipidomics approaches were obtained in a single extraction procedure from tears of patients affected by MuS and healthy controls. Tear lipidomics showed 30 phospholipids significantly modulated and, notably, many sphingomyelins resulted lower in MuS. Moreover, the metabolomics approach carried out both on tears and serum highlighted the diagnostic potential of specific aminoacids and acylcarnitines. In conclusion, the metabolic profiling of tears appears to reflect the pathological conditions of the central nervous system, suggesting that the molecular repository of tears can be considered as a source of potential biomarkers for MuS.

Simultaneous early-onset severe autoimmune hemolytic anemia and albuminuria during alemtuzumab treatment for multiple sclerosis.

BACKGROUND: Alemtuzumab, approved for multiple sclerosis (MS), can cause secondary autoimmune adverse events including thyroid disorders, immune thrombocytopenia (ITP), and glomerular nephropathies. Non-ITP autoimmune cytopenias are rarely reported. OBJECTIVE: To report a case of autoimmune hemolytic anemia (AIHA) and nephropathy in a MS patient treated with alemtuzumab. CASE REPORT: A 34-year-old man with MS developed albuminuria and AIHA after the first and only alemtuzumab treatment, with positive Coombs' direct and indirect tests and IgG autoantibodies. Both AIHA and nephropathy resolved 1 month after treatment with steroids and intravenous immunoglobulins. CONCLUSION: Our report adds to literature on AIHA and nephropathy after alemtuzumab treatment and suggests to add Coombs' tests to the screening panel required for alemtuzumab treatment.

Metabolomic Signature in Sera of Multiple Sclerosis Patients during Pregnancy.

Multiple sclerosis (MuS) is an autoimmune disease of the central nervous system characterized by neuroinflammation, neurodegeneration, and degradation of the myelin sheath. Epidemiological studies have shown that the female gender is more susceptible than the male gender to MuS development, with a female-to-male ratio of 2:1. Despite this high onset, women have a better prognosis than men, and the frequency of the relapsing phase decreases during pregnancy, while it increases soon after birth. Therefore, it is interesting to investigate hormonal fluctuations during pregnancy and whether they correlate with metabolic signatures. To gain a deeper inside into the biochemical mechanism of such a multifactorial disease, we adopted targeted metabolomics approaches for the determination of many serum metabolites in 12 pregnant women affected by MuS by mass spectrometry analysis. Our data show a characteristic hormonal fluctuation for estrogens and progesterone, as expected. They also highlight other interesting hormonal alterations for cortisol, corticosterone, 11-deoxycortisol, 4-androstene-3,17-dione, testosterone, and 17α-hydroxyprogesterone. Furthermore, a negative correlation with progesterone levels was observed for amino acids and for acylcarnitines, while an imbalance of different sphingolipids pathways was found during pregnancy. In conclusion, these data are in agreement with the characteristic clinical signs of MuS patients during pregnancy and, if confirmed, they may add an important tessera in the complex mosaic of maternal neuroprotection.

Guidelines on the clinical use for the detection of neutralizing antibodies (NAbs) to IFN beta in multiple sclerosis therapy: report from the Italian Multiple Sclerosis Study group.

Interferon beta (IFNß) was the first specific disease-modifying treatment licensed for relapsing-remitting multiple sclerosis, and is still one of the most commonly prescribed treatments. A strong body of evidence supports the effectiveness of IFNß preparations in reducing the annual relapse rate, magnetic resonance (MRI) disease activity and disease progression. However, the development of binding/neutralizing antibodies (BAbs/NAbs) during treatment negatively affects clinical and MRI outcomes. Therefore, guidelines for the clinical use for the detection of NAbs in MS may result in better treatment of these patients. In October 2012, a panel of Italian neurologists from 17 MS clinics convened in Milan to review and discuss data on NAbs and their clinical relevance in the treatment of MS. In this paper, we report the panel's recommendations for the use of IFNß Nabs detection in the early identification of IFNß non-responsiveness and the management of patients on IFNß treatment in Italy, according to a model of therapeutically appropriate care.

Oxidative modifications of cerebral transthyretin are associated with multiple sclerosis.

Transthyretin (TTR) is a homotetrameric protein of the CNS that plays a role of as the major thyroxine (T4) carrier from blood to cerebrospinal fluid (CSF). T4 physiologically helps oligodendrocyte precursor cells to turn into myelinating oligodendrocytes, enhancing remyelination after myelin sheet damage. We investigated post-translational oxidative modifications of serum and CSF TTR in multiple sclerosis subjects, highlighting high levels of S-sulfhydration and S-sulfonation of cysteine in position ten only in the cerebral TTR, which correlate with an anomalous TTR protein folding as well as with disease duration. Moreover, we found low levels of free T4 in CSF of multiple sclerosis patients, suggestive of a potential role of these modifications in T4 transport into the brain.

Patient adherence to and tolerability of self-administered interferon ß-1a using an electronic autoinjection device: a multicentre, open-label, phase IV study.

BACKGROUND: Achieving good adherence to self-injected treatments for multiple sclerosis can be difficult. Injection devices may help to overcome some of the injection-related barriers to adherence that can be experienced by patients. We sought to assess short-term adherence to, and tolerability of, interferon (IFN) ß-1a administered via electronic autoinjection device in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: BRIDGE (RebiSmart to self-inject Rebif serum-free formulation in a multidose cartridge) was a 12-week, multicentre, open-label, single-arm, observational, Phase IV study in which patients self-administered IFN ß-1a (titrated to 44 µg), subcutaneously (sc), three times weekly, via electronic autoinjection device. Patients were assessed at baseline and 4-weekly intervals to Week 12 or early termination (ET) for: physical examinations; diary card completion (baseline, Weeks 4, 8 only); neurological examinations (baseline, Week 12/ET only); MS Treatment Concern Questionnaire (MSTCQ; Weeks 4, 8, 12 only); Convenience Questionnaire (Week 12 only); Hospital Anxiety and Depression Scale (HADS); and Paced Auditory Serial Addition Task (PASAT; baseline only). Adherence was defined as administration of ≥ 80% of scheduled injections, recorded by the autoinjection device. RESULTS: Overall, 88.2% (105/119; intent-to-treat population) of patients were adherent; 67.2% (80/119) administered all scheduled injections. Medical reasons accounted for 35.6% (31/87) of missed injections, forgetfulness for 20.6% (18/87). Adherence did not correlate with baseline Expanded Disability Status Scale (P = 0.821) or PASAT (P = 0.952) scores, or pre-study therapy (P = 0.303). No significant changes (baseline-Week 12) in mean HADS depression (P = 0.482) or anxiety (P = 0.156) scores were observed. 'Overall convenience' was the most important reported benefit of the autoinjection device. Device features associated with handling and ease of use were highly rated. Mean MSTCQ scores for 'flu-like' symptoms (P = 0.022) and global side effects (P = 0.002) significantly improved from Week 4-12. Mean MSTCQ scores for pain at injection site and injection pain increased from Week 4-12 (P < 0.001). Adverse events were mild/moderate. No new safety signals were identified. CONCLUSION: Convenience and ease of use of the autoinjection device may improve adherence and, therefore, outcomes, in patients with RRMS receiving sc IFN ß-1a. TRIAL REGISTRATION: EU Clinical Trials Register (EU-CTR; http://www.clinicaltrialsregister.eu): 2009-013333-24.

Relation between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disorder. Since acetylcholine (ACh) is known to participate in the inflammatory response, we investigated the possible relationship between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis (RR-MS) patients. Levels of ACh and pro-inflammatory cytokines IL1-β and IL-17 were measured both in cerebrospinal fluid (CSF) and sera of 22 RR-MS patients in the relapsing phase and in 17 control subjects affected by other non-neurological diseases (OND). We observed higher levels of pro-inflammatory cytokines such as IL-1β and IL-17 in both CSF and serum of RR-MS patients compared to control subjects. Moreover, ACh levels were lower in CSF and serum of RR-MS patients compared to levels of control subjects. Although the relationship between high inflammatory cytokine levels and low ACh levels need to be further investigated in the future, our data suggest that IL-1β, and cytokines induced by it, such as IL-17 and ACh, may be involved in the pathogenesis of MS.

Data of safety in a single-center alemtuzumab treated population.

Alemtuzumab is approved for highly active MS and, in Europe, can be employed after other disease-modifying treatments (DMTs) as an escalation approach or first therapeutic option. The occurrence of secondary autoimmune adverse events and infections differs depending on the employed approach. In the manuscript entitled "Alemtuzumab treatment of multiple sclerosis in real-world clinical practice: report from a single Italian center" by di Ioia M. and collaborators, efficacy and safety data of alemtuzumab were evaluated in a real-world MS population. The aim of the article is to describe in detail the unexpected serious adverse events which occurred in this cohort during and after the administration of the alemtuzumab treatment. Adverse events were observed in 45,7% of the patients. These events were ranked as severe in 23% of the patients. We reported, in particular, cases of autoimmune hemolytic anemia (AIHA), pancytopenia, viral hepatitis E and noninfectious meningo-encephalomyelitis.

Enhanced release of acid sphingomyelinase-enriched exosomes generates a lipidomics signature in CSF of Multiple Sclerosis patients.

Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS.

Integration of metabolomics and proteomics in multiple sclerosis: From biomarkers discovery to personalized medicine.

Personalized medicine is the science of individualized prevention and therapy. In the last decade, advances in high-throughput approaches allowed the development of proteomic and metabolomic studies in evaluating the association of genetic and phenotypic variability with disease sensitivity and analgesic response. These considerations have more value in case of multiple sclerosis (MuS), a multifactorial disease with high heterogeneity in clinical course and treatment response. In this review, we reported and updated about proteomic and metabolomic studies for the research of new candidate biomarkers in MuS, and difficulties in their clinical applications. We focused especially on the description of both "omics" approaches that, once integrated, may synergically describe pathophysiology conditions. To prove this assumption, we rebuilt interaction between proteins and metabolites described in the literature as potential biomarkers for MuS, and a pathway analysis of these molecules was performed. The result of such speculation demonstrated a strong convergence of proteomic and metabolomic results in this field, showing also a poorness of available tools for incorporating "omics" approaches. In conclusion, the integration of Metabolomics and Proteomics may allow a more complete characterization of such a heterogeneous disease, providing further insights into personalized healthcare.

Unraveling the molecular repertoire of tears as a source of biomarkers: beyond ocular diseases.

Proteomics and metabolomics investigations of body fluids present several challenges for biomarker discovery of several diseases. The search for biomarkers is actually conducted in different body fluids, even if the ideal biomarker can be found in an easily accessible biological fluid, because, if validated, the biomarker could be sought in the healthy population. In this regard, tears could be considered an optimum material obtained by noninvasive procedures. In the past years, the scientific community has become more interested in the study of tears for the research of new biomarkers not only for ocular diseases. In this review, we provide a discussion on the current state of biomarkers research in tears and their relevance for clinical practice, and report the main results of clinical proteomics studies on systemic and eye diseases. We summarize the main methods for tear samples analyses and report recent advances in "omics" platforms for tears investigations. Moreover, we want to take stock of the emerging field of metabolomics and lipidomics as a new and integrated approach to study protein-metabolites interplay for biomarkers research, where tears represent a still unexplored and attractive field.

An integrated metabolomics approach for the research of new cerebrospinal fluid biomarkers of multiple sclerosis.

Multiple Sclerosis (MuS) is a disease caused due to an autoimmune attack against myelin components in which non proteic mediators may play a role. Recent research in metabolomics and lipidomics has been driven by rapid advances in technologies such as mass spectrometry and computational methods. They can be used to study multifactorial disorders like MuS, highlighting the effects of disease on metabolic profiling, regardless of the multiple trigger factors. We coupled MALDI-TOF-MS untargeted lipidomics and targeted LC-MS/MS analysis of acylcarnitines and aminoacids to compare cerebrospinal fluid metabolites in 13 MuS subjects and in 12 patients with Other Neurological Diseases (OND). After data processing and statistical evaluation, we found 10 metabolites that significantly (p < 0.05) segregate the two clinical groups. The most relevant result was the alteration of phospholipids levels in MuS and the correlation between some of them with clinical data. In particular lysophosphatidylcholines (m/z = 522.3 Da, 524.3 Da) and an unidentified peak at m/z = 523.0 Da correlated to the Link index, lysophosphatidylinositol (m/z = 573.3 Da) correlated to EDSS and phosphatidylinositol (m/z = 969.6 Da) correlated to disease duration. We also found high levels of glutamate in MuS. In conclusion, our integrated mass spectrometry approach showed high potentiality to find metabolic alteration in cerebrospinal fluid. These data, if confirmed in a wider clinical study, could open the door for the discovery of novel candidate biomarkers of MuS.

Risk-benefit considerations in the treatment of relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and mainly affects young adults. Its natural history has changed in recent years with the advent of disease-modifying drugs, which have been available since the early 1990s. The increasing number of first-line and second-line treatment options, together with the variable course of the disease and patient lifestyles and expectations, makes the therapeutic decision a real challenge. The aim of this review is to give a comprehensive overview of the main present and some future drugs for relapsing-remitting MS, including risk-benefit considerations, to enable readers to draw their own conclusions regarding the risk-benefit assessment of personalized treatment strategies, taking into account not only treatment-related but also disease-related risks. We performed a Medline literature search to identify studies on the treatment of MS with risk stratification and risk-benefit considerations. We focused our attention on studies of disease-modifying, immunomodulating, and immunosuppressive drugs, including monoclonal antibodies. Here we offer personal considerations, stemming from long-term experience in the treatment of MS and thorough discussions with other neurologists closely involved in the care of patients with the disease. MS specialists need to know not only the specific risks and benefits of single drugs, but also about drug interactions, either in simultaneous or serial combination therapy, and patient comorbidities, preferences, and fears. This has to be put into perspective, considering also the risks of untreated disease in patients with different clinical and radiological characteristics. There is no single best treatment strategy, but therapy has to be tailored to the patient. This is a time-consuming task, rich in complexity, and influenced by the attitude towards risk on the parts of both the patient and the clinical team. The broader the MS drug market becomes, the harder it will be for the clinician to help the patient decide which therapeutic strategy to opt for.

Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune demyelinating disease affecting young adults. The aetiology still remains a mystery and diagnosis is impaired by the lack of defined molecular markers. Autoimmune response remains the main topic under investigation and recent studies suggest additional non-proteic mediators of brain inflammation such as lipids. We carried out an LC-MS based lipidomics approach to highlight serum lipids profiling in MS. Method was optimised and applied in a preliminary clinical cross-sectional investigation of MS patients vs Healthy Controls (HC) and patients with Other Neurological Diseases (OND). Ten significant metabolites were highlighted and tentatively identified by accurate mass and MS/MS experiments. Our most relevant data show altered level of lyso-glycerophosphatidylcholine (lysoPC) and glycerophosphatidylcholine (PC) species. Total lysoPC/PC ratio showed significant decrease in pathological groups (MS, OND) and, in addition, MS subjects had a relevant decrease of this ratio also in respect to OND. These findings suggest that there may be an altered phospholipid metabolism in MS that can be evaluated in serum. Some of these features are distinctive and may be considered specific for MS. Our lipidomics data show, for the first time, evidence in serum of a relationship between LysoPC/PC ratio and MS.

Cleavage of cystatin C is not associated with multiple sclerosis.

Recently, Irani and colleagues proposed a C-terminal cleaved isoform cystatin C (12.5 kDa) in cerebrospinal fluid as a marker of multiple sclerosis. In this study, we demonstrate that the 12.5 kDa product of cystatin C is formed by degradation of the first eight N-terminal residues. Moreover, such a degradation is not specific in the cerebrospinal fluid of multiple sclerosis, but rather is given by an inappropriate sample storage at -20 degrees C. We conclude that the use of the 12.5 kDa product of cystatin C in cerebrospinal fluid might lead to a fallacious diagnosis of multiple sclerosis. Preanalytical validation procedure is mandatory for proteomics investigations.